doi:10.1093/plcell/koab189
THE PLANT CELL 2021: 33: 3207–3234
Return of the Lemnaceae: duckweed as a model plant
system in the genomics and postgenomics era
Review
1
2
3
4
5
6
7
8
9
,3
Department of Plant Biology, Rutgers the State University of New Jersey, New Brunswick, NJ 08901, USA
Plant Physiology, Matthias Schleiden Institute, University of Jena, Jena 07737, Germany
The Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben D-06466, Germany
Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
School of Biological, Earth and Environmental Sciences, Environmental Research Institute, University College Cork, Cork T23 TK30, Ireland
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
Department of Environmental Science, Central University of Kerala, Periye 671320, India
Institute for Evolution and Biodiversity, University of Münster, Münster 48149, Germany
Plant Molecular and Cellular Biology Laboratory, The Salk Institute of Biological Studies, La Jolla, California 92037, USA
* Author for correspondence: eric.lam@rutgers.edu (E.L.), toddpmichael@gmail.com (T.P.M.)
Senior authors.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the
Instructions for Authors (https://academic.oup.com/plcell) are: Eric Lam (eric.lam@rutgers.edu) and Todd P. Michael (toddpmichael@gmail.com)
†
Abstract
The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms
known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed
has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants
from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established
community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate
knowledge gained for field deployment to address some of society’s pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative
studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.
Introduction
The duckweed family Lemnaceae belongs to the monocot
order Alismatales (Figure 1) and consists of 36 recognized
species representing five genera: Spirodela (Sp.), Landoltia
(La.), Lemna (Le.), Wolffiella (We.), and Wolffia (Wo.) (Les et
al., 2002; Bog et al., 2020b). The common name duckweed
derives from their global distribution along with waterfowl
such as ducks (Silva et al., 2018) and their prodigious growth
Received December 14, 2020. Accepted June 18, 2021. Advance access publication July 17, 2021
C American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com
V
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Kenneth Acosta ,1 Klaus J. Appenroth ,2 Ljudmilla Borisjuk ,3 Marvin Edelman ,4
Uwe Heinig ,4 Marcel A.K. Jansen ,5 Tokitaka Oyama ,6 Buntora Pasaribu ,1 Ingo Schubert
Shawn Sorrels ,1 K. Sowjanya Sree,7 Shuqing Xu ,8 Todd P. Michael 9,*,† and Eric Lam 1,*,†
3208
| THE PLANT CELL 2021: 33: 3207–3234
K. Acosta et al.
rates. Some duckweeds are commonly referred to as water
lentil (Lemna spp.) or water meal (Wolffia spp.). The individual plant can range in size from 1.5 cm (Sp. polyrhiza) to
<1 mm (Wo. angusta) and is composed of a leaf–stem
structure called a frond, with some genera having roots,
such as Spirodela, Landoltia, and Lemna (Figure 2).
Before Arabidopsis thaliana was adopted as a model plant
in the genomics era, duckweed was an important experimental system for plant physiology and biochemistry. While
a core group of researchers has continued to study duckweeds since the 1950s, the era of genomics has opened new
opportunities to build tools for the broader community.
Like Arabidopsis, Sp. polyrhiza has a small genome of 158
Mb, yet it only has 19,000 annotated genes, which represent a conserved set of angiosperm genes without large
paralog expansions (Wang et al., 2014; Michael et al., 2017).
Moreover, the further loss of genetic redundancy in the 354
Mb Wo. australiana genome, which has even fewer genes
(15,000) with remarkable attrition in pathways such as disease resistance and organogenesis, provides a unique opportunity to define gene functions in a minimalist plant
(Michael et al., 2021). As aquatic plants, duckweeds also present an important opportunity to define the molecular
mechanisms underlying the biochemistry, metabolism, and
interactions with microbial symbionts (Acosta et al., 2020).
The development of tools such as robust transformation
methods and multiomics database resources will make the
duckweed platform more accessible and a versatile component of the plant molecular biology toolkit once again.
As we show in this model system review, the types of
experiments which unique features of duckweeds will enable
and facilitate, as well as their growing commercial applications, make it an exciting model plant in the postgenomic
era. For instance, the reduced anatomical features of duckweed coupled to its clonal reproduction should make it possible to track all cells with current single-cell capture
methods and high-throughput omics technologies. There is
also growing interest in the commercial sector to farm duckweed as a staple food and as a source of protein for plantbased ingredients that are sustainable and resilient to climate change. In this review, we provide an overview of the
species in the Lemnaceae family, how they can be used for
commercial and research applications, current and future
tool development, and available resources that again make
these aquatic monocots an attractive model for plant
research.
Phylogenetic history and traits
Taxonomic position
Closely connected with the simple morphology of duckweeds is the question of whether these aquatic plants represent the end of an evolutionary reduction process
(Hegelmaier, 1868). The emergence of molecular taxonomy
revolutionized the systematics of duckweeds and their position within angiosperms (Les et al., 2002), and demonstrated
that duckweeds are closely related to the Araceae
(Nauheimer et al., 2012). Extending these studies, several
reports deployed plastidic barcodes on a larger number of
clones for each species to determine their phylogenetic relationships within the Lemnaceae (Wang et al., 2010; Borisjuk
et al., 2015; Tippery et al., 2015; Bog et al., 2019). More
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Figure 1 The Lemnaceae family is a sister lineage to other extant monocot families that has readapted to an aquatic lifestyle. (A) Phylogenetic relationship between the greater duckweed (Spirodela, Lemnaceae, Alismatales) and other branches of the angiosperms. The genus names are
shown immediately to the right of the tree diagram, followed by the family names. WGD events are shown in black circles at the approximate
time during their evolutionary history. “A field in a flask”: a culture of Wo. globosa (B) or Sp. polyrhiza (C) plants in a flask of growth medium. (D)
Turions are a dormant form of many Lemnaceae species that enable these simple plants to overwinter at the bottom of their resident water bodies. Pictured is a clone of Sp. polyrhiza under low phosphate conditions in the growth medium. A typical growing frond cluster is shown in the
middle, surrounded by turions. DF, daughter frond; MF, mother frond; T, turion.
Duckweed for plant systems studies
THE PLANT CELL 2021: 33: 3207–3234
| 3209
recently, amplified fragment length polymorphisms
(AFLPs) and the application of genotyping-by-sequencing
helped to resolve problematic species that were difficult
to distinguish based on plastidic sequences alone (Bog
et al., 2020a, 2020b, 2020c). In contrast to the Angiosperm
Phylogeny Group III definition (APG, 1998), which characterizes duckweeds as a subfamily (Lemnoideae) of the
Araceae, many researchers in the field now consider duckweeds to be a separate family (Lemnaceae), which is in
agreement with general taxonomic rules (Bog et al., 2020a;
Tippery and Les, 2020).
Phylogenetic analysis with both nuclear and plastidic
markers showed that Spirodela and Landoltia represent older
phylogenetic branches from the common ancestor than the
more recently derived genera of Lemna, Wolffiella, and
Wolffia. When comparing species belonging to each of the
five genera of Spirodela (2 species), Landoltia (1), Lemna
(12), Wolffiella (10), and Wolffia (11), a reduction of organismic complexity from Spirodela toward Wolffia is observed,
which is quantified by the so-called “degree of primitivity”
(Landolt, 1986), and is generally accompanied by a higher
nuclear DNA content (Figure 3). These observations suggest
an evolutionary trajectory from a more to less differentiated
plant body during adaptation to the aquatic lifestyle
through a series of morphological reductions (Landolt,
1986). While there is general concordance at the genus level
between taxonomic trees created with morphological characteristics (Landolt, 1986) and those generated from
molecular data (Borisjuk et al., 2015; Bog et al., 2019), the
latter has a much higher resolving power at the species
level.
Anatomy, morphology, and growth characteristics
The adaptation of duckweeds to a floating aquatic lifestyle
apparently led to morphological and biochemical properties
distinct from those normally found in land plants (Figure 2).
Examples of such features are their meristem structure and
low lignin content in the cell wall. Endemic species such as
We. denticulata and We. gladiata have evolved distinctive
morphologies compared to the more cosmopolitan pioneer
species, such as Sp. polyrhiza and Le. minor (Figure 2). The
simplified vegetative body of a duckweed is called a frond or
thallus (Hillman, 1961). Depending on the genus, duckweed
fronds can be roundish to obovate (Spirodela, Landoltia, and
Lemna), hemispherical or boat-shaped (Wolffia) or sickleshaped, tongue-shaped or ovate (Wolffiella). The fronds of
subsequent generations are held together, in some cases
even after maturity, thus resulting in colonies of connected
fronds (see Le. trisulca in Figure 2). The smallest colonies
consist of two fronds (Wolffia and some Wolffiella) and
the largest range up to 50, such as in We. gladiata and
Le. trisulca (Landolt, 1986; Bog et al., 2020a). The fronds are
physically held together with the help of an elongated stipe.
Abscission zones (two in Sp. polyrhiza and one in Wo.
microscopica) are present on the stipe, facilitating the separation of daughter fronds from the mother frond (Landolt,
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Figure 2 Morphological variations among diverse genera and species of duckweed. Six different species from four genera of duckweeds are shown
to illustrate the various sizes and shapes of these aquatic plants. The genome sequences of three of these clones (Sp. polyrhiza 9509, Le. minor
5500, and Wo. australiana 8730) are currently available.
3210
| THE PLANT CELL 2021: 33: 3207–3234
K. Acosta et al.
1986; Kim, 2016). The stipe originates from the base at the
ventral side of the mother frond from where the cells further divide and grow (Landolt, 1986; Sree et al., 2015a). This
could be considered intercalary growth at the base of the
frond. The stipe has been speculated to function in the
transport of nutrients and substances from the mother
frond to the daughter frond (Kim, 2016).
A transparent waxy cuticle encloses the entire duckweed
body and fortifies its epidermis against mechanical injury
and solar radiation (Borisjuk et al., 2018). It may also serve
as a barrier for gas and solute exchange controlled by the
epidermis. The cell walls of epidermal cells in duckweed
have distinct morphologies depending on the genus and are
bent (Spirodela), undulated (Landoltia, Lemna), or straight
(Wolffiella, Wolffia). While stomata are found on the epidermis of duckweeds, these aquatic plants do not form trichomes or root hairs (Landolt, 1986). Differentiation of
stomata is restricted to the dorsal epidermis and may depend on growth conditions, such as light and temperature
(Klich et al., 1986). In contrast to land plants, stomata remain open in Lemna (McLaren and Smith, 1976) even upon
prolonged exposure to the phytohormone abscisic acid
(ABA). The ventral epidermis is involved in nutrient uptake
(Cedergreen and Madsen, 2002) and may provide an active
surface for interactions with aquatic bacteria (Duong and
Tiedje, 1985).
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Figure 3 Phylogeny and variations in genome size of different Lemnaceae species. Left: Evolutionary relationships between Lemnaceae species
based on maximum likelihood analysis of concatenated alignment of 139 atpF-atpH and psbK-psbI intergenic spacer sequences from all 36
Lemnaceae species with taro (Colocasia esculenta) as an outgroup. Numbers in parentheses represent the number of clones analyzed. Species that
could not be confidently resolved into a single clade were collapsed into a multispecies clade. One interesting observation is that the plastidic barcode sequences of Wo. brasiliensis consistently showed higher similarity to those of We. hyalina and We. rotunda, while morphologically it is distinctly a Wolffia species. This apparent discrepancy could be due to potential hybridization events in the past that resulted in the transfer of
plastid genome sequences from a Wolffiella ancestor to a Wolffia lineage. Future genome sequencing of relevant species that may be involved will
help clarify this issue. For a detailed methods description, see https://github.com/kenscripts/tpc_dw_review/. Right: The genome sizes for 28 selected species from six groups representing all five genera were estimated using several methods, and in some cases the genome sizes for a significant number of clones from the same species were measured (Sp. polyrhiza, Sp. Intermedia, La. punctata, and Le. minor). Genome size estimates
were carried out by flow cytometry (FC, black outline), which requires the inclusion of accurate controls, or by K-mer frequency analysis (kmer,
red outline), which relies on high quality short-read sequencing data. Numbers in red depict the number of clones used for each species in genome
size estimations. The genome size of We. neotropica was estimated based on K-mer frequency.
Duckweed for plant systems studies
| 3211
Turions are thought to represent overwintering, dormant
duckweed derived from the meristematic pocket in place of
normal proliferating daughter fronds (Figure 1D). As it
matures, the color of a dormant turion can change from
green to purple due to hyperaccumulation of anthocyanin;
the mature turion eventually detaches from the mother
frond colony and sinks to the bottom of the water body.
When proper light, temperature, and nutrient conditions return, such as in spring, turions can germinate and resume
metabolic activities (Landolt and Kandeler, 1987; Appenroth
and Augsten, 1990). During the early phase of germination,
the low-molecular weight carbohydrate reserve is consumed,
but starch degradation is not observed. Storage starch, however, is used to support the rapid growth of newly germinated fronds, allowing them to cover the water surface
quickly in spring (Appenroth et al., 2013).
Compared to fronds, turions have smaller cells, lack aerenchyma and plasmodesmata, and have thicker cell walls
(Jacobs, 1947; Kim, 2013). Turion cells are densely packed
with starch grains (Smart and Trewavas, 1983b), with starch
content in turions exceeding 70% dry weight (Dolger et al.,
1997). In addition to the Lemnaceae, turion-producing species have been reported in 11 genera of aquatic vascular
plants (Adamec 2018), indicating that this may be a common strategy for adaptation to aquatic habitats. In the
Lemnaceae, turions are observed in almost all Wolffia species
(not reported for Wo. microscopica), Le. turionifera, Le. perpusilla, Le. aequinoctialis (Landolt, 1986), and Sp. polyrhiza
(Figure 1D). Abiotic turion-inducing factors include limiting
nutrient levels (phosphate, nitrate, sulfate) and low temperatures (Appenroth et al., 1989; Appenroth, 2002; Appenroth
and Nickel, 2010). Turion formation rates for different clones
of Sp. polyrhiza were shown to be linked to local climatic
conditions (Kuehdorf et al., 2014). The ‘specific turion yield’
is a quantitative trait that can be predicted for an adapted
population of Sp. polyrhiza by using five different local climatic parameters. This remarkable observation indicates
that the production of turions in Sp. polyrhiza is a key trait
for the species’ survival in a particular climate. In addition
to nutrient limitation in the growth medium, ABA is a
powerful experimental tool to induce the formation of
turions (Smart and Trewavas, 1983a) and could function as
an intracellular mediator of other turion-inducing factors
(Smart et al., 1995).
Vegetative reproduction, such as the budding of clonal
daughter fronds from a mother frond (Figure 1), is the most
common mode of duckweed propagation. Fronds display a
limited lifespan of a few weeks and produce a finite number
of daughter fronds whose area progressively diminishes
(Ashby et al., 1949). Therefore, growth in duckweeds
includes an increase in cell size, the number of individual
plants, and the number of daughter fronds produced by
each plant. Growth can thus be measured in terms of
either biomass (such as fresh weight and dry weight) or the
number of fronds. A standard measure of relative yield for
duckweed was recently introduced (Ziegler et al., 2015). In
nature, growth responses of different duckweed species to
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
The bulk of the frond consists of parenchyma cells with a
central vacuole, contributing to the high (up to 95%) water
content of the tissues. Dorsal cell layers contain a higher
density of chloroplasts (Figure 2) and perform the function
of the chlorenchyma (White and Wise, 1998; Kwak and Kim,
2008). The photosynthetic properties of chloroplasts and
their rearrangements in response to light can be associated
with distinct light-utilization strategies in different species
(Paolacci et al., 2018a). A loose tissue structure, increased
cell size, and formation of gas/air spaces (Jones et al., 2021)
are indicative of the aerenchyma. The aerenchyma in duckweed supports the exchange of gases between the dorsal
and ventral portion of the body. Importantly, the presence
of the aerenchyma may also allow fronds to control their
degree of flotation on or under the water surface by regulating the air space volume within fronds (Landolt and
Kandeler, 1987).
The duckweed root is an adventitious organ found in
Spirodela, Landoltia, and Lemna species (Bellini et al., 2014)
that develops on the lower side of the frond, next to the
budding pouches, and is subtended by both the epidermal
sheath at the junction and by the root-cap at the root tip.
Apical root growth is followed by the differentiation of epidermis, cortex, tracheary elements, and phloem cells
(Melaragno and Walsh, 1976; Echlin et al., 1982; Landolt,
1986; Kim, 2007). Because of the considerable length and
the thread-like structure with high cytoplasmic density at
the root tip, the root may act like a pendulum to attenuate
dynamic loads from water and wind motion.
The frond meristem is composed of densely packed, proliferating mitotic cells that are significantly smaller than their
neighboring parenchyma cells. Frond meristem cells contain
small vacuoles and proplastids with only a few thylakoids
(McCormac and Greenberg, 1992; Kim, 2011). Meristems
usually localize to the ventral side of the frond body inside a
small cavity (vegetative pouch) where clonal daughters bud
and detach (Landolt, 1986; Lemon and Posluszny, 2000).
Wolffia and Wolffiella contain only one basal pouch, whereas
Spirodela, Landoltia, and Lemna possess two lateral pouches.
At the abscission stage, the young daughter frond usually
contains at least two successive generations of vegetative
buds (Rimon and Galun, 1968; Sree et al., 2015a). While differentiation of the meristem is less understood in duckweeds and rather unlike the differentiation of the canonical
shoot apical meristem of land plants, the frond itself can be
botanically described as a juvenile tissue (Landolt, 1986).
Interactions between meristem cells and functionally differentiated tissues of the frond mainly occur via symplastic
connections. The arrangement of plasmodesmata and their
dynamic features appear to be sufficient for communication
and metabolite transport. In general, a vascular system is
completely absent (Wolffia and Wolffiella) or fairly simplified
(Spirodela, Landoltia, and Lemna) in duckweeds (Landolt,
1986).
A prominent morphological feature that facilitates the
survival of many duckweed species under unfavorable environments is the formation of turions (Landolt, 1986).
THE PLANT CELL 2021: 33: 3207–3234
3212
| THE PLANT CELL 2021: 33: 3207–3234
Chromosomes and genomics of duckweeds
Variations in chromosome number and genome size
While the most common diploid chromosome number in
duckweeds is 2n ¼ 40 (Hoang et al., 2019), highly variable
chromosome numbers have been reported within some
duckweed species. For example, 20–84 chromosomes have
been reported for Le. aequinoctialis (Urbanska, 1980; Geber,
1989; Wang et al., 2011; Hoang et al., 2019). Even considering
the predominantly asexual propagation of duckweed species,
such high variability in chromosome number appears unusual. However, in cases where previously variable numbers
were reported and the corresponding clones are still available, deviating chromosome numbers could not always be
confirmed (Hoang et al., 2019). Studies with some of the
cultured clones indicated apparent autotetraploidy (e.g. Le.
aequinoctialis, 2n ¼ 42 and 84), which might have occurred
spontaneously or could be chemically induced, such as in
La. punctata 5562 with 2n ¼ 46 and 92 (Vunsh et al., 2015;
Hoang et al., 2019).
Genome size across 28 duckweed species with available
data (Figure 3) ranges from 158 Mb in the phylogenetically oldest genus Spirodela up to 2,203 Mb in Wo. arrhiza,
a species in the phylogenetically most recent genus (Wang
et al., 2011; Smarda et al., 2014; Van Hoeck et al., 2015; Bog
et al., 2015, 2020c; Hoang et al., 2019; Michael et al., 2021).
The largest intrageneric variation was found in the most derived genus Wolffia, spanning from 354 to 2,203 Mb. Among
eleven species sampled across the five genera of duckweed,
genome size was positively correlated with guard cell and
nucleus volume, in parallel with progressive organ size reduction. In contrast, no correlation between genome size
and the number of chromosomes was observed (Hoang et
al., 2019). More surprising is the considerable variation in estimated genome size for clones within some species. For 27
Le. minor clones, genome size varied from 356 to 604 Mb
(Wang et al., 2011), while two verified diploid genomes from
this species displayed size estimates of 409 and 481 Mbp by
K-mer analysis for clones 5500 and 8623, respectively (Van
Hoeck et al., 2015; Hoang et al., 2019). With Le. aequinoctialis
and La. punctata, a possible explanation is spontaneous
whole-genome duplication (WGD) in clones of these species
(Hoang et al., 2019). Further, cytogenomic investigations and
whole-genome resequencing would be helpful for resolving
whether clone-specific WGD occurred, followed by an extremely rapid genome size reduction, or whether a subset of
clones comprise interspecific hybrids or cryptic species.
Genome sequences and features suggest novel modes of
regulation
The first duckweed genome sequenced was that of Sp. polyrhiza due to its small genome size of 158 Mb and basal
position in the Lemnaceae. The initial Sp. polyrhiza reference
genome draft (20 coverage) was carried out with clone
7498 (Sp7498) from Durham, NC, USA. The genome sequence revealed two lineage-specific ancient WGD events
(Figure 1A) and the smallest gene repertoire of any plant sequenced at the time of its publication, with 19,623 proteincoding genes (Wang et al., 2014), which is 30% fewer than
reported for A. thaliana. While the Sp. polyrhiza genome
shares most of the core gene families found in plants, its reduced gene content reflects the lowest gene expansion and
copy number, which is consistent with the aggressive removal of duplicated genes after the two WGD events.
Therefore, the Sp. polyrhiza genome is ideal for gene
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
abiotic factors such as light and temperature are closely
linked to the range of their geographical distribution.
Consistent with geobotanical data, some S. polyrhiza clones
can withstand temperatures of up to 38 C, unlike Le. minor
clones, which typically display growth arrest at 32 C.
Similarly, Le. minor clones can often withstand temperatures
down to 5 C, while Sp. polyrhiza clones typically only tolerate temperatures as low as 12 C before growth arrest
(Docauer, 1983; Landolt, 1986). Like turion formation,
growth rate also displays a high level of clonal dependence
(Sree et al., 2015b; Ziegler et al., 2015).
Although vegetative clonal division is most common in
duckweeds, they can also propagate generatively through
sexual reproduction. When flowering occurs, the floral
organs are located in a cavity on the dorsal surface of the
frond in Wolffiella and Wolffia spp. or in a membranous,
sac-like spathe within a lateral budding pouch in Spirodela,
Landoltia, and Lemna spp. (Landolt, 1986; Lemon and
Posluszny, 2000). Flower size and morphology are minimized
to a male (androecium) and female (gynoecium) floral organ, while a corolla and calyx are absent. Flowers are bisexual, usually protogynous, and smallest in Wolffia and
Wolffiella. Modulation of various abiotic factors and the addition of different chemical molecules to duckweeds have
been used as inducers of flowering under in vitro conditions.
Exposure to low temperature (22 C) was found to induce
flowering in Wo. microscopica (Rimon and Galun, 1968),
while the effects of phytohormones, chelators, heavy metal
ions, and photosynthetic products on flowering initiation
have been investigated in other species (Landolt and
Kandeler, 1987). Among these, ethylenediamine-di-ohydroxyphenylacetic acid (EDDHA) and salicylic acid (SA)
were reported to be floral inducers in duckweeds
(Maheshwari and Seth, 1966; Cleland and Ajami, 1974), even
under noninductive conditions in terms of day length
(Landolt and Kandeler, 1987). For example, EDDHA could
induce flowering in short-day plants (Maheshwari and Seth,
1966), long-day plants (Pieterse and Müller, 1977), and dayneutral plants (Khurana and Maheshwari, 1986). EDDHA
was initially thought to act by chelating metal ions that
might be required for floral induction in duckweeds. It was
later suggested that the breakdown of EDDHA may release
SA-like active molecules (Tanaka et al., 1979; Pieterse, 2013).
In line with the current understanding of the role of SA in
plant defense, it was suggested that flowering could be a
stress response and that endogenous SA produced in
stressed plants leads to flower induction (Pieterse, 2013).
K. Acosta et al.
Duckweed for plant systems studies
| 3213
high-quality genome assemblies revealed that Wa7733
and Wa8730 have a further reduction in the number of
predicted protein-coding genes compared to Sp. polyrhiza,
with 15,312 and 14,324, respectively. This finding is consistent with the minimal tissue types and lack of roots in Wo.
australiana (Michael et al., 2021). The Wo. australiana genome also contains 50% repetitive sequences, which is less
than that of Le. minor (61%) but approximately twice that
of Sp. polyrhiza (25%). Consistent with having the smallest
genome in the Wolffia genus, transposable elements have
been actively purged from Wo. australiana, resulting in an
even higher solo-to-intact ratio than in Sp. polyrhiza. The reduction in circadian, light signaling, developmental, rootrelated, and disease resistance genes in the Wo. australiana
genome is apparently linked to the proliferation and purging
of transposable elements, which provides a unique opportunity to explore the genesis and genomic architecture of a
highly derived plant.
These initial genomics studies in the Lemnaceae represent
an important resource for the plant biology community.
With their basal position in the monocot lineage, the
Lemnaceae provide valuable information about the genome
structures of the early common ancestors of grasses and
other grain crops. In addition, as an angiosperm family that
has adapted to an aquatic habitat, the distinct developmental attributes in the Lemnaceae offer unique opportunities
to associate gene presence or absence to the gain and loss
of traits. Thus, high-quality genome assemblies are being
completed for additional duckweed species in diverse genera. For instance, the genomes of two Sp. intermedia clones
(Si8410 and Si7747) were recently sequenced, shedding light
on chromosomal dynamics between Sp. intermedia with 18
chromosomes (2n ¼ 36) and Sp. polyrhiza with 20 chromosomes (2n ¼ 40) (Hoang et al., 2020). Also, draft genomes
for Le. minuta, Le. turionifera, Le. japonica, La. punctata,
and We. neotropica have been generated: requests can be
made for the data from the corresponding authors. These
resources and additional genome assemblies covering the
remaining duckweed species should provide an essential
foundation for understanding the relationships between
genera and species in Lemnaceae, as well as the evolutionary
path that enables these tiny plants to adapt to aquatic
niches in a wide geographic range.
Ecology and biogeography
Duckweed biota
Duckweeds typically inhabit relatively small and shallow water bodies in areas ranging from tropical to boreal regions
(Supplemental Table S1). The freshwater ecosystems in
which Lemnaceae can be found include small rivers, lakes,
ditches, and wetlands. Lemnaceae represent cornerstone
species in aquatic food webs, as they comprise an essential
food resource for many organisms (Landolt, 1986). On the
other hand, the growth of Lemnaceae can also reduce the
abundance of other macrophytes and microphytes due to
their competition for nutrients and light in the water
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
discovery and functional analysis of highly conserved core
pathways in plants, since it likely contains fewer redundancies that often confound reverse genetic approaches.
To improve the Sp. polyrhiza genome assembly and to
help uncover the genetic basis for variations in turion formation, clone 9509 (Sp9509) from Lotschen, Germany was
chosen for assembly of a high-quality genome due to its
lower specific turion yield compared to Sp7498 (Kuehdorf et
al., 2014). Sp9509 was sequenced using a combination of
high coverage (100) Illumina short read libraries containing
small (500 bp) to large (20 kbp) genome fragment
inserts and single-molecule BioNano Genomics optical maps
for additional chromosome scaffolding (Michael et al., 2017).
The higher resolution Sp9509 reference genome revealed the
highest solo-to-intact ratio of any plant genome tested to
date at the time, indicating that this species is actively purging active long terminal repeat retrotransposons as well as
other sequences to maintain or further decrease its genome
size. This finding is consistent with the reduced proteincoding gene content after two WGD events and is further
supported by the observation that Sp. polyrhiza has retained
only 20% of the ribosomal DNA repeats found in similarly
sized plant genomes. In addition, the Sp9509 genome has
the lowest DNA methylation level of any known flowering
plant, specifically in the syntenic regions with retained
paralogs involved in growth control and photosynthesis having little to no DNA methylation (Michael et al., 2017).
Comparison of the two Sp. polyrhiza reference genomes also
revealed a surprisingly low level of nucleotide diversity and
highly conserved chromosomal structure. Resequencing of
additional Sp. polyrhiza populations from across the globe
further extended this observation and revealed a very low
level of nucleotide variation in this species yet a large population size, which possibly reflects its rapid clonal propagation and local adaptation (Ho et al., 2019; Xu et al., 2019).
More recently, additional orthogonal technologies have been
applied to further improve the end-to-end assemblies and
gene annotation for the 20 chromosomes in these two
Sp. polyrhiza reference genomes by several groups (Hoang et
al., 2018; An et al., 2019; Harkess et al., 2021). These are excellent resources for duckweed research specifically and
more broadly for comparative genomics in plant research.
Lemna minor (Lm5500) was the first duckweed species to
be sequenced after Sp. polyrhiza, revealing a slightly larger
gene repertoire of 22,382 protein-coding genes and a much
higher repeat content of 61% (Van Hoeck et al., 2015).
Lm5500 is diploid, in contrast to another sequenced Le. minor clone (Lm8627), which is polyploid. The assembled
sequences for both Lm8627 and Le. gibba clone 7742
(Lg7742) are currently available to the community (lemna.org). The intraspecific differences in genome size and
chromosome number that have been observed in Le. minor
clones (Figure 3; Wang et al., 2011) contrast with the relatively stable and highly conservative genome of Sp. polyrhiza.
Since Wo. australiana has the smallest genome of the
Wolffia genus at 354 Mb, reference genomes were recently
completed for two clones (Wa7733 and Wa8730). These
THE PLANT CELL 2021: 33: 3207–3234
3214
| THE PLANT CELL 2021: 33: 3207–3234
Duckweed dispersal and distribution
Lemnaceae, which rarely undergo sexual reproduction, are
model species to study the dispersal of vegetative propagules. The local dispersal of Lemnaceae can be facilitated by
streaming water and occasionally by strong winds, while dispersal over longer distances (>10 km), as well as dispersal
between separate, isolated waterbodies, is often facilitated
by a dispersing organism. Birds, in particular, have long been
flagged as epizoochorous dispersers of many aquatic plants,
including the Lemnaceae (Darwin, 1859; Coughlan et al.,
2017). The mechanism underlying the attachment to a disperser species is rarely studied, but nonspecific entanglement is often assumed. It was recently suggested that the
stickiness of roots may play a key role in attaching colonies
of rooted Lemnaceae species to disperser surfaces (Cross,
2017). Once attached to a disperser, dehydration and the associated loss of viability of the propagule are major constraints for the survival of the dispersed plant (Landolt and
Kandeler, 1987). However, mallard ducks can effectively
transfer Le. minuta across significant distances (up to 250
km), likely due to the humid microclimate that exists between the feathers of dispersing birds (Coughlan et al., 2015;
Coughlan et al., 2017). Perhaps unexpectedly, endozoochorous dispersal has also been reported. Viable Wo. columbiana propagules were identified in the feces of ducks and
swans, indicating that plants can survive passage through
the guts of some waterfowls (Silva et al., 2018).
Endozoochory may also be relevant for the dispersal of relatively well-protected turions (Landolt and Kandeler, 1987).
Thus, epi- and endozoochorous transport can realistically facilitate the dispersal of Lemnaceae over distances of several
hundred kilometers. The dispersal of Lemnaceae over longer
distances most likely occurs through a “step-by-step” process
involving a series of intermediate waterbodies with birds acting as dispersing agents (Coughlan et al., 2017), which might
explain the disjointed population distribution for some species of Lemnaceae (Les et al., 2003). While the likelihood
that a propagule survives a long flight may be low, staggering numbers of birds covering large distances as part of their
annual migration (sometimes in excess of 10,000 km) could
provide a potential transport highway, with a significant
number of successful dispersals of viable individuals.
The extent of natural dispersal of Lemnaceae is debated,
and contradictory information can be found in the literature. It might be surmised that the extent of dispersal could
potentially be revealed by the genetic structure of a population. Studies of the intraspecific genetic variation within a
local population of Le. minor showed that such populations
are made up of just a small number of genetically distinct
individuals, while substantial intraspecific diversity has been
observed between closely located populations of Le. minor
(Cole and Voskuil, 1996; Martirosyan et al., 2008; El-Kholy et
al., 2015). These data suggest low levels of gene flow between populations. In some cases, such limitations in gene
flow were attributed to geographic barriers. Resequencing
and comparative study of 68 genomes of Sp. polyrhiza suggested that the Himalayas represent one of the barriers preventing the dispersal of this species in Southeast Asia (Xu et
al., 2019), while a smaller study of 23 clones of Sp. polyrhiza
suggested that the Hungarian population is isolated from
other European genotypes, possibly due to the mountainous
borders surrounding this region (Chu et al., 2018).
Alien, invasive duckweeds
Plants, animals, and other organisms that disperse to new
locations and negatively affect the ecosystems and ecosystem services of their new environment are defined as alien
invasive species. Many species of Lemnaceae have dispersed
widely beyond their natural distribution range and are considered to be more invasive than other species based on
traits such as rapid vegetative propagation (Moodley et al.,
2016). A prime example of an alien invasive species is Le.
minuta. This species is native throughout the temperate
zones of the Americas, but it dispersed widely throughout
Eurasia in the 1950–1960s through natural means, such as
bird-mediated dispersal (Ceschin et al., 2018; Lucey, 2003;
Mifsud, 2010). Le. minuta is not the only alien Lemnaceae in
Europe, since evidence is emerging that both Le. turionifera
and Le. valdiviana are invasive in parts of Eurasia (Iberite et
al., 2011), as is Wo. columbiana (Ardenghi et al., 2017). La.
punctata is another alien species in both Europe and North
America. Florida in the United States, with its extensive
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
column (Scheffer et al., 2003). Thus, changes in duckweed
growth and distribution can drastically influence the diversity and stability of freshwater ecosystems. Because of anthropogenic activities, nitrogen and phosphorus levels in the
water column as well as average water temperature have increased globally. The combination of these factors changes
the growth dynamics and equilibrium between duckweeds
and their biota. Using mesocosms (controlled outdoor experimental systems), Feuchtmayr et al. (2009) showed that
both warming water and increased nutrient levels in the water column favor duckweeds over phytoplankton, one of the
major competitors of duckweeds (Scheffer et al., 2003).
Studies on the interactions between Le. minor and moth larvae (Cataclysta lemnata), a natural herbivore of duckweeds,
showed that increased temperature reduced the grazing
pressure of Le. minor by the insect (Van Der Heide et al.,
2006). Consistently, long-term monitoring of Dutch ditches
showed that higher temperatures and increased water nutrient levels increased the risk of duckweed dominance, which
can result in a reduction in biodiversity (Peeters et al., 2013).
Therefore, models that can forecast different stable states
between duckweeds and their biota can be useful for developing sustainable strategies that prevent the loss of biodiversity in freshwater ecosystems in the future (Scheffer et al.,
2003). To this end, systematic and quantitative measures of
the ecological consequences of duckweed dominance are
needed. Also, the current long-term and global freshwater
ecosystems monitoring system (https://www.sdg661.app/
home) would benefit from the inclusion of Lemnaceae.
K. Acosta et al.
Duckweed for plant systems studies
aquatic habitats, is home to at least six nonnative species of
Lemnaceae (Ward and Hall, 2010). However, La. punctata is
the only species to exert a strong enough impact on ecosystems to be considered an alien, invasive species.
Comparative analysis of congeneric plant species with similar morphological structures and lifecycles is a powerful tool
to identify plant traits related to invasiveness. This makes
the Lemnaceae excellent model organisms for the study of
dispersal and invasiveness, two processes that are particularly relevant in a world experiencing climate change.
The potential commercial applications of duckweed have
attracted investigators from both the basic and applied sectors for more than 50 years. Aside from their prodigious
growth rates, other unique qualities of duckweed that compare favorably to traditional crop plants are their natural
aquatic habitat (which obviates the need for arable land),
their small size, and a floating lifestyle that enables easy harvesting. Duckweeds can produce relative yields (i.e. the
amount of biomass after 7 days of cultivation starting with
1 g of initial biomass) of up to 50 g in the case of some
clones of Wo. microscopica under optimized conditions
(Sree et al., 2015b). With these attractive qualities and an
urgent need for additional crops that can be produced
sustainably as well as economically, there are great opportunities to develop the Lemnaceae into a novel agriculture
platform that can augment traditional farming systems.
While there are engineering challenges for growing duckweeds reliably at scale, in addition to potential hurdles involved in creating a market for duckweed-related products,
there are also encouraging advances in delineating numerous applications that duckweeds are well suited for. Here,
we summarize these applications to illustrate how basic research in duckweed could have societal impacts in the near
term.
Human nutrition and animal feed
Duckweeds, especially Wo. globosa, have traditionally been
used as human food source in some Asian countries such as
Thailand, Laos, and Cambodia (Bhanthumnavin and
McGarry, 1971). Rusoff et al. (1980) reported four duckweed
species with the remarkably high protein content of
35%–40% of dry weight and an essential amino acid spectrum for the human diet that compares well with soybean
(Glycine max). Representatives from all five known genera
(Appenroth et al., 2017), and especially from Wolffia
(Edelman and Colt, 2016), including all 11 species of the genus (Appenroth et al., 2018), were analyzed for their starch,
protein, fat, mineral, vitamin, and phytosterol content, as
well as amino acid and fatty acid spectra. All data showed
that their contents in duckweeds were in good agreement
with the recommended levels for human nutrition by the
World Health Organization. Moreover, no toxic effects on
three different human cell lines were detected in extracts of
different duckweeds spanning all five genera (Sree et al.,
| 3215
2019). While some duckweed species such as those in the
Lemna genus have been known to contain significant levels
of calcium oxalate in the form of raphides (Landolt, 1986),
which may be linked to health issues such as kidney stones,
raphides are not found in the rootless duckweeds such as
Wolffiella spp. (Appenroth et al., 2017). Over the past decade, several companies (e.g. Parabel, Hinoman, GreenOnyx,
and Plantible) have emerged that aim to popularize duckweed as a food and protein source, while products derived
from Lemna and Wolffia species have been granted
Generally Recognized as Safe status by the US Food and
Drug Administration. These activities are thus paving the
way for the large-scale use of duckweed-derived products
for human consumption.
The use of duckweed as animal feed also has a long tradition. Animals such as cows, chicken, pigs, ram, sheep, horses,
and especially a broad spectrum of fishes were reported to
feed on duckweeds (Landolt and Kandeler, 1987; Sonta et
al., 2019). Comparisons between duckweed and corn (Zea
mays) revealed that duckweed can be a better feedstock for
animals than corn kernels due to its high protein content of
up to 30% (Lee et al., 2016). However, the commercial application of duckweeds for livestock feeding might be a challenge since the current price of animal feed is generally low.
Nonetheless, duckweed could attract a higher price when
used as a nutritious pet food, making this a more realistic
possibility. As a source of either food or animal feed, a high
protein content in duckweed biomass is desirable, since adequate protein intake is a major requirement for the proper
development of young animals as well as proper health in
adults.
Feedstocks for biofuel and biogas production
Duckweeds can accumulate up to 50% starch on a dryweight basis, with increased starch accumulation during
wastewater cleaning (Cheng and Stomp, 2009), making
them a potential feedstock for bioenergy production (Ma et
al., 2018). Several treatments can induce starch accumulation in duckweed, such as abiotic stressors, nutrient limitation (Cui and Cheng, 2015; Guo et al., 2020), the addition of
the phytohormone ABA (Liu et al., 2018), and treatment
with heavy metals or salt (Sree et al., 2015c; Shao et al.,
2020). Duckweed starch can then be degraded to sugars and
fermented to bioethanol or higher alcohols, such as butanol
(Cui and Cheng, 2015). In addition to starch and soluble
sugars, the cell wall material from duckweed, comprising
more than 30% of its biomass, is more easily converted to
fermentable sugars compared to the cell wall material of the
energy plant sugarcane due to its low lignin content
(Sowinski et al., 2019; Pagliuso et al., 2020). Duckweed biomass can also be used to produce biogas via anaerobic digestion (Ren et al., 2018) and can be combined with
saccharification and fermentation to bioalcohols to significantly enhance the total energy output (Calicioglu and
Brennan, 2018; Kaur et al., 2019). In addition, wastewater purification could be readily combined, at least in principle,
with the production of bioalcohols or biogas (Xu et al.,
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
What are duckweeds good for?
THE PLANT CELL 2021: 33: 3207–3234
3216
| THE PLANT CELL 2021: 33: 3207–3234
2012; Cui and Cheng, 2015). The high rate of biomass production by duckweed, assimilation of CO2, and its benefit
for carbon credit should be economically relevant in view of
its potential to mitigate climate change via carbon
sequestration.
Phytoremediation
Phytotoxicity testing
The toxic effects on duckweeds upon exposure to different
concentrations of heavy metals can be measured based on
common physiological parameters, such as relative growth
rate, chlorophyll, or carotenoid content under standardized
conditions. Using dose–response curves, toxicity parameters
for effective doses at an effect level of 50%, 20%, or 10% can
be calculated (Naumann et al., 2007). This procedure is
called biomonitoring, which can be used to quantify the
toxicity of the substances present or to evaluate the health
of water bodies in environmental monitoring (Ziegler et al.,
2016). The well-known phenotyping company LemnaTec developed an automated method for monitoring the phytotoxic effects of substances by visually tracking duckweed
growth (Perera et al., 2019). While Le. minor growth has
been deployed as a standardized assay by the Environmental
Protection Agency of the United States and the
International Organization for Standardization to monitor
the presence of toxic substances (USEPA, 1996; ISO, 2005),
the nature of a causal agent is difficult to ascertain without
any predetermined target(s). One proposed approach is to
identify highly specific plant responses for each toxic substance of interest, making it possible to recognize the type
of contaminants based on a number of chosen markers to
monitor (Ziegler et al., 2018). This may be possible using
metabolomics to generate chemically induced metabolite
fingerprints in duckweed that could be used to evaluate the
causal agent for the stress responses (Kostopoulou et al.,
2020). However, resources would have to be invested to create a comprehensive reference data library in order to test
this possibility.
Production of biopolymers, proteins, and vaccines
Biopolymers such as polylactic acid and polyhydroxybutyrate
can be produced from different plant components.
Duckweed powder made from complete fronds of Lemna
species has been used to produce bioplastics by mixing it
with glycerol and polyethylene (Zeller et al., 2013).
Genetically modified duckweed could be used as an expression system for valuable products such as high-value monoclonal antibodies or antibodies with humanized
glycosylation patterns, as well as virus-like particles for vaccine platforms (reviewed in Cross, 2015). While products
from transgenic duckweed have yet to make it into the
commercial space, genetic modification of duckweed for
commercial applications remains an interesting prospect. It
has been shown that the E1 gene from the bacterium A. cellulolyticus, encoding a hydrolytic enzyme used in fuel production (Sun et al., 2007), as well as therapeutic monoclonal
antibodies against CD20, CD30, and a 2b interferon (Cox et
al., 2006) can be successfully expressed in Le. minor.
One benefit of duckweed’s aquatic growth habitat is the
secretion of target proteins directly into the growth medium
for easier purification. Aprotinin, a medically important protease inhibitor, has been expressed in La. punctata, secreted
into the medium, and successfully purified using an immunoaffinity column (Rival et al., 2008). Adequate vaccine production is a growing challenge in a globalized world where
pathogens can spread with greater ease than ever before.
Duckweeds may provide an efficient and safe system for
vaccine production to help tackle the rising demand for vaccines, especially in the developing world. Protective antigens
have been developed against porcine epidemic diarrhea virus
(Ko et al., 2011) and tuberculosis (Peterson et al., 2015).
Several studies have shown the potential of using duckweeds to express antigens from the avian influenza virus
H5N1 (Thu et al., 2015; Bertran et al., 2015), while Firsov et
al. (2018) also successfully expressed a part of the M2e surface protein of H5N1 with the mucosal adjuvant ricin lectin
subunit B (RTB) in duckweed. Mice orally immunized with
the RTB-M130 protein produced specific antibodies against
M2e peptide. Transgenic duckweed plants thus appear to be
a promising route for producing quality antigens as edible
vaccines that may provide affordable control of H5N1 in
animals.
History of duckweed in plant biology
research
The first recorded scientific studies of the Lemnaceae concentrated on describing their morphology and histology. A
monograph focused on the Lemnaceae was published in
1839 by the botanist Matthias J. Schleiden (Schleiden, 1839),
one of the originators of the cell theory. Later, Christoph F.
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Phytoremediation refers to the cleaning of the environment
via the uptake or degradation of pollutants using plants.
Duckweeds are especially useful for this purpose in aquatic
locales because most of the surfaces of these fast-growing
plants are in direct contact with water. Water purification
by duckweeds can be facilitated by the uptake of heavy
metals, uptake and metabolism of xenobiotics and pharmaceutical drugs, or uptake of macroelements such as nitrates
and phosphates from eutrophic water (Ziegler et al., 2016).
Experiments on nutrient removal by duckweed cultivation
have been carried out in several pilot scale studies with
promising results (Xu et al., 2012; Zhao et al., 2015). More
recently, the applications of duckweed for remediation of
crude oil contaminants and polyester manufacturing wastewater have been reported (Ekperusi et al., 2020; Osama et
al., 2020). While these studies have documented the effectiveness of duckweed to aid in water remediation, how
duckweed biomass is utilized after harvest will be determined by the particular contaminant’s lifecycle in duckweed
tissues. Effective solutions to manage and utilize the duckweed harvested from contaminated sources would open this
platform for large-scale applications in many communities.
K. Acosta et al.
Duckweed for plant systems studies
| 3217
characterization of auxin biosynthesis pathways in plants.
Using intact duckweed plants to carry out isotopic compound loading to avoid confounding issues of other studies
that rely on excised plant parts, the first clear biochemical
evidence for the existence of a tryptophan (Trp)-independent indole-3-acetic acid (IAA) biosynthesis pathway in Le.
gibba G3 was demonstrated (Baldi et al., 1991). This conclusion was soon supported by the isolation and study of Trpauxotrophs from A. thaliana (Normanly et al., 1993). The
relative importance of the two IAA biosynthesis pathways
was also shown to be influenced by temperature in Le. gibba
G3, with the Trp-independent pathway predominating at
higher growth temperatures, such as 30 C (Rapparini et al.,
2002). While the Trp-dependent pathway has been well
characterized over the past two decades (Mano and
Nemoto, 2012), the first breakthrough for the Trpindependent pathway finally came with the report of indole
synthase (INS) as a key branchpoint toward IAA from
indole-3 glycerol phosphate (Wang et al., 2015). However,
the pathway from indole produced by INS in the cytosol to
IAA remains to be elucidated.
Finally, perhaps the most impactful contribution of duckweed to plant biology is the discovery of the function and
roles of the D1 protein in photosystem II (PSII) of the thylakoids, also known as the 32 kDa herbicide-binding protein.
Mattoo et al. in the Edelman laboratory produced seminal
results describing the lifecycle (Mattoo and Edelman, 1987)
of this highly unstable protein that acts as a critical primary
electron acceptor in the PSII complex (Mattoo et al., 1984).
Pulse labeling of La. punctata plants with 35S-methionine enabled the discovery of the translocation of precursor D1
protein from its synthesis on the stromal lamellae to its site
of action in the granal lamellae of the thylakoids, among
other findings (Mattoo and Edelman, 1987).
Coinciding with the rise of Arabidopsis genetics and molecular biology beginning in the mid-1980s, basic research
using duckweeds subsided over the period of 1990–2010.
While some of the first cloned plant genes were from
Lemna, and the dissection of promoter functions had been
carried out in duckweed using transient assays (Stiekema et
al., 1983; Kehoe et al., 1994), the duckweed research community remained small during this boom time of plant molecular genetics. Over the past decade, however, the rapid
accumulation of genomics tools coupled with advances in
analytical and computational technologies have set the stage
for a renaissance of duckweed research (Lam et al., 2014).
The many advantages of duckweed as an experimental platform, such as its aquatic habitat and rapid clonal propagation, coupled with its smaller gene repertoire, should enable
it once again to help illuminate novel pathways and paradigms in plant biology.
Hot topics in duckweed research
Propelled in large part by recent advances in genomics tools,
several areas of duckweed research are beginning to contribute new knowledge to key areas of plant biology. In the
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Hegelmaier published “The Lemnaceae: a monographic
study” (Hegelmaier, 1868) with detailed drawings that are
still relevant today. By the turn of the 20th century, it had
been established that the family Lemnaceae comprised “four
well-defined genera and about twenty-eight species, distributed throughout the torrid and temperate zones”
(Thompson, 1898). The family has since been expanded to 5
genera with 36 species (Bog et al., 2020a, 2020b; Figure 3).
Modern studies of the Lemnaceae can be dated to the
mid-1950s, with the movement of William Hillman and
others away from the descriptive toward more quantitative
biochemical mechanisms (Hillman, 1957). As pointed out by
Hillman (1961) and expanded on by Landolt and Kandeler
(1987), the use of duckweed as a model system for physiological and photosynthetic studies is based on its ease of
manipulation and maintenance. Since duckweeds are small,
morphologically reduced, fast-growing, easily cultivated under aseptic conditions, and particularly suited to biochemical
studies involving isotope labeling, they were considered to
be an ideal system for plant research (Hillman, 1976). For example, much of what we know about photoperiodic flowering responses came from fundamental studies conducted
with Lemna by Hillman at the Brookhaven National
Laboratory. This included diversification of the time measurement systems among different photoperiodic species
both within the Lemnaceae and among different geographical isolates of a single species. In the era of plant molecular
biology (since the 1980s), this valuable physiological information from early studies involving duckweed served to inspire
researchers utilizing model plants with better genetic tools
or stronger commercial interests, such as A. thaliana and
rice.
The Lemnaceae are especially well suited for in vivo biochemical research compared to other plants. Their facile uptake of solutes from defined growth media and easy
handling under controlled conditions have made them attractive for whole-plant biochemical research (Figure 1, B
and C). Anthony J. Trewavas, one of the first to recognize
this, used radiolabeled compounds to study nucleic acid and
protein turnover in Le. minor plants (Trewavas, 1970, 1972).
In a series of papers, Trewavas et al. also explored the effects
of ABA on turion development, including changes in protein
and mRNA synthesis (Smart and Trewavas, 1984) and the
downregulation of cell wall polysaccharide synthesis by
UDP-apiose/UDP-xylose synthase during turion development
(Longland et al., 1989). Similarly, Datko et al. (1978a) used
Le. perpusilla 6746 to carry out in vivo labeling studies with
radiolabeled sulfate and other sulfur-containing compounds
to quantify sulfur assimilation by plants into various products under defined conditions. A phytostat system (analogous to the powerful chemostat platform for the study of
microbial metabolism networks) was established for the first
time with plants to enable quantitative measurement of the
effects from changing concentrations of a single component
in the system (Datko et al., 1978b).
Another exceptional achievement leveraging the in vivo
labeling capacity of the duckweed system was the early
THE PLANT CELL 2021: 33: 3207–3234
3218
| THE PLANT CELL 2021: 33: 3207–3234
following sections, we describe research topics that exemplify how the physical characteristics of duckweeds help to
simplify experimental systems. We also illustrate how
Lemnaceae could provide a novel window into the diversity
of evolved strategies that have helped shape genome structures as well as their associated metabolic, physiological, and
evolutionary processes.
Plant–microbiota interactions
2020; Fitzpatrick et al., 2020; Huang et al., 2020).
Furthermore, many of the bacterial taxa associated with
duckweed appear to be enriched in the surrounding water
when compared to the original inoculum (Acosta et al.,
2020), as has been observed for the recruitment of rootassociated communities (Edwards et al., 2015). Therefore,
similar structuring principles may be found between the
duckweed microbiome and terrestrial plant microbiome.
While community profiling studies are essential for providing a comprehensive understanding of the ecological
processes and structural aspects of the plant microbiome,
reductionist-based approaches involving gnotobiotic plants
and synthetic microbial consortia are used to establish
causality and gain a mechanistic understanding of the roles
various microbial community members may play in this ecosystem (Vorholt et al., 2017; Durán et al., 2018). Prerequisites
for this approach include being able to isolate representative
microbial community members as well as having reference
genomes for these community members and the plant host.
Fortunately, high-quality genomes are available for different
duckweed species and a significant portion of the DAB community can be cultivated (Matsuzawa et al., 2010; Tanaka et
al., 2018), with an increasing number of DAB genomes being
sequenced. Functional studies with DAB isolates have shown
that they affect duckweed growth under defined conditions
(Toyama et al., 2017; Ishizawa et al., 2019b), facilitate the removal of nutrients (Zhao et al., 2015), enhance bioremediation of pollutants (Toyama et al., 2006; Yamaga et al., 2010;
Ogata et al., 2013; Xie et al., 2014), produce phytohormones
(Gilbert et al., 2018), and affect plant development (Huang
et al., 2020). The ability to readily analyze duckweed exudates and collect microbial community members from the
growth medium allows researchers to examine which classes
of exudates play key roles in community assembly and to
characterize how microbiota respond to these compounds.
For example, certain duckweed exudates, such as fatty acid
esters and amides, were found to stimulate the nitrogenremoval efficiency of bacteria by activating nitrate and nitrite reductases (Sun et al., 2016), the methane oxidation activity of methanotrophs (Iguchi et al., 2019), or the
pollutant degradation activity of bacteria (Xu et al., 2015).
Analysis of the community dynamics revealed by inoculating
different plant-growth-promoting bacteria onto duckweed
demonstrated that the colonization of bacteria can change
in the presence of other microbes even though these bacteria occupy different plant niches (Yamakawa et al., 2018)
and irrespective of inoculation density or inoculation order
(Ishizawa et al., 2019a). Furthermore, exogenous DABs with
plant-growth-promoting ability can be displaced from the
host by native DAB communities (Ishizawa et al., 2020),
highlighting the resiliency of the indigenous community
against invaders.
The emergence of duckweed genomics will advance the
exploration of the mechanistic underpinnings from the plant
host perspective using transcriptomic approaches. These
advances, together with the ability to precisely define media
conditions and construct synthetic bacterial communities,
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
A rapidly growing area of plant biology is plant microbiome
research, which ultimately aims to understand and manipulate plant-associated microbial communities toward beneficial agricultural outcomes (Busby et al., 2017). This research
is hindered in terrestrial plant model systems, where both
biological factors such as complex plant development
(Beilsmith et al., 2021) and technical factors such as the
need to manipulate soil conditions (Kremer et al., 2021)
complicate reductionist approaches used to gain a mechanistic understanding of plant microbiome processes
(Vorholt et al., 2017). In contrast, duckweed presents a facile
model plant microbiome system for dissecting mechanisms
underlying plant–microbiota interactions. Compared to soil,
the liquid growth medium used for duckweed allows for
an experimental system with minimal heterogeneity and
ready access to exudates and microbe(s) of interest. An exudate trapping system (Lu et al., 2014) and whole microbial
community capture method (Ishizawa et al., 2017) have
been developed to analyze these exudates and microbial
communities. When testing interactions between bacteria
isolates and gnotobiotic plants, the aquatic lifestyle and
small size of duckweed greatly facilitate the functional analysis of plant–microbe interactions under defined conditions.
To begin to dissect the mode of interaction between
duckweed associated bacteria (DABs) and duckweed, a
PCR-based attachment assay and methods for reintroduction of microbes with gnotobiotic plants have been reported
(Huang et al., 2020; Acosta et al., 2020; Ishizawa et al., 2020).
These studies should help pave the way for the application
of reductionist approaches to the duckweed system, as
have been shown in Arabidopsis (Durán et al., 2018).
Together, these tools could be leveraged to build a tractable
high-throughput model plant microbiome system with
duckweed.
One question that arises is whether the microbiome profiles found in terrestrial plants are related to those found in
duckweed. To this end, several high-throughput amplicon
sequencing studies have generated community profiles and
characterized the assembly of the DAB community from
field sites and reconstitution experiments using gnotobiotic
plants. The DAB community is primarily composed of
Proteobacteria, followed by Bacteroidetes (Xie et al., 2015;
Acosta et al., 2020), and most closely resembles the profile
from terrestrial leaf microbiomes (Acosta et al., 2020). In addition, duckweed serves as a distinct habitat for bacteria
compared to the surrounding environmental community,
demonstrating the selection of particular bacteria, as observed in terrestrial plants (Xie et al., 2015; Acosta et al.,
K. Acosta et al.
Duckweed for plant systems studies
should promote our understanding of DAB community establishment at the systems level. Future directions for duckweed microbiome research should include characterizing
communities of other microorganisms associated with duckweed, including fungi and algae (Watanabe et al., 2016).
While this floating macrophyte is evolutionarily distant from
terrestrial models, investigation of the duckweed microbiome may assist in the discovery of conserved principles
underlying plant microbiota interactions that can be translated to economically important crops.
Disease resistance genes in duckweed
| 3219
the smaller gene set of the Sp. polyrhiza genome (Michael et
al., 2017). A more dramatic decrease to only 3–4 NLR-related genes is observed in the two clones of Wo. australiana
(Michael et al., 2021). Since the Wo. australiana genomes
are 375 Mb in size, similar to the rice genome, this low
number of NLR genes indicates that a significant repertoire
of varied NLRs is not needed by Wo. australiana to provide
immune functions for the survival of this species.
Interestingly, examining the number of PRR genes encoding
proteins with a kinase domain, which likely include genes in
the PTI pathway, both Wo. australiana genomes exhibit an
increase in the number of genes in this category compared
to the two Spirodela genomes. In contrast, the number of
PRR-related genes without a kinase domain showed the opposite trend with the two Wolffia genomes displaying twoto three-fold lower numbers, which reflects a similar trend
for most of the other gene families in this species compared
to other sequenced Lemnaceae species (Michael et al.,
2021). Strikingly, while both Spirodela genomes are missing
LysM-type PRRs, they can be found in the Wo. australiana
genomes. This observation indicates that these LysM-type
PRRs must have been present in the common ancestor of
Sp. polyrhiza and Wo. australiana. Altogether, these results
suggest that in Wo. australiana, the basal immunity functions mediated by PRR genes could play a more dominant
role in the pathogen response in contrast to Sp. polyrhiza,
where the NLR genes could play a more significant role.
Finally, of the eight classes of AMPs that were examined,
the class of AMPs with the highest number of genes in
duckweeds is in the lipid transfer protein (LTP) category,
followed by the Snakin and Defensin classes (Table 1). While
this analysis did not reveal a significant increase in the number of AMP-encoding genes in these duckweed genomes,
the persistent numbers of LTP, Snakin, and Defensin genes
in Wo. australiana suggest that they are indispensable for
this minimalist species where the total gene set has
decreased to 15,000 genes. These observations raise the
possibility that these AMPs could be downstream effectors
from the LRR-RK-type PRRs in Wolffia to provide major
immune functions in comparison to Spirodela. It would be
interesting to test predictions of this hypothesis, such as the
induction of AMP-encoding genes upon the challenge of
Wolffia with microbial pathogens. Elucidating the mechanisms of induced immunity to potential invaders in duckweeds is likely to uncover novel disease resistance strategies
and could be a key advance for a successful cropping system
to be designed using these versatile plants.
Cell autonomy and transcriptome studies of
circadian rhythms in duckweeds
The small size, relatively flat surface of the fronds, and simple architecture of intact duckweed are ideal for long-term,
automated microscopic observations, which can be used to
track and quantify plant physiological processes at high spatial resolution. One such process is the circadian clock, an
internal biological timing device for adjusting various
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
As duckweeds are aquatic plants that have adapted to
thrive in organics-rich environments with high microbial
loads, these plants might be expected to have heightened
disease resistance functions. It is thus surprising that the
number of nucleotide-binding leucine-rich repeat domain
genes (NLRs), which encode many of the proteins involved
in disease resistance, was found to be significantly lower in
the Sp. polyrhiza 9509 (Sp9509) genome than in other
model plants (Michael et al., 2017). Interestingly, similar attrition of these NLRs was observed in the eelgrass Zostera
marina and in carnivorous plants of the Utricularia and
Genlisea genera (Baggs et al., 2020). These observations raise
this intriguing question: What is the basis for the broadspectrum resistance in the Lemnaceae? In a recent study of
the Sp7498 genome, it was suggested that there may be an
amplification of antimicrobial protein (AMP)-encoding genes
due in part to the presence of many of these loci with duplicated AMPs (An et al., 2019). The heightened expression
of some of these AMPs in Sp7498 was hypothesized to provide enhanced immunity in this duckweed.
Using the latest available annotations from high-quality
reference genome assemblies for two clones each of Sp. polyrhiza (Hoang et al., 2018; Harkess et al., 2021) and Wo. australiana (Michael et al., 2021), NLR genes were curated using
the NB-ARC (nucleotide-binding adaptor shared by APAF-1,
certain R gene products, and CED-4) conserved motif and
their gene numbers compared to those from well-curated
genomes of Brachypodium distachyon BD21, rice (Oryza sativa), A. thaliana Col-0, and alfalfa (Medicago truncatula).
Genes encoding pattern-recognition receptors (PRRs), which
include genes involved in microbial-associated molecular
pattern-triggered immunity (PTI), were also identified. Those
that contain a kinase domain (LRR-RK, LysM-RK) were separately curated from those that do not (LRR-RP, LysM-RP).
Lastly, genes encoding members from eight families of
known plant AMPs (Hammami et al., 2009) in these same
genomes were identified as well. Using a relatively stringent
informatics pipeline, 169 NLR genes were found in A. thaliana with a genome size of 150 Mb (Table 1). The number
of NLR-related genes increased in the other model terrestrial
plant genomes with larger genome sizes. In contrast, both
Sp. polyrhiza genome assemblies produced sets of 20–35
NLR genes. Since Sp. polyrhiza has approximately the same
genome size as A. thaliana, this lower NLR gene count is unlikely to be due to a smaller genome even when factoring in
THE PLANT CELL 2021: 33: 3207–3234
3220
| THE PLANT CELL 2021: 33: 3207–3234
K. Acosta et al.
Table 1 Comparison of defense-related genes between sequenced duckweeds and those of other model plant species. Genes encoding AMPs,
PRRs containing LRR (LRR-RK, LRR-RP) or LysM (LysM-RK, LysM-RP) domains, and NLR proteins were curated for Sp9509 (Hoang et al., 2018),
Sp7498 (Harkess et al., 2021), Wo. australiana 8730, and Wo. australiana 7733 (Michael et al., 2021). For comparison, B. distachyon, O. sativa, A.
thaliana, and M. truncatula were analyzed with the same pipeline using proteomes from the Monocot and Dicot PLAZA 4.5 database (Van Bel
et al., 2018). For a detailed methods description, see https://github.com/kenscripts/tpc_dw_review/.
Sp. polyrhiza
7498
Wo. australiana
8730
Wo. australiana
7733
B. distachyon
O. sativa
A. thaliana
NB-ARC
20
35
3
4
346
483
169
797
PRRs
LRR-RK
LRR-RP
LysM-RK
LysM-RP
56
78
0
0
36
80
0
0
95
20
2
5
78
32
2
5
401
111
3
3
375
143
3
7
356
162
5
4
434
332
19
9
AMPs
Cyclotide
Defensin
Hevein
Knottins
LTP
Snakin
Thionin
Vicilin
0
5
0
0
25
9
2
3
0
5
0
0
26
8
1
2
1
9
6
0
28
12
0
0
1
9
5
0
28
13
0
0
0
44
16
2
67
15
12
2
0
23
10
2
79
15
12
0
1
60
10
0
61
19
4
2
1
85
8
0
91
28
1
1
Gene Type
NLRs
functions to the cyclic natural environment. Once entrained,
physiological outputs of the clock display circadian rhythms
even under constant conditions. These rhythms are usually
synchronized to the day–night cycles in the natural environment. Core circadian clock genes encoding single MYB domain transcription factors form conserved transcription–
translation feedback loops that generate circadian oscillations in plants. While circadian regulation of plant genes has
been well studied at the organismal and tissue levels,
whether the clock can function cell autonomously was unknown. To observe circadian rhythms of individual cells in
live duckweed plants, bioluminescence imaging of Le. gibba
fronds that were transfected with the AtCCA1:LUC reporter
by particle bombardment was used to monitor rhythmic
gene expression of multiple cells simultaneously (Muranaka
et al., 2013). Analyses of the circadian clock’s behavior at a
single-cell level in these intact plants revealed quantitative
parameters for the heterogeneity and stability of individual
cellular clocks, spontaneous local coupling of cellular clocks,
and the stochastic synchronization patterns of cellular clocks
under non-24-h light/dark cycles (reviewed in Muranaka
and Oyama, 2018). Using the experience and tools generated
from these studies, bioluminescence monitoring at singlecell resolution was successfully applied to detached
Arabidopsis leaves (Kanesaka et al., 2019). Although the
gradual growth with 3D distortion of Arabidopsis leaves was
a disadvantage for long-term studies of more than several
days, the bioluminescence of individual cells was traceable
to detect cellular circadian rhythms. Thus, the bioluminescence monitoring system originally established in duckweed
could be applied to other plants of interest to examine cell
autonomous behaviors in different species.
Plant growth is controlled by the coordination of time-ofday (TOD) pathways to specific times in a day by the circadian clock (Michael et al., 2008a). In fact, global TOD gene
regulation is a common feature in plants, with 30%–50%
M. truncatula
and 10%–20% of genes displaying peak expression or phased
expression at specific times over the day under diurnal and
circadian free-run conditions, respectively (Michael et al.,
2008b; Filichkin et al., 2011). All core circadian clock orthologs cycle in Spirodela with the expected phases as in other
plants, and most fundamental biological processes such as
photosynthesis, the cell cycle, protein synthesis, and metabolism are phased to a conserved TOD. Strikingly, only 13% of
genes in Wo. australiana were found to display TOD expression under the same diurnal LDHH conditions (Michael et
al., 2021). While all the core circadian clock orthologs have
conserved TOD expression, many pathways are not phased
to a specific time of day in Wolffia. However, the core photosynthetic and chloroplast-associated pathways still cycle in
a TOD fashion, which is consistent with the notion that anticipation of the light–dark transitions to optimize these
light-dependent, energy-related functions is still being maintained. The loss of TOD regulation for most pathways in
Wolffia apparently results from the loss of key developmental and light-regulated pathways, which parallels its highly
reduced body plan and loss of roots. The contrasts between
the control of global gene expression patterns among duckweeds should provide an exciting model to further understand growth-related pathways in plants as well as
mechanisms for energy use optimization via strategic minimization of gene control.
Duckweed metabolomics: plant secondary
metabolites
Duckweeds have been recognized as an excellent model system to study biosynthetic pathways and metabolic mechanisms in large part due to their simplified structure and the
rapid uptake of labeled precursor compounds under defined
growth conditions. With the rapid advance in analytical instrumentation and growth of databases for compound identification over the past decades, the time is ripe for using
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Sp. polyrhiza
9509
Defense-Related
Gene Category
Duckweed for plant systems studies
| 3221
acid amides that might be involved in plant/bacteria interactions (Sun et al., 2016). Semipolar metabolites from duckweeds have been analyzed by liquid chromatography
coupled with mass spectrometry (LC–MS) techniques, and
distinct patterns of metabolite profiles have been found between different genera and species (Figure 4A). Flavonoids,
compounds used for nutraceutical, medicinal, and cosmetic
purposes, have also been characterized in some duckweeds
(Qiao et al., 2011; Ren et al., 2016; Böttner et al., 2020).
Currently, the major bottleneck in mass spectrometry-based
metabolomics is metabolite identification. Shahaf et al.
(2016) developed a unique analysis platform that enables
matching of mass spectra from plant extracts against a natural product library containing several thousand entries of
secondary metabolite standards (Figure 4B). This technique
was used for analysis of LC–MS chromatograms from Le.
gibba, La. punctata, Sp. polyrhiza, and Wo. globosa, which
Figure 4 Integration of metabolomics and genomics analysis in the Lemnaceae. (A) Comprehensive mass spectrometry-based metabolomics as a
tool for pathway elucidation and characterization of biosynthetic enzymes and genes. Plant extracts are analyzed using high-resolution LC–MS
(HR-LC–MS). Raw chromatograms typically consist of several thousand mass signals, and data can be deconvoluted using computation tools. (B)
One method for compound identification from complex mixtures involves matching the mass spectra from an extract corresponding to a peak
onto an entry in spectral libraries. An example for metabolite NP006950 (Weizmass library) is shown, illustrating that chromatographic retention,
accurate mass, and mass fragmentation of a peak fraction in a Lemna gibba extract (upper black peak) matched a library entry (lower red peak)
that can be assigned with high confidence (Shahaf et al., 2016). (C) These techniques, in combination with chemical classification, allow the metabolic landscape of Lemnaceae to be defined. (D) This knowledge can help identify metabolic genes/enzymes using either structure-based reaction
prediction or genome mining approaches (such as metabolic gene cluster prediction that hints at possible biosynthetic pathways) as well as possible secondary metabolites (such as by correlation with the metabolomics dataset).
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
the duckweed platform to delineate the intricate metabolite
network of a complete plant at the systems level.
Characterizing these metabolites will also help to explore
and optimize the use of duckweed beyond its role as a
model organism in the laboratory, since these plants could
have commercial potential.
Secondary metabolites and small molecules have been less
studied in the Lemnaceae than in other model plants, but
this is now rapidly changing. Using gas-chromatography coupled with mass spectrometry (GC–MS ), the profile of epicuticular waxes was analyzed in Sp. polyrhiza (Borisjuk et al.,
2018). While in land plants, sterols are not present on the
surface of leaves or stems but in the cuticle, high levels of
sterols were found on the surface of Spirodela fronds, which
is thought to function as a sunscreen for these plants.
Furthermore, GC–MS analysis of exudates of Sp. polyrhiza
also led to the identification of several fatty acids and fatty
THE PLANT CELL 2021: 33: 3207–3234
3222
| THE PLANT CELL 2021: 33: 3207–3234
strategies for linking metabolites to their biosynthetic genes/
enzymes. Figure 4D illustrates how whole-genome data can
be used by the PlantiSMASH analysis pipeline to identify
plant BGCs (Kautsar et al., 2017). Candidate BGC contents
were compared using reference genomes of Sp. polyrhiza
and Wo. australiana. Sp. polyrhiza had a lower number of
candidate clusters (five), while the Wo. australiana genome
showed eleven candidate BGCs. Interestingly, this analysis
revealed more unique gene clusters between the two duckweed genera than common ones. Although the data are
preliminary, the occurrence of clusters and the resulting
metabolites that can be produced from them might help explain important phenotypic differences between the genera.
In summary, the Lemnaceae display a diverse and unique
metabolite profile between genera. Their metabolites and
pathways show potential for varied biological activity and
might help explain the lifestyles of the different members of
the Lemnaceae family in their natural aquatic environment.
The combination of metabolic profiling and gene discovery
could be a powerful tool to characterize the chemical space
in these aquatic plants and to investigate how metabolites
might mediate the interactions of duckweeds with their
aquatic environment.
Experimental evolution and eco-evolutionary
dynamics
Their short generation times and small size also make duckweed an ideal model system to tackle problems in evolutionary ecology (Laird and Barks, 2018). Here, we highlight
two emerging fields that can greatly benefit from using
duckweed as a model system. Experimental evolution, in
which phenotypic and genotypic changes are measured under controlled selection regimes, is a powerful approach to
study the processes and mechanisms of adaptive evolution
in real-time (Teotonio et al., 2009; Schlotterer et al., 2015).
Plant scientists have used long-term experiments to investigate the phenotypic responses of plants to herbivory under
natural conditions (either herbivore exclusion or addition to
the plant population) for decades (Detling and Painter,
1983). They have convincingly demonstrated that herbivores
can drive the contemporary evolution of plant traits, such
as the levels of defense metabolites (Züst et al., 2012), plant
phenology (Agrawal et al., 2012), and growth rates (Turley
et al., 2013). However, due to the long lifecycles of many
plants, most of these studies only investigated the evolutionary process during a few generations, which limits the power
of detecting traits under selection (Kofler and Schlotterer,
2014). In addition, such studies are difficult to replicate in
many other laboratories, as they require a large planting
area and long durations. The short generation times (within
a few days) and small size (1–15 mm) of duckweeds are excellent attributes for experimental evolution studies to address many questions that are not as tractable with other
systems. For example, under indoor conditions, one can use
the “select and resequence” approach to study >100 generations of plants that are evolving under different stress
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
led to the identification of 29 metabolites from 8 different
natural product classes with high confidence, providing the
first glimpse of the “natural product repertoire” for the
Lemnaceae (Shahaf et al., 2016; Figure 4C). While several of
the identified molecule classes are typically found in monocots, such as flavonoids related to the apigenin and luteolin
type, the Lemnaceae metabolite profile also showed several
molecules/metabolite classes previously not described in
these plants, such as gingerglycolipid “A” and its derivatives.
These compounds were only previously described in ginger
(Zingiber officinale) and were believed to be specialized
metabolites unique to this plant.
Metabolite identification not only provides leads for the
search and characterization of derivatives for these compounds, but it also facilitates the prediction of metabolic
pathways as well as biosynthetic genes/enzymes, and hence
the metabolic network of duckweed. One approach to defining metabolic pathways involving a biosynthetic precursor
of interest is called DLEMMA (Feldberg et al., 2009). In this
method, the duckweed is fed different stable isotope-labeled
variants of a precursor compound to facilitate the subsequent use of the labeling pattern of detected compounds
for intermediate identification. For example, feeding of Sp.
polyrhiza with differentially labeled tyrosine led to the identification of 59 tyrosine-derived metabolites, all of them new
for Lemnaceae (Feldberg et al., 2018). Furthermore, the identification of these derivatives enabled the construction of
entirely new metabolic pathway models based only on metabolite data without using genetic information. Knowledge
about the metabolic landscape of duckweed, especially
metabolites identified with high confidence, allows one to
predict enzymatic reactions necessary to form a specific
structure. These hypothetical reactions can in turn hint at
enzymes and genes that are involved in biosynthesis and for
which candidate genes with the predicted enzyme activity
may be identified from transcriptome and/or genome data.
The production of secondary metabolites is often under
environmental control, with a particular stress or condition
modulating the pathway for the metabolite. The use of metabolite extraction techniques under a single environmental
condition will likely not be sufficient to characterize all the
potential compounds a duckweed clone can produce.
Mining the genome for potential metabolic pathways (i.e.
the gene space) can help establish the repertoire of potential
enzymes in one duckweed clone needed to generate a specific secondary metabolite. Genes encoding enzymes involved in successive steps of biosynthetic pathways that
form secondary metabolites in plants are sometimes found
in clusters (Osbourn, 2010), termed biosynthetic gene clusters (BGCs). Identifying duckweed BGCs by analyzing wellannotated reference genomes in combination with metabolite extraction and identification creates a powerful tool that
can be used to help discover biosynthetic pathways in duckweed. As described earlier, reference genomes are rapidly being completed for the Lemnaceae (see “Chromosomes and
genomics of duckweeds”). These resources enable the use of
K. Acosta et al.
Duckweed for plant systems studies
Duckweed research tools, technologies, and
resources
Critical for successful model plant systems are tools, technologies, and resources that allow researchers to fully explore
their questions. Key technologies include a genotyping platform, transformation system, and genetic methods. Also at
the heart of a vibrant community is an organizing committee and international meetings that provide researchers opportunities to share research ideas and findings. Finally,
resources such as genomics databases (and, in the case of
duckweed, clone collections) will allow interested laboratories to quickly adopt a new model and explore its potential.
Genotyping
Several universal plastidic barcode sequences, among those
recommended by the Consortium for the Barcode of Life
plant-working group (Hollingsworth et al., 2009), have been
used to distinguish 31 of the 37 duckweed species
| 3223
recognized at the time (Borisjuk et al., 2015), with more
challenging species found within the Lemna, Wolffiella, and
Wolffia genera. AFLP technology, although technically challenging and more expensive, was used to further resolve
most of the species within these genera (Bog et al., 2010,
2013). More recently, genotyping-by-sequencing (Bog et al.,
2020c) and a tubulin-based polymorphism method (Braglia
et al., 2021) demonstrated the feasibility of resolving interspecific differences amongst closely related duckweed species. As more genome sequences become available, these
technologies can be refined and serve as high-resolution
methods for rapid species identification in duckweed.
Besides distinguishing duckweed species, biotechnological
applications will require identification at the clonal level,
since significant physiological and biochemical differences
can exist between clones of the same species (Sree et al.,
2015b). Facile genotyping methods with the capability of resolving intraspecific variations would be invaluable. A novel
bioinformatic pipeline using so-called polymorphic NB-ARCrelated genes was successfully tested using a training set of
genome sequences from nine clones of Sp. polyrhiza to identify primer sets with the ability to distinguish these nine
clones from each other as well as 11 additional Sp. polyrhiza
clones, illustrating this strategy’s efficacy (Chu et al., 2018).
Sp. polyrhiza was selected for this proof-of-concept study
due to its low intraspecific variations based on whole-genome sequencing studies (Michael et al., 2017; Ho et al.,
2019; Xu et al., 2019). A limitation of this technique is that
it requires the generation of multiple whole-genome assemblies for the investigated species as a training set for the
algorithm. Fortunately, more and more high-quality genome
assemblies for numerous duckweed species and clones are
being generated and made available through large-scale sequencing studies.
Transformation and its application in duckweed
Transformation has been demonstrated for almost all duckweed genera, with the fastest methods taking 2–3 months,
like in other model crops. The first demonstration of transgenic gene expression in duckweed was reported in 1991
by transfecting Le. gibba fronds with a transgene using the
biolistic particle gun system to demonstrate phytochrome
regulation of a sequence upstream of the SSU5B gene by
transient assays (Rolfe and Tobin, 1991). Conditions for transient expression using Agrobacterium-mediated gene transfer
were subsequently described for Wo. columbiana (Boehm et
al., 2001). Stable transformation was demonstrated in the
late 1990s (Stomp and Rajbhandari 1999; Edelman et al.,
1999). Some of the key challenges for development of stable
transformation systems were the high inter- and intraspecific
variability observed in attempts to standardize tissue culture
conditions for plant regeneration from callus tissue (Chang
and Chiu, 1978; Chang and Hsing, 1978; Liu et al., 2019). The
effects of medium composition and light intensity on callus
induction, growth, and frond regeneration were optimized
for Le. gibba (Moon and Stomp,1997; Li et al., 2004), while
carbohydrate and phytohormone requirements for callus
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
conditions within 1 year (Burghardt et al., 2018). A critical
component for such studies is to have diverse genotypes at
the beginning of the study, as evolution in such experiments
will likely be affected by initial genetic variations in the population. To this end, the currently available duckweed collections and their ongoing sequencing efforts described here
should provide the resources and reference material critical
for such studies.
It is now apparent that evolution can take place within
ecological timescales and alter population dynamics
(Lavergne et al., 2010), which in turn can modify evolutionary trajectories and create eco-evolutionary feedbacks
(Kinnison and Hairston, 2007; Pelletier et al., 2009).
Understanding how these feedbacks alter the evolution of
species interactions is one of the major challenges in evolutionary ecology. Addressing this challenge requires the interacting organisms to be specifically manipulated at the
ecological and evolutionary levels (Bailey et al., 2009; Hendry,
2019), which is challenging for many study systems.
However, recent work has demonstrated the potential of using duckweeds to study eco-evolutionary feedbacks. Using a
combination of theoretical modeling and experimental manipulation of duckweed evolution, rapid evolution was
shown to affect the dynamics of competing species and
eco-evolutionary interactions to shape the trajectory of
species coexistence (Hart et al., 2019). In another recent
study, eco-evolutionary dynamics were characterized for
duckweed–microbiome interactions where microbiome presence apparently led to rapid evolutionary change in bacterial
community members, which in turn affected the bacterial
species composition and host fitness (Tan et al., 2021).
The advent of duckweed genomics will put these tiny plants
in a position to further advance research on ecoevolutionary dynamics by providing quantitative genomic
data to help unravel the genomic underpinnings and
the underlying molecular mechanisms of these complex
processes at the systems level (Rudman et al., 2018).
THE PLANT CELL 2021: 33: 3207–3234
3224
| THE PLANT CELL 2021: 33: 3207–3234
Genetics and crossing systems
Flower-induction and cross-pollination protocols will help to
facilitate genetic studies in the Lemnaceae. While duckweed
propagation is mainly vegetative, many studies have investigated flowering induction in duckweed to reveal triggers of
sexual reproduction in these plants (see “Anatomy, morphology, and growth characteristics”). Among these, SA and
the chelating agent EDDHA can promote flowering in many
duckweed species when added to the culture medium
(Pieterse, 2013). A recent systematic comparison of the
effects of these chemicals, daylength, and other culture conditions on the frequency of flower induction and seed production was reported for clones of Sp. polyrhiza, Le. gibba,
and Wo. microscopica (Fourounjian et al., 2021). Although
floral induction is possible in duckweed, flowers are often
aborted and do not form seeds, as observed in some clones
of Le. gibba. Fu et al. (2017) found that the application of
SA can prevent seed abortion in Le. gibba clone 7741 but
not in clone 5504. The anthers did not dehisce in clone
5504, and artificially released pollen grains did not germinate, suggesting male sterility. This hypothesis of male sterility was supported by cross-pollination between the two
clones, since pollination of clone 5504 with pollen grains
from clone 7741 yielded seeds. Analysis of the resulting
progeny using intersimple sequence repeat markers suggested that cross-pollination occurred, resulting in the
production of intraspecific hybrids (Fu et al., 2017). While it
would be important to verify the hybrid nature of these
putative F1 plants by examining the F2 lines for segregation
of the two sets of chromosomes, the data so far indicate
that outcrossing between duckweed clones might be possible using this strategy. If validated, this approach could
open the gateway for facile methods of transformation and
genetic studies that are commonly performed in other
model plants.
Missing duckweed technologies
To further enhance the field of duckweed research as a
model plant system, the development and advances in several key technologies are highly desirable. One such missing
technology is a reliable genetic system where routine flowering induction and hybridization between strains or backcrosses can be performed. As discussed above, there are
several species that have clones known to flower routinely,
and good progress is being made to establish the necessary
knowledge base for environmental and hormonal cues that
induce flowering in a few key duckweed species. The current
lack of such a reliable and facile genetic protocol has hindered the development of basic research in duckweed.
While mutant induction and identification in duckweed
were shown to be feasible, predominantly via the deployment of X-ray bombardment or treatment with nitrosomethyl urea as a mutagen to generate so-called “aberrants”
that have auxotrophic or growth phenotypes (Smith and
Castle, 1960; Posner, 1962; Tam et al., 1995), the genetic basis of the phenotypes observed in these lines has never been
resolved. For example, Strain 1073 derived from Lemna perpusilla 6746 was found to exhibit differences in its flowering
response to light and is blocked in its photosynthetic activities (Eames and Posner, 1974). This was subsequently traced
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
induction, callus growth, and frond regeneration were
established for clones of La. punctata 5562 (Li et al., 2004;
formerly named Sp. oligorrhiza) and more recently, for Wo.
arrhiza (Khvatkov et al., 2015).
Duckweed can be transformed through calli or directly
using fronds or roots. Duckweed callus transformation (DCT)
protocols generally involve the selection/treatment of an explant, callus induction, callus maintenance and growth, callus
transformation, transformant selection, and frond regeneration (Li et al., 2004), with many of these steps requiring a different set of conditions for different clones (Huang et al.,
2016; Yang et al., 2017). Different DCT protocols require specific conditions because duckweed clones, even within the
same species, can respond differently to transformation conditions (Chang and Hsing, 1978; Liu et al., 2019). Furthermore,
only certain clones produce callus under a particular condition, which also limits the generality of each DCT protocol
(Yang et al., 2018a; Liu et al., 2019). As an alternative, direct
in planta transformation (DIPT) of fronds or roots has been
used to produce stable transformants of duckweed (Ko et al.,
2011; Balaji et al., 2016; Yang et al., 2018b). Duckweed DIPT
involves the selection/treatment of explants, Agrobacterium
inoculation, cocultivation, selection, and frond regeneration.
DIPT protocols with Lemna have used wounded fronds and
separated mother and daughter fronds as explants (Ko et al.,
2011; Yang et al., 2018b), while unwounded Spirodela fronds
may be required for transformation (Balaji et al., 2016). Like
DCT protocols, Agrobacterium inoculation and cocultivation
in DIPT protocols involve the addition of acetosyringone (Ko
et al., 2011; Balaji et al., 2016; Yang et al., 2018b), while selection and frond regeneration can also be combined in DIPT
protocols to speed up transformation (Ko et al., 2011; Yang
et al., 2018b). Monitoring transgene expression for a period of
10 months showed stable transformation with a DIPT protocol (Balaji et al., 2016). Transformation protocols also exist for
Wolffia species, albeit with lower transformation efficiency
compared to protocols for other species (Khvatkov et al.,
2015; Heenatigala et al., 2018).
In summary, the short transformation time required in
DIPT protocols makes them an attractive alternative to DCT
protocols for duckweed research. Currently, researchers can
adopt two transformation strategies depending on their research objective: (1) transformation conditions can be optimized for a particular clone from a species of interest that
has already been identified as amenable to transformation
or (2) several clones from a species of interest can be
screened using a particular transformation protocol to identify ones with a higher frequency of success. With the introduction of miRNA silencing (Cantó-Pastor et al., 2015) and
precise genome-editing technologies using the CRISPR/Cas9
method (Liu et al., 2019), a routine transformation protocol
should allow for hypothesis-driven studies in the duckweed
model system.
K. Acosta et al.
Duckweed for plant systems studies
| 3225
coupled to long read sequencing such as Oxford Nanopore
Technologies to define the sites of transgene insertions.
Sequencing should reveal the genome locations of the insertion events and whether they are likely to disrupt the function of a duckweed gene.
Lastly, in order to use insertion lines to study gene function, it is important to keep in mind that most insertions
are initially hemizygous. Unless the insertion resulted in a
dominant phenotype, which will probably be a rare event,
the transgenic lines will not show a mutant phenotype in
the T0 primary transformants. Thus, readily induced flowering and setting of viable seeds will be highly desirable traits
for users of this resource as well.
Community organization, newsletter, and
international meetings
Steering committees have been instrumental in guiding, coordinating, and communicating research for successful plant
model systems such as Arabidopsis, maize, and
Brachypodium (Parry et al., 2020). The International Steering
Committee on Duckweed Research and Applications
(ISCDRA) was founded in 2013 to provide similar functions
for the duckweed community. The ISCDRA releases a
quarterly newsletter (Duckweed Forum) that represents a
comprehensive resource for new and current researchers as
well as application specialists, providing announcements, research updates, guidelines, protocols, and discussion topics
such as nomenclature standards that are relevant for the
community. Past and present issues of the Duckweed Forum
can be freely accessed via the website of the Rutgers
Duckweed Stock Cooperative (RDSC; ruduckweed.org). The
ISCDRA organizes a biennial international meeting that
brings together researchers from all over the world, with the
next meeting scheduled for 2022 in Germany (previous
meetings were held in China, the United States, Japan, India,
and Israel). In addition, each year at the Plant and Animal
Genome meeting in San Diego, CA, there is a workshop titled “Duckweed Research and Applications,” which has been
running since 2018.
Clone collections
The first major duckweed clone collection in the community
was amassed and maintained from 1953 to 2012 by Elias
Landolt at the Geobotanical Institute of SFIT Zurich. At its
peak, this collection comprised over 1,000 clones and served
as a source of duckweed germplasm for duckweed researchers around the world as well as starting material for later
collections (Lämmler and Bogner, 2014). Currently, several
duckweed clone collections exist around the world, including the United States, Germany, Switzerland, and China (see
Lam et al., 2020, for locations and contacts for these facilities). Researchers can request duckweed clones from these
collections by directly contacting the respective director of
the collection. To identify and track duckweed clones, a 4digit numbering system was first adopted by Elias Landolt.
Clones from the original Landolt Collection maintain this
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
to a loss of expression of the nuclear gene encoding the
Rieske Fe–S protein (Lam and Malkin, 1985), which led to
rapid turnover of the other proteins that make up the cytochrome b6/f complex in the thylakoid membrane (Bruce and
Malkin, 1991). The question of whether the flowering phenotype in the Le. perpusilla 1073 mutant is related to its photosynthesis defect could potentially be dissected using classical
genetic approaches such as backcrossing to wild-type
parents. Unfortunately, this clone has not been maintained.
Another important technology that is missing is systematic
mutant library generation to facilitate functional analysis of
all genes encoded in the genomes of a representative species.
With the completion of multiple high-quality assemblies for a
growing number of duckweed species, the availability of
indexed insertion libraries in these species would provide a
powerful resource to query gene function rapidly, as in
Arabidopsis (Matus et al., 2014) and the green alga
Chlamydomonas reinhardtii (Li et al., 2016). To enable the deployment of this approach, several accessory technologies are
needed: (1) a facile transformation technology that is easily
scalable; (2) robust and reliable transformant identification
and curation; (3) high-throughput insert sequencing and
indexing; and (4) protocols for selfing of transformants to
produce homozygotes for phenotyping. To generate a large
number of random insertions, an efficient transformation system would need to be deployed, and a more rapid method
such as DIPT would be preferable (Yang et al., 2018b). The recent advent of in planta transformation using nanomaterials
as DNA carriers could also be an attractive method to develop for duckweeds to further improve the throughput of
transgenic plant line production (Wang et al., 2019). In addition to generating stable transformants in large numbers, this
approach could also be applied for rapid, high-throughput
transient expression assays to test gene functions.
Creating and maintaining a large library of transgenic duckweed lines presents another challenge. While the incorporation of a transgene can be readily detected using PCR-based
screening strategies after selecting duckweed plants on the
appropriate antibiotics, continuously propagating thousands
of transgenic duckweed lines as living plant clones in culture
would be laborious and costly. A better approach for maintaining the selected transgenic lines may be to produce seeds
from the identified transgenic lines and store them until they
are needed. Thus, the selection of a proper duckweed clone
that is amenable to viable seed production is an important
consideration. Systematic attempts at cryopreservation of
duckweeds have only been marginally successful (Sauter 1993;
https://patents.google.com/patent/WO2011005502A3), so it is
not a viable option at this time .
Insertion site identification is facilitated by rapid advances
in high-throughput sequencing technologies that reduce
costs while increasing the accuracy of DNA sequencing.
Techniques such as TAILED-PCR (Liu and Chen, 2007) can
be used to amplify DNA from regions adjacent to the inserts
embedded in the genome of the transformed duckweed. A
next generation approach would be to employ CRISPR
THE PLANT CELL 2021: 33: 3207–3234
3226
Table 2 Lemnaceae germplasm and data resources
University of Jena
Landolt Stock Collection
RDSC
University of Münster
Chengdu Institute of
Biology
Jena, Germany
Zurich, Switzerland
New Brunswick, NJ, USA
Münster, Germany
Chengdu, China
Klaus J. Appenroth
Walter Laemmler
Eric Lam
Shuqing Xu
Hai Zhao
550
400
740
260
800
Klaus.Appenroth@uni-jena.de
wlaemmler@duckweed.ch
ruduckweed.org
shuqing.xu@uni-muenster.de
zhaohai@cib.ac.cn
Type
Clone
Technology
Assembly
Protein Coding Genes
Identifier
Reference
Nuclear
Sp. polyrhiza 7498
PacBio
139 Mb
18,708
CoGe: 55812
Sp. polyrhiza 9509
ONT, mcFISH, BioNano
138 Mb
20,661
CoGe: 51364
Sp. intermedia 7747
Sp. intermedia 8410
Le. minor 5500
Wo. australiana 8730
Wo. australiana 7733
Sp. polyrhiza 7498
Sp. polyrhiza 7498
Le. minor
We. lingulata 7289
Wo. australiana 7733
La. punctata (5 clones)
PacBio
ONT, Illumina
Illumina
PacBio, Illumina
PacBio, Illumina
SOLiD
PacBio
ABI
SOLiD
SOLiD
Illumina
148 Mb
137 Mb
472 Mb
354 Mb
393 Mb
228 Kb
168 Kb
166 Kb
169 Kb
168 Kb
166–171 Kb
22,245
21,594
22,382
14,324
15,312
57
78
78
–
–
80
PRJEB35514
PRJEB35634
CoGe: 27408
CoGe: 56605
CoGe: 56606
NC_017840.1
MN419335.1
NC_010109.1
JN160604.1
JN160605.1
(See reference)
Harkess et al. (2021); An et
al. (2019)
Hoang et al. (2018);
Michael et al. (2017)
Hoang et al. (2020)
Hoang et al. (2020)
Van Hoeck et al. (2015)
Michael et al. (2021)
Michael et al. (2021)
Wang et al. (2012)
Zhang et al. (2020)
Mardanov et al. (2008)
Wang and Messing (2011)
Wang and Messing (2011)
Ding et al. (2017)
Type
Clone
Technology
Description
Data size
Identifier
Reference
Transcriptome
Sp. polyrhiza 7498
Sp. polyrhiza 7498
Le. aequinoctialis 6000
Le. gibba 7741
La. punctata 6001
Sp. polyrhiza (2 clones)
Sp. polyrhiza 7498
69 Sp. polryhiza ecotypes
39 Sp. polyrhiza ecotypes
Sp. polyrhiza 7498
La. punctata 0202
Wo. australiana
Spirodela, Lemna, Wolffia
PacBio
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
iTRAQ-LC–MS/MS
Illumina
Illumina
492,435 isoforms
196 million reads
202 million reads
953 million reads
419 million reads
57 million reads
911 million reads
3,491 million reads
532 million reads
459 million reads
6955 unique spectra
34 samples
64 samples
SRX5321175
PRJNA557001
PRJNA368628
PRJNA631155
PRJNA361433
PRJNA473779
PRJNA473779
PRJNA476302
PRJNA552613
PRJNA563960
–
PRJNA219151
PRJNA561628
An et al. (2019)
An et al. (2019)
Yu et al. (2017)
Fu et al. (2020b)
Xu et al. (2018)
Fourounjian et al. (2019)
Fourounjian et al. (2019)
Xu et al. (2019)
Ho et al. (2019)
Fu et al. (2020a)
Huang et al. (2015)
Xie et al. (2015)
Acosta et al. (2020)
Le. aequinoctialis, Rice
Illumina
Multiple conditions
ABA treatment
Nitrogen starvation
SA-induced flowering
Cadmium treatment
miRNA identification
Degradome sequencing
Genetic diversity
Genetic diversity
Salt stress
Nutrient starvation
Reconstituted
Natural and
reconstituted
Natural
57 samples
PRJNA545325
Huang et al. (2020)
Mitochondrial
Chloroplast
Datasets
sRNA and degradome
Population
ssRNA sequencing
Proteomics
Bacterial community
Director
Number of Clones
Contact
K. Acosta et al.
Location
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Genomes
Affiliation
| THE PLANT CELL 2021: 33: 3207–3234
Germplasm
Duckweed for plant systems studies
Genomic resources
The duckweed community is growing and generating valuable resources for genome discovery at an increasing rate. A
growing number of datasets are available at the National
Library of Medicine (NCBI) and the Short Read Archive
(SRA). Currently, the Sp. polyrhiza reference genomes
(Sp7498v2; Sp9509v3) can be accessed from multiple locations including phytozome (https://phytozome-next.jgi.doe.
gov/), PLAZA (https://bioinformatics.psb.ugent.be/plaza/),
SpirodelaGenome (http://spirodelagenome.org/), and NCBI.
The reference genomes for two Sp. intermedia clones, Si7747
and Si8410, can be obtained from the European Nucleotide
Archive and raw data from NCBI SRA (Hoang et al., 2020).
Additional Lemna and Wolffia genomes can also be found
at lemna.org and CoGe (https://genomevolution.org/coge/).
One of the first duckweed datasets at NCBI contains fulllength cDNA clones for Wo. arrhiza 8872a, Wo. australiana
7733, and La. punctata 9264 sequenced by the Waksman
Student Scholars Program at Rutgers University in New
Jersey since 2009 (https://wssp.rutgers.edu/research/previ
ous). In addition to genome sequencing data, multiple transcriptome, proteome, and bacterial DNA amplicon datasets
from studies involving duckweeds have been deposited in
publicly accessible databases. These duckweed-related
resources are summarized in Table 2, along with their access
information and references.
| 3227
Outlook
There is a need for new models to tackle complex molecular
and ecological processes in plant biology using multidisciplinary approaches. Duckweeds are well-suited to play an important role in these endeavors, as their clonal mode of
reproduction, transformation systems, minimal core gene
content, rapid growth, small size, easy uptake of labeled
compounds, fewer cell types, and unique lifestyle provide
new opportunities for the discovery of novel traits and pathways. Two advanced technologies that we believe will be especially exciting to apply to the duckweed model system are
single-cell genomics and X-ray computed microtomography
(microCT). Both of these methodologies have recently been
successfully applied to excised plant organs or tissues to
demonstrate their ability to reveal new information on root
cell biology in the case of single-cell transcriptomics (Rhee
et al., 2019) and leaf air-space quantification in intact leaf
samples by microCT (Mathers et al., 2018). In fact, as this review was being prepared, the first report describing the application of microCT to duckweed was published (Jones et
al., 2021). Because of its diminutive size, the duckweed
model system should allow these approaches to be applied
at the whole plant level and provide quantitative information on discrete lineages of cell populations or 3D parameters for plant architecture as a function of growth and the
environment. These types of discoveries will be required for
a future in which improved crops will have to withstand
large temperature swings, droughts, and floods and there
will be an increasing need for higher yields and nutritional
qualities. As aquatic plants that have adapted to habitats
that are distinct from those of land plants, the Lemnaceae
appears to deploy different strategies for defense and growth
regulation. Elucidating these alternative pathways may provide insights on novel strategies for designing future crops
by the next generation of farmers, scientists, and climate
warriors.
Supplemental data
The following materials are available in the online version of
this article.
Supplemental Table S1. Examples of habitats and growth
habits of duckweeds.
Acknowledgments
We would like to acknowledge the amazing work of Elias
Landolt who amassed and organized the first large-scale
duckweed collection in the community, as well as his monumental monographs that summarized all-things-duckweed
up to the 1980s, which served as his legacy to our community. These are truly the cornerstones on which progress has
been built upon up to the current time. We apologize to
researchers whose relevant work we have not cited due to
space limitations.
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
identification system, with numbers for more recent clone
isolates assigned by the RDSC. However, with the increasing
number of investigators interested in population studies involving duckweed over the past decade, newer clones in collections can also adopt an individualized identification
system consisting of an investigator’s initials followed by digits (Lam et al., 2020). An accepted clone identification system
is critical for replicating experimental results between laboratories and extending prior work. It thus serves as a foundation for the community in which results from various
reported studies can be integrated and leveraged together based on knowledge of the biological material being tested. As noted earlier in this review, duckweeds
have historically been used to study various aspects of
plant biology. In some cases, keynote clones have been
extensively used in a particular field of study. This
includes Le. minor 8627 and Le. aequinoctialis 6002 for
transformation studies (Cantó-Pastor et al., 2015; Liu et
al., 2019); Le. perpusilla 6746 (now renamed as Le. aequinoctialis 6746) and Le. gibba G3 to study flowering
(Hillman, 1959; Fu et al., 2017); Le. minor 5512 as well as
Le. minor 5576 to study plant–microbe interactions
(Ishizawa et al., 2020; Acosta et al., 2020); Le. minor 5500
for ecological and toxicological studies (Paolacci et al.,
2018b); La. punctata 5562 (previously named Sp. oligorrhiza) to study the D1 protein of PSII (Mattoo and
Edelman, 1987); and Sp. polyrhiza clones 7498 and 9509
for genomic studies (Wang et al., 2014; Michael et al.,
2017; Hoang et al., 2018).
THE PLANT CELL 2021: 33: 3207–3234
3228
| THE PLANT CELL 2021: 33: 3207–3234
Funding
Duckweed research at the Lam laboratory is supported in
part by a grant from the Department of Energy (DESC0018244), a Hatch project (#12116), and a Multi-State
Capacity project (#NJ12710) from the New Jersey
Agricultural Experiment Station at Rutgers University.
Duckweed research at the Xu laboratory is funded by the
German Science Foundation (DFG, #427577435 and
#438887884) and the University of Münster.
Conflict of interest statement. None declared.
Acosta K, Xu J, Gilbert S, Denison E, Brinkman T, Lebeis S, Lam E
(2020) Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome. PloS One 15: p.e0228560
Adamec L (2018) Ecophysiological characteristics of turions of
aquatic plants: a review. Aquat Bot 148: 64–77
Agrawal AA, Hastings AP, Johnson MT, Maron JL, Salminen JP
(2012) Insect herbivores drive real-time ecological and evolutionary
change in plant populations. Science 338: 113–116
An D, Zhou Y, Li C, Xiao Q, Wang T, Zhang Y, Wu Y, Li Y, Chao,
D-Y, Messing J et al. (2019) Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of
Spirodela. Proc Natl Acad Sci U S A 116: 18893–18899
APG (1998) An ordinal classification for the families of flowering
plants. Ann Missouri Bot Gard 85: 531–553
Appenroth KJ, Hertel W, Jugnickel F, Augsten H (1989) Influence
of nutrient deficiency and light on turion formation in Spirodela
polyrhiza (L.) Schleiden. Biochem Physiol Pflanzen 184: 395–403
Appenroth KJ, Augsten H (1990) Photophysiology of turion germination in Spirodela polyrhiza (L.) Schleiden-V. Demonstration of a
calcium-requiring phase during phytochrome-mediated germination. Photochem Photobiol 52: 61–65
Appenroth KJ (2002) Co-action of temperature and phosphate in inducing turion formation in Spirodela polyrhiza (greater duckweed).
Plant Cell Environ 25: 1079–1085
Appenroth KJ, Nickel G (2010) Induction of turion formation in
Spirodela polyrhiza under close-to-nature conditions: the environmental signals that induce the developmental process in nature.
Physiol Plant 138: 312–320
Appenroth KJ, Palharini L, Ziegler P (2013) Low-molecular weight carbohydrates modulate dormancy and are required for post-germination
growth in turions of Spirodela polyrhiza. Plant Biol 15: 284–291
Appenroth KJ, Sree KS, Böhm V, Hammann S, Vetter W, Leiterer
M, Jahreis G (2017) Nutritional value of duckweeds (Lemnaceae)
as human food. Food Chem 217: 266–273
Appenroth KJ, Sree KS, Bog M, Ecker J, Seeliger C, Bohm V,
Lorkowski S, Sommer K, Veeter W, Tolzin-Banasch K, et al. (2018)
Nutritional value of the duckweed species of the genus Wolffia
(Lemnaceae) as human food. Front Chem 6: 483
Ardenghi NM, Armstrong WP, Paganelli D (2017) Wolffia columbiana (Araceae, Lemnoideae): first record of the smallest alien flowering plant in southern Europe and Italy. Bot Lett 164: 121–127
Ashby E, Wangermann E, Winter EJ (1949) Studies in the morphogenesis of leaves. III. Preliminary observations on vegetative growth
in Lemna minor. New Phytol 48: 374–381
Baggs EL, Monroe GJ, Thanki AS, O’Grady R, Schudoma C, Haerty
W, Krasileva KV (2020) Convergent loss of an EDS1/PAD4 signaling pathway in several plant lineages reveals coevolved components of plant immunity and drought response. Plant Cell 32:
2158–2177
Bailey JK, Hendry AP, Kinnison MT, Post DM, Palkovacs EP,
Pelletier F, Harmon LJ, Schweitzer JA (2009) From genes to
ecosystems: an emerging synthesis of eco-evolutionary dynamics.
New Phytol 184: 746–749
Balaji P, Satheeshkumar PK, Venkataraman K, Vijayalakshmi MA
(2016) Expression of anti-tumor necrosis factor alpha (TNFa) single-chain variable fragment (scFv) in Spirodela punctata plants
transformed with Agrobacterium tumefaciens. Biotechnol Appl
Biochem 63: 354–361
Baldi BG, Maher BR, Slovin JP, Cohen JD (1991) Stable isotope labeling, in vivo, of D- and L-tryptophan pools in Lemna gibba and the
low incorporation of label into IAA. Plant Physiol 95: 1203–1208
Beilsmith K, Perisin M, Bergelson J (2021) Natural bacterial assemblages in Arabidopsis thaliana tissues become more distinguishable
and diverse during host development. Mbio 12: e02723–20
Bellini C, Pacurar DI, Perrone I (2014) Adventitious roots and lateral roots: similarities and differences. Annu Rev Plant Biol 65:
639–666
Bertran K, Thomas G, Guo X, Bublot M, Pritchard N, Regan JT,
Cox KM, Gasdaska JR, Dickey LF, Kapczynski DR, et al. (2015)
Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity
avian influenza virus challenge in immunized chickens. Vaccine 33:
3456–3462
Bhanthumnavin K, McGarry MG (1971) Wolffia arrhiza as a possible source of inexpensive protein. Nature 232: 495
Boehm R, Kruse C, Voeste D, Barth S, Schnabl H (2001) A transient transformation system for duckweed (Wolffia columbiana)
using Agrobacterium-mediated gene transfer. J Appl Bot 75:
107–111
Bog M, Baumbach H, Schween U, Hellwig F, Landolt E,
Appenroth KJ (2010) Genetic structure of the genus Lemna L.
(Lemnaceae) as revealed by amplified fragment length polymorphism. Planta 232: 609–619
Bog M, Schneider P, Hellwig F, Sachse S, Kochieva EZ,
Martyrosian E, Landolt E, Appenroth KJ (2013) Genetic characterization and barcoding of taxa in the genus Wolffia Horkel ex
Schleid. (Lemnaceae) as revealed by two plastidic markers and amplified fragment length polymorphism (AFLP). Planta 237: 1–13
Bog M, Lautenschläger U, Landrock M F., Landolt E, Fuchs J, Sree
K S., Oberprieler C, Appenroth KJ (2015) Genetic characterization and barcoding of taxa in the genera Landoltia and Spirodela
(Lemnaceae) by three plastidic markers and amplified fragment
length polymorphism (AFLP). Hydrobiologia 749: 169–182
Bog M, Appenroth KJ, Sree KS (2019) Duckweed (Lemnaceae): its
molecular taxonomy. Front Sustain Food Syst 3: 117
Bog M, Appenroth KJ, KS Sree (2020a) Key to the determination of
taxa of Lemnaceae: an update. Nord J Bot 38: e02658
Bog M, Sree KS, Fuchs J, Hoang PTN., Schubert I, Kuever J,
Rabenstein A, Paolacci S, Jansen MAK, Appenroth KJ (2020b) A
taxonomic revision of Lemna sect. Uninerves (Lemnaceae). Taxon
69: 56–66
Bog M, Xu S, Himmelbach A, Brandt R, Wagner F, Appenroth KJ,
Sree KS (2020c) Genotyping-by-sequencing for species delimitation
in Lemna section Uninerves Hegelm. (Lemnaceae). In XH Cao, P
Fourounjian, W Wang, eds, The Duckweed Genomes. Springer,
Berlin, Heidelberg, pp. 115–123
Borisjuk N, Chu P, Gutierrez R, Zhang H, Acosta K, Friesen N,
Sree KS, Garcia C, Appenroth KJ, Lam E (2015) Assessment,
validation and deployment strategy of a two-barcode protocol for
facile genotyping of duckweed species. Plant Biol 17: 42–49
Borisjuk N, Peterson AA, Lv J, Qu G, Luo G, Shi L, Chen G,
Kishchenko O, Zhou Y, Shi J (2018) Structural and biochemical
properties of duckweed surface cuticle. Front Chem 6: 317
Böttner L, Grabe V, Gablenz S, Böhme N, Appenroth KJ,
Gershenzon J, Huber M (2020) Differential localization of flavonoid glucosides in an aquatic plant implicates different functions
under abiotic stress. Plant Cell Environ 44: 900–914
Braglia L, Lauria M, Appenroth KJ, Bog M, Breviario D, Grasso A,
Gavazzi F, Morello L (2021) Duckweed species genotyping and
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
References
K. Acosta et al.
Duckweed for plant systems studies
| 3229
Detling JK, Painter EL (1983) Defoliation responses of western
wheatgrass populations with diverse histories of prairie dog grazing. Oecologia 57: 65–71
Ding Y, Fang Y, Guo L, Li Z, He K, Zhao Y, Zhao H (2017)
Phylogenetic study of Lemnoideae (duckweeds) through complete
chloroplast genomes for eight accessions. PeerJ 5: e4186
Docauer DM (1983) A nutrient basis for the distribution of the
Lemnaceae. PhD thesis. University of Michigan, USA
Dolger C, Tirlapur UK, Appenroth KJ (1997) Phytochromeregulated starch degradation in germinating turions of Spirodela
polyrhiza. Photochem Photobiol 66: 124–127
Duong TP, Tiedje JM (1985) Nitrogen fixation by naturally occurring
duckweed—cyanobacterial associations. Can J Microbiol 31:
327–330
Durán P, Thiergart T, Garrido-Oter R, Agler M, Kemen E,
Schulze-Lefert P, Hacquard S (2018) Microbial interkingdom interactions in roots promote Arabidopsis survival. Cell 175: 973–983
Eames C, Posner HB (1974) Abnormal flowering responses and a
lack of photosynthesis in a mutant of Lemna perpusilla. Plant
Physiol 55: S11
Echlin P, Lai CE, Hayes TL (1982) Low-temperature X-ray microanalysis of the differentiating vascular tissue in root tips of Lemna
minor L. J Microsc 126: 285–306
Edelman M, Perl A, Flaishman M, Blumenthal A (1999) Transgenic
Lemnaceae. WO 99/19497
Edelman M, Colt M (2016) Nutrient value of leaf vs. seed. Front
Chem 4: 32
Edwards J, Johnson C, Santos-Medellı́n C, Lurie E, Podishetty NK,
Bhatnagar S, Eisen JA, Sundaresan V (2015) Structure, variation,
and assembly of the root-associated microbiomes of rice. Proc Natl
Acad Sci U S A 112: E911–E920
Ekperusi AO, Nwachukwu EO, Sikoki FD (2020) Assessing and
modeling the efficacy of Lemna paucicostata for the phytoremediation of petroleum hydrocarbons in crude oil-contaminated wetlands. Sci Rep 10: 8489
El-Kholy AS, Youssef MS, Eid EM (2015) Genetic diversity of Lemna
gibba L. and L. minor L. populations in Nile delta based on biochemical and ISSR markers. Egypt J Exp Biol Bot 11: 11–19
Feldberg L, Venger I, Malitsky S, Rogachev I, Aharoni A (2009)
Dual labeling of metabolites for metabolome analysis (DLEMMA):
a new approach for the identification and relative quantification of
metabolites by means of dual isotope labeling and liquid
chromatography-mass spectrometry. Anal Chem 81: 9257–9266
Feldberg L, Dong Y, Heinig U, Rogachev I, Aharoni A (2018)
DLEMMA-MS-imaging for identification of spatially localized
metabolites and metabolic network map reconstruction. Anal
Chem 90: 10231–10238
Feuchtmayr H, Moran R, Hatton K, Connor L, Heyes T, Moss B,
Harvey I, Atkinson D (2009) Global warming and eutrophication:
effects on water chemistry and autotrophic communities in experimental hypertrophic shallow lake mesocosms. J Appl Ecol 46:
713–723
Filichkin SA, Breton G, Priest HD, Dharmawardhana P, Jaiswal P,
Fox SE, Michael TP, Chory J, Kay SA, Mockler TC (2011) Global
profiling of rice and poplar transcriptomes highlights key conserved circadian-controlled pathways and cis-regulatory modules.
PLoS One 6: e16907
Firsov A, Tarasenko I, Mitiouchkina T, Shaloiko L, Kozlov O,
Vinokurov L, Rasskazova E, Murashev A, Vainstein A, Dolgov S
(2018) Expression and immunogenicity of M2e peptide of avian influenza virus H5N1 fused to ricin toxin B chain produced in duckweed plants. Front Chem 6: 22
Fitzpatrick CR, Salas-González I, Conway JM, Finkel OM, Gilbert
S, Russ D, Teixeira PJPL, Dangl JL (2020). The plant microbiome:
from ecology to reductionism and beyond. Ann Rev Microbiol 74:
81–100
Fourounjian P, Tang J, Tanyolac B, Feng Y, Gelfand B, Kakrana A,
Tu M, Wakim C, Meyers BC, Ma J, Messing J (2019)
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
interspecific hybrid discovery by tubulin-based polymorphism fingerprinting. Front Plant Sci 12: 625670
Bruce BD, Malkin R (1991) Biosynthesis of the chloroplast cytochrome b6-f complex: studies in a photosynthetic mutant of
Lemna. Plant Cell 3: 203–212
Burghardt LT, Epstein B, Guhlin J, Nelson MS, Taylor MR, Young
ND, Sadowsky MJ, Tiffin P (2018) Select and resequence reveals
relative fitness of bacteria in symbiotic and free-living environments. Proc Natl Acad Sci U S A 115: 2425–2430
Busby PE, Soman C, Wagner MR, Friesen ML, Kremer J, Bennett
A, Morsy M, Eisen JA, Leach JE, Dangl JL (2017) Research priorities for harnessing plant microbiomes in sustainable agriculture.
PLoS Biol 15: p.e2001793
Calicioglu O, Brennan RA (2018) Sequential ethanol fermentation
and anaerobic digestion increases bioenergy yields from duckweed.
Bioresour Technol 257: 344–348
Cantó-Pastor A, Mollá-Morales A, Ernst E, Dahl W, Zhai J, Yan Y,
Meyers BC, Shanklin J, Martienssen R (2015) Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant
Biol 17: 59–65
Cedergreen N, Madsen TV (2002) Nitrogen uptake by the floating
macrophyte Lemna minor. New Phytol 155: 285–292
Ceschin S, Abati S, Ellwood NTW, Zuccarello V (2018) Riding invasion waves: spatial and temporal patterns of the invasive Lemna
minuta from its arrival to its spread across Europe. Aquat Bot 150:
1–8
Chang WC, Chiu PL (1978) Regeneration of Lemna gibba G3
through callus culture. Z Pflanzenphysiol 89: 91–94
Chang WC, Hsing YI (1978) Callus formation and regeneration of
frond-like structures in Lemna perpusilla 6746 on a defined medium. Plant Sci Lett 13: 133–136
Cheng JJ, Stomp AM (2009) Growing duckweed to recover nutrients
from wastewaters and for production of fuel ethanol and animal
feed. Clean Soil Air Water 37: 17–26
Chu P, Wilson GM, Michael TP, Vaiciunas J, Honig J, Lam E (2018)
Sequence-guided approach to genotyping plant clones and species using polymorphic NB-ARC-related genes. Plant Mol Biol 98: 219–231
Cleland CF, Ajami A (1974) Isolation and identification of the
flower-inducing factor from aphid honeydew as being salicylic acid.
Plant Physiol 54: 904–906
Cole CT, Voskuil MI (1996) Population genetic structure in duckweed (Lemna minor, Lemnaceae). Can J Bot 74: 222–230
Coughlan NE, Kelly TC, Jansen MAK (2015) Mallard duck (Anas
platyrhynchos)-mediated dispersal of Lemnaceae: a contributing
factor in the spread of invasive Lemna minuta? Plant Biol 17:
108–114
Coughlan NE, Kelly TC, Jansen MAK (2017) “Step by step”: high frequency short-distance epizoochorous dispersal of aquatic macrophytes. Biol Invasions 19: 625–634
Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG,
Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, et al.
(2006) Glycan optimization of a human monoclonal
antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:
1591–1597
Cross JW (2015) The rise and fall of a duckweed biotechnology firm:
what can it tell us? ISCDRA Duckweed Forum 3: 83–88
Cross JW (2017) Duckweed roots: their role in vegetative dispersal.
ISCDRA Duckweed Forum 5: 58–59
Cui W, Cheng JJ (2015) Growing duckweed for biofuel production: a
review. Plant Biol 17: 16–23
Darwin C (1859) On the Origin of Species by Means of Natural
Selection, Vol 167. John Murray, London
Datko AH, Mudd HS, Giovanelli J, MacNicol PK (1978a)
Sulfur-containing compounds in Lemna perpusilla 6746 grown at a
range of sulfate concentrations. Plant Physiol 62: 629–635
Datko AH, Mudd HS, MacNicol PK, Giovanelli J (1978b) Phytostat
for the growth of Lemna in semicontinuous culture with low sulfate. Plant Physiol 62: 622–628
THE PLANT CELL 2021: 33: 3207–3234
3230
| THE PLANT CELL 2021: 33: 3207–3234
integrating cytogenetic maps, PacBio and Oxford Nanopore libraries. Sci Rep 10: 19230
Hollingsworth PM, Forrest LL, Spouge JL, Hajibabaei M,
Ratnasingham S, van der Bank M, Chase MW, Cowan RS,
Erickson DL, Fazekas AJ, et al. (2009) A DNA barcode for land
plants. Proc Natl Acad Sci U S A 106: 12794–12797
Huang M, Fang Y, Liu Y, Jin Y, Sun J, Tao X, Ma X, He K, Zhao H
(2015) Using proteomic analysis to investigate uniconazoleinduced phytohormone variation and starch accumulation in
duckweed (Landoltia punctata). BMC Biotechnol 15: 1–13
Huang M, Fu L, Sun X, Di R, Zhang J (2016) Rapid and highly efficient callus induction and plant regeneration in the starch-rich
duckweed strains of Landoltia punctata. Acta Physiol Plant 38: 122
Huang W, Gilbert S, Poulev A, Acosta K, Lebeis S, Long C, Lam E
(2020) Host-specific and tissue-dependent orchestration of microbiome community structure in traditional rice paddy ecosystems.
Plant Soil 452: 379–395
Iberite M, Iamonico D, Abati S, Abbate G (2011) Lemna valdiviana
Phil. (Araceae) as a potential invasive species in Italy and Europe:
taxonomic study and first observations on its ecology and distribution. Plant Biosyst 145: 751–757
Iguchi H, Umeda R, Taga H, Oyama T, Yurimoto H, Sakai Y
(2019) Community composition and methane oxidation activity of
methanotrophs associated with duckweeds in a freshwater lake. J
Biosci Bioeng 128: 450–455
Ishizawa H, Kuroda M, Morikawa M, Ike M (2017) Evaluation of
environmental bacterial communities as a factor affecting the
growth of duckweed Lemna minor. Biotechnol Biofuels 10: 62
Ishizawa H, Kuroda M, Inoue K, Inoue D, Morikawa M, Ike M
(2019a) Colonization and competition dynamics of plant
growth-promoting/inhibiting bacteria in the phytosphere of the
duckweed Lemna minor. Microbiol Ecol 77: 440–450
Ishizawa H, Tada M, Kuroda M, Inoue D, Ike M (2019b)
Performance of plant growth-promoting bacterium of duckweed
under different kinds of abiotic stress factors. Biocatal Agric
Biotechnol 19: 101146
Ishizawa H, Kuroda M, Inoue D, Morikawa M, Ike M (2020)
Community dynamics of duckweed-associated bacteria upon inoculation of plant growth-promoting bacteria. FEMS Microbiol Ecol
96: fiaa101
ISO (2005) Water quality – determination of the toxic effect of water
constituents and waste to duckweed (Lemna minor) – Duckweed
growth inhibition test. International Organization for Standardization,
Geneva, Switzerland
Jacobs DL (1947) An ecological life-history of Spirodela polyrrhiza
(greater duckweed) with emphasis on the turion phase. Ecol
Monogr 17: 437–469
Jones DH, Atkinson BS, Ware A, Sturrock CJ, Bishopp A, Wells
D.M. (2021) Preparation, scanning and analysis of duckweed using
X-Ray computed microtomography Front. Plant Sci 11: 2140
Kanesaka Y, Okada M, Ito S, Oyama T (2019) Monitoring
single-cell bioluminescence of Arabidopsis leaves to quantitatively
evaluate the efficiency of a transiently introduced CRISPR/Cas9 system
targeting the circadian clock gene ELF3. Plant Biotechnol 36: 187–193
Kaur M, Kumar M, Singh D, Sachdeva S, Puri SK (2019) A sustainable biorefinery approach for efficient conversion of aquatic weeds
into bioethanol and biomethane. Energy Convers Manag 187:
133–147
Kautsar SA, Duran HGS, Blin K, Osbourn A, Medema MH (2017)
plantiSMASH: automated identification, annotation and expression
analysis of plant biosynthetic gene clusters. Nucleic Acids Res 45:
W55–W63
Kehoe DM, Degenhardt J, Winicov I, Tobin EM (1994) Two 10-bp
regions are critical for phytochrome regulation of a Lemna gibba
Lhcb gene promoter. Plant Cell 6: 1123–1134
Khurana JP, Maheshwari SC (1986) A comparison of the effects of
chelates, salicylic acid and benzoic acid on growth and flowering of
Spirodela polyrrhiza. Plant Cell Physiol 27: 919–924
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Post-transcriptional adaptation of the aquatic plant Spirodela polyrhiza under stress and hormonal stimuli. Plant J 98: 1120–1133
Fourounjian P, Slovin J, Messing J (2021) Flowering and seed production across the Lemnaceae. Int J Mol Sci 22: 2733
Fu L, Huang M, Han B, Sun X, Sree KS, Appenroth KJ, Zhang J
(2017). Flower induction, microscope-aided cross-pollination, and
seed production in the duckweed Lemna gibba with discovery of a
male-sterile clone. Sci Rep 7: 1–13
Fu L, Ding Z, Tan D, Han B, Sun X, Zhang J (2020a) Genome-wide
discovery and functional prediction of salt-responsive lncRNAs in
duckweed. BMC Genomics 21: 1–14
Fu L, Tan D, Sun X, Ding Z, Zhang J (2020b) Transcriptional analysis reveals potential genes and regulatory networks involved in salicylic acid-induced flowering in duckweed (Lemna gibba). Plant
Physiol Biochem 155: 512–522
Geber G (1989) Zur Karyosystematik der Lemnaceae. PhD thesis.
University of Vienna, Vienna, Austria
Gilbert S, Xu J, Acosta K, Poulev A, Lebeis S, Lam E (2018)
Bacterial production of indole related compounds reveals their
role in association between duckweeds and endophytes. Front
Chem 6: 265
Guo L, Jin Y, Xiao Y, Tan L, Tian X, Ding Y, He K, Du A, Li J, Yi Z,
et al. (2020) Energy-efficient and environmentally friendly production of starch-rich duckweed biomass using nitrogen-limited cultivation. J Clean Prod 251: 119726
Hammami R, Ben Hamida J, Vergoten G, Fliss I (2009) PhytAMP: a
database dedicated to antimicrobial plant peptides. Nucleic Acids
Res 37: D963–D968
Harkess A, McLaughlin F, Bilkey N, Elliot K, Emenecker R,
Mattoon E, Miller K, Czymmek K, Vierstra R, Meyers BC, et al.
(2021) Improved Spirodela polyrhiza genome and proteomic analyses reveal a conserved chromosomal structure with high abundance of chloroplastic proteins favoring energy production. J Exp
Bot 72: 2491–2500
Hart SP, Turcotte MM, Levine JM (2019) Effects of rapid evolution
on species coexistence. Proc Natl Acad Sci 116: 2112–2117
Heenatigala PPM., Yang J, Bishopp A, Sun Z, Li G, Kumar S, Hu S,
Wu Z, Lin W, Yao L, Duan P (2018) Development of efficient
protocols for stable and transient gene transformation for Wolffia
globosa using Agrobacterium. Front Chem 6: 227
Hegelmaier F (1868) Die Lemnaceen. Eine monographische
Untersuchung. Engelmann, Leipzig, Germany
Hendry AP (2019) A critique for eco-evolutionary dynamics. Funct
Ecol 33: 84–94
Hillman WS (1957) Nonphotosynthetic light requirement in
Lemna minor and its partial satisfaction by kinetin. Science 126:
165–166
Hillman WS (1959) Experimental control of flowering in Lemna I.
General methods. Photoperiodism in Lemna perpusilla 6746. Am J
Bot 46: 466–473
Hillman WS (1961) The Lemnaceae, or duckweeds. A review of the
descriptive and experimental literature. Bot Rev 27: 221–287
Hillman WS (1976) Calibrating duckweeds—light, clocks, metabolism, flowering. Science 193: 453–458
Ho EKH., Bartkowska M, Wright SI, Agrawal AF (2019) Population
genomics of the facultatively asexual duckweed Spirodela polyrhiza.
New Phytol 224: 1361–1371
Hoang PTN., Michael TP, Gilbert S, Chu P, Motley ST, Appenroth
KJ, Schubert I, Lam E (2018) Generating a high-confidence reference genome map of the Greater Duckweed by integration of
cytogenomic, optical mapping, and Oxford Nanopore technologies.
Plant J 96: 670–684
Hoang PTN, Schubert V, Meister A, Fuchs J, Schubert I (2019)
Variation in genome size, cell and nucleus volume, chromosome
number and rDNA loci among duckweeds. Sci Rep 9: 3234
Hoang PTN, Fiebig, A, Novak P, Macas J, Cao HX, Stepaneko A,
Chen G, Borisjuk N, Scholz U, Schubert I (2020) Chromosomescale genome assembly for the duckweed Spirodela intermedia,
K. Acosta et al.
Duckweed for plant systems studies
| 3231
Les DH, Crawford DJ, Landolt E, Gabel JD, Kimball RT (2002)
Phylogeny and systematics of Lemnaceae, the duckweed family.
Syst Bot 27: 221–240
Les DH, Crawford DJ, Kimball RT, Moody ML, Landolt E (2003)
Biogeography of discontinuously distributed hydrophytes: a molecular appraisal of intercontinental disjunctions. Int J of Plant Sci
164: 917–932
Li J, Jain M, Vunsh R, Vishnevetsky J, Hanania U, Flaishman M,
Perl A, Edelman M (2004) Callus induction and regeneration in
Spirodela and Lemna. Plant Cell Rep 22: 457–464
Li X, Zhang R, Patena W, Gang SS, Blum SR, Ivanova N, Yue R,
Robertson JM, Lefebvre PA, Fitz-Gibbon ST, Grossman AR,
Jonikas MC (2016) An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas
reinhardtii. Plant Cell 28: 367–387
Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.
Biotechniques 43: 649–650
Liu Y, Chen X, Wang X, Fang Y, Huang M, Guo L, Zhang Y, Zhao
H (2018) Improving biomass and starch accumulation of bioenergy
crop duckweed (Landoltia punctata) by abscisic acid application.
Sci Rep 8: 9544
Liu Y, Wang Y, Xu S, Tang X, Zhao J, Yu C, He G, Xu H, Wang S,
Tang Y, et al. (2019) Efficient genetic transformation and
CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.
Plant Biotechnol J 17: 2143–2152
Longland JM, Fry SC, Trewavas AJ (1989) Developmental control of
apiogalacturonan biosynthesis and UDP-apiose production in a
duckweed. Plant Physiol 90: 972–976
Lu Y, Zhou Y, Nakai S, Hosomi M, Zhang H, Kronzucker HJ, Shi
W (2014) Stimulation of nitrogen removal in the rhizosphere of
aquatic duckweed by root exudate components. Planta 239:
591–603
Lucey J (2003) Lemna minuta Kunth (Least Duckweed) in E. Cork
(VC H5). Irish Bot News 13: 5–8
Ma YB, Zhu M Yu CJ, Wang Y, Liu Y, Li ML, Sun YD, Zhao JS,
Zhou GK (2018) Large-scale screening and characterization of
Lemna aequinoctialis and Spirodela polyrhiza strains for starch production. Plant Biol 20: 357–364
Mano Y, Nemoto K (2012) The pathway of auxin biosynthesis in
plants. J Exp Bot 63: 2853–2872
Maheshwari SC, Seth PN (1966) Induction of flowering in
Wolffia microsporica by iron salt by ethylenediamine-di-Ohydroxyphenilacetic acid (FE-EDDHA). Z Pl Physiol 55: 89–91
Mardanov AV, Ravin NV, Kuznetsov BB, Samigullin TH, Antonov
AS, Kolganova TV, Skyabin KG (2008) Complete sequence of the
duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms. J Mol
Evol 66: 555–564
Martirosyan EV, Ryzhova NN, Skryabin KG, Kochieva EZ (2008)
RAPD analysis of genome polymorphism in the family Lemnaceae.
Russ J Genet 44: 360–364
Mathers AW, Hepworth C, Baillie AL, Sloan J, Jones H, Lundgren
M, Fleming AJ, Mooney S, Sturrock CJ (2018) Investigating the
microstructure of plant leaves in 3D with lab-based X-ray computed tomography. Plant Methods 14: 99
Matsuzawa H, Tanaka Y, Tamaki H, Kamagata Y, Mori K (2010)
Culture-dependent and independent analyses of the microbial
communities inhabiting the giant duckweed (Spirodela polyrrhiza)
rhizoplane and isolation of a variety of rarely cultivated organisms
within the phylum Verrucomicrobia. Microbes Environ 25:
302–308
Mattoo AK, Hoffman-Falk H, Marder JB, Edelman M (1984)
Regulation of protein metabolism: Coupling of photosynthetic
electron transport to in vivo degradation of the rapidlymetabolized 32-kilodalton protein of the chloroplast membranes.
Proc Natl Acad Sci U S A 81: 1380–1384
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Khvatkov P, Chernobrovkina M, Okuneva A, Pushin A, Dolgov S
(2015) Transformation of Wolffia arrhiza (L.) Horkel ex Wimm.
Plant Cell Tissue Organ Cult 123: 299–307
Kim I (2007) Development of the root system in Spirodela polyrhiza
(L.) schleiden (Lemnaceae). J Plant Biol 50: 540–547
Kim, I (2011) Features of plastids within Spirodela polyrhiza. Korean J
Microsc 41: 55–60
Kim I (2013) Cellular features of the fronds and turions in Spirodela
polyrhiza. Korean Soc Microsc 43:140–145
Kim I (2016) Structural differentiation of the connective stalk in
Spirodela polyrhiza (L.) Schleiden. Korean Soc Microsc Appl
Microsc 46: 83–88
Kinnison MT, Hairston NG (2007) Eco-evolutionary conservation biology: contemporary evolution and the dynamics of persistence.
Funct Ecol 21: 444–454
Klich G, Mújica MB, Fernández OA (1986) Stomatal morphology
and ontogeny in Spirodela intermedia W. Koch. Aquat Bot 26:
155–164
Ko SM, Sun HJ, Oh MJ, Song IJ, Kim MJ, Sin HS, Goh CH, Kim
YW, Lim PO, Lee HY, Kim SW (2011) Expression of the protective
antigen for PEDV in transgenic duckweed, Lemna minor. Horticult
Environ Biotechnol 52: 511
Kofler R, Schlotterer C (2014) A guide for the design of evolve and
resequencing studies. Mol Biol Evol 31: 474–483
Kostopoulou S, Ntatsi G, Arapis G, Aliferis KA (2020) Assessment
of the effects of metribuzin, glyphosate, and their mixtures on the
metabolism of the model plant Lemna minor L. applying metabolomics. Chemosphere 239: 124582
Kremer JM, Sohrabi R, Paasch BC, Rhodes D, Thireault C,
Schulze-Lefert P, Tiedje JM, He SY (2021) Peat-based gnotobiotic
plant growth systems for Arabidopsis microbiome research. Nat
Protoc 16: 2450–2470
Kuehdorf K, Jetschke G, Ballani L, Appenroth KJ (2014) The clonal
dependence of turion formation in the duckweed Spirodela polyrhiza - an ecogeographical approach. Physiol Plant 150: 46–54
Kwak M, Kim I (2008) Turion as dormant structure in Spirodela polyrhiza. Korean J Microsc 38: 307–314
Laird RA, Barks PM (2018) Skimming the surface: duckweed as a
model system in ecology and evolution. Am J Bot 105: 1962–1966
Lam E, Malkin R (1985) Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex. Biochim
Biophys Acta 810: 106–109
Lam E, Appenroth KJ, Michael T, Mori K, Fakhoorian T (2014)
Duckweed in bloom: the 2nd International Conference on
Duckweed Research and Applications heralds the return of a plant
model for plant biology. Plant Mol Biol 84: 737–742
Lam E, Appenroth KJ, Ma Y, Shoham T, Sree KS (2020)
Registration of duckweed clones—future approach. Duckweed
Forum 8: 35–37
Lä mmler W, Bogner J (2014) Elias Landolt and the duckweeds.
Aroideana 37: 81–88
Landolt E (1986) Biosystematic investigations in the family of duckweeds (Lemnaceae) (Vol 2). The family of Lemnaceae—a monographic study. Veroeffentlichungen des Geobotanischen Instituts
der ETH, Vol 1. Stiftung Ruebel, Switzerland
Landolt E, Kandeler R (1987) The family of Lemnaceae—a monographic study, 2. Biosystematic Investigations in the Family of
Duckweeds (Lemnaceae). Veröffentlichungen des Geobotanischen
Instutes der ETH. Stiftung Rübel, Zurich.
Lavergne S, Mouquet N, Thuiller W, Ronce O (2010) Biodiversity
and climate change: integrating evolutionary and ecological responses
of species and communities. Annu Rev Ecol Evol Syst 41: 321–350
Lee CJ, Yangcheng HY, Cheng JJ, Jane JL (2016) Starch characterization and ethanol production of duckweed and corn kernel.
Starch-Staerke 68: 348–354
Lemon C D, Posluszny U (2000) Comparative shoot development
and evolution in the Lemnaceae. Int J Plant Sci 161: 733–748
THE PLANT CELL 2021: 33: 3207–3234
3232
| THE PLANT CELL 2021: 33: 3207–3234
Osbourn A (2010) Secondary metabolic gene clusters: evolutionary
toolkits for chemical innovation. Trends Genet 26: 449–457
Pagliuso D, Grandis A, Lam E, Buckeridge MS (2020) High saccharification, low lignin, and high sustainability potential make
duckweeds adequate as bioenergy feedstocks. BioEnergy Res DOI:
10.1007/s12155-020-10211-x [Online publication date: November 2,
2020]
Paolacci S, Harrison S, Jansen MA (2018a) The invasive
duckweed Lemna minuta Kunth displays a different light utilization strategy than native Lemna minor Linnaeus. Aquat Bot 146:
8–14
Paolacci S, Jansen MAK., Harrison S (2018b) Competition between
Lemna minuta, Lemna minor, and Azolla filiculoides. Growing fast
or being steadfast? Front Chem 6: 207
Parry G, Provart NJ, Brady SM, Uzilday B, Multinational
Arabidopsis Steering Committee, Adams K, Araújo W, Aubourg
S, Baginsky S, Bakker E, et al. (2020) Current status of the multinational Arabidopsis community. Plant Direct 4: p.e00248
Peeters ETH.M., van Zuidam JP, van Zuidam BG, Van Nes EH,
Kosten S, Heuts PGM, Roijackers RMM, Netten JJC, Scheffer M
(2013) Changing weather conditions and floating plants in temperate drainage ditches. J Appl Ecol 50: 585–593
Pelletier F, Garant D, Hendry AP (2009) Eco-evolutionary dynamics.
Philos T R Soc B 364: 1483–1489
Perera WH, Meepagala KM, Fronczek FR, Cook DD, Wedge DE,
Duke SO (2019) Bioassay-guided isolation and structure elucidation of fungicidal and herbicidal compounds from Ambrosia salsola
(Asteraceae). Molecules 24: 835
Peterson AA, Vasylenko MY, Matvieieva NA, Kuchuk MV (2015)
Accumulation of recombinant fusion protein-secretory analog of
Ag85B and ESAT6 Mycobacterium tuberculosis proteins-in transgenic Lemna minor L. plants. Biotechnol Acta 8: 39–48
Pieterse AH, Müller LJ (1977) Induction of flowering in Lemna gibba
G3 under short-day conditions. Plant Cell Physiol 18: 45–53
Pieterse AH (2013) Is flowering in Lemnaceae stress-induced? A review. Aquat Bot 104: 1–4
Posner HB (1962) Characteristics of X-ray-induced aberrants of
Lemna perpusilla 6746. Plant Cell Physiol 3: 275–284
Qiao X, He W, Xiang C, Han J, Wu L, Guo D, Ye M (2011)
Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by high-performance liquid chromatography coupled with
mass spectrometry. Phytochem Anal 22: 475–483
Rapparini F, Tam YY, Cohen JD, Slovin JP (2002) Indole-3-acetic
acid metabolism in Lemna gibba undergoes dynamic changes in response to growth temperature. Plant Physiol 128: 1410–1416
Ren D, Han B, Xin Z, Liu W, Ma S, Liang Y, Yi L (2016)
Computation-aided separation of seven components from
Spirodela polyrrhiza (L.) via counter-current chromatography. Sep
Purif Technol 165: 160–165
Ren H, Jiang N, Wang T, Omar MM, Ruan W, Ghafoor A (2018)
Enhanced biogas production in the duckweed anaerobic digestion
process. J Energy Res Technol Trans ASME 140: 041805
Rhee SY, Birnbaum KD, Ehrhardt DW (2019) Toward building a
plant cell atlas. Trends Plant Sci 24: 303–310
Rimon D, Galun E (1968) Morphogenesis of Wolffia microscopica:
frond and flower development. Phytomorphology 18: 364–372
Rival S, Wisniewski JP, Langlais A, Kaplan H, Freyssinet G,
Vancanneyt G, Vunsh R, Perl A, Edelman M (2008) Spirodela
(duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res 17: 503–513
Rolfe SA, Tobin EM (1991) Deletion analysis of a phytochromeregulated monocot RBCS promotor in a transient assay system.
Proc Natl Acad Sci 88: 2683–2686
Rudman SM, Barbour MA, Csillery K, Gienapp P, Guillaume F,
Hairston Jr NG, Hendry AP, Lasky JR, Rafajlovic M, Rasanen K,
et al. (2018) What genomic data can reveal about
eco-evolutionary dynamics. Nat Ecol Evol 2: 9–15
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Mattoo KA, Edelman M (1987) Intramembrane translocation and
posttranslational palmitoylation of the chloroplast 32kDa herbicide
binding protein. Proc Natl Acad Sci U S A 84: 1497–1501
Matus JT, Ferrier T, Riechmann JL (2014) Identification of
Arabidopsis knockout lines for genes of interest. Methods Mol Biol
1110: 347–362
McLaren JS, Smith, H (1976) The effect of abscisic acid on growth,
photosynthetic rate and carbohydrate metabolism in Lemna minor
L. New Phytol 76: 11–20
McCormac DJ, Greenberg BM (1992) Differential synthesis of photosystem cores and light-harvesting antenna during proplastid to
chloroplast development in Spirodela oligorrhiza. Plant Physiol 98:
1011–1019
Melaragno JE, Walsh MA (1976) Ultrastructural features of developing sieve elements in Lemna minor L.—the protoplast. Am J Bot
63: 1145–1157
Michael TP, Breton G, Hazen SP, Priest H, Mockler TC, Kay SA,
Chory J (2008a) A morning-specific phytohormone gene
expression program underlying rhythmic plant growth. PLoS Biol 6:
e225
Michael TP, Mockler TC, Breton G, McEntee C, Byer A, Trout JD,
Hazen SP, Shen R, Priest HD, Sullivan CM, et al. (2008b)
Network discovery pipeline elucidates conserved time-of-day–specific cis-regulatory modules. PLoS Genet 4: e14
Michael TP, Bryant D, Gutierrez R, Borisjuk N, Chu P, Zhang H,
Xia J, Zhou J, Peng H, El Baidouri M, et al. (2017)
Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies. Plant J 89: 617–635
Michael TP, Ernst E, Hartwick N, Chu P, Bryant D, Gilbert S,
Ortleb S, Baggs EL, Sree KS, Appenroth KJ, et al. (2021) Genome
and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.
Genome Res 31: 225–238
Mifsud S (2010) First occurences of Lemna minuta Kunth (fam.
Lemnaceae) in the Maltese islands. Cent Medit Nat 5: 1–4
Moodley D, Procheş Ş, Wilson JR (2016) A global assessment of a
large monocot family highlights the need for group-specific analyses of invasiveness. AoB Plants 8: plw009
Moon HK, Stomp AM (1997) Effects of medium components and
light on callus induction, growth, and frond regeneration in Lemna
gibba (Duckweed). In Vitro Cell Dev Biol Plant 33: 20–25
Muranaka T, Kubota S, Oyama T (2013) A single-cell bioluminescence imaging system for monitoring cellular gene expression in a
plant body. Plant Cell Physiol 54: 2085–2093
Muranaka T, Oyama T (2018) Monitoring circadian rhythms of individual cells in plants. J Plant Res 131: 15–21
Nauheimer L, Metzler D, Renner SS (2012) Global history of the ancient monocot family Araceae inferred with models accounting for
past continental positions and previous ranges based on fossils.
New Phytol 195: 938–950
Naumann B, Eberius M, Appenroth KJ (2007) Growth rate based
dose-response relationships and EC-values of ten heavy metals using the duckweed growth inhibition test (ISO 20079) with Lemna
minor L. clone St. J Plant Physiol 164: 1656–1664
Normanly J, Cohen JD, Fink GR (1993) Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for
indole-3-acetic acid. Proc Natl Acad Sci U S A 90: 10355–10359
Ogata Y, Toyama T, Yu N, Wang X, Sei K, Ike M (2013)
Occurrence of 4-tert-butylphenol (4-t-BP) biodegradation in an
aquatic sample caused by the presence of Spirodela polyrhiza and
isolation of a 4-t-BP-utilizing bacterium. Biodegradation 24:
191–202
Osama R, Awadc HM, Ibrahima MG, Tawfikd A (2020)
Mechanistic and economic assessment of polyester wastewater
treatment via baffled duckweed pond. J Water Process Eng 35:
101179
K. Acosta et al.
Duckweed for plant systems studies
| 3233
1,5-bisphosphate carboxylase from Lemna gibba L. G3. Nucleic
Acid Res 11: 8051–8061
Stomp AM, Rajbhandari N (1999) Genetically engineered duckweed.
WO 1999007210
Sun Y, Cheng JJ, Himmel ME, Skory CD, Adney WS, Thomas SR,
Tisserat B, Nishimura Y, Yamamoto YT (2007) Expression and
characterization of Acidothermus cellulolyticus E1 endoglucanase in
transgenic duckweed Lemna minor 8627. Bioresour Technol 98:
2866–2872
Sun L, Lu Y, Kronzucker HJ, Shi W (2016) Quantification and
enzyme targets of fatty acid amides from duckweed root exudates
involved in the stimulation of denitrification. J Plant Physiol 198:
81–88
Tam YY, Slovin JP, Cohen JD (1995) Selection and characterization
of a-methyltryptophan-resistant lines of Lemna gibba showing a
rapid rate of indole-3-acetic acid turnover. Plant Physiol 107:
77–85
Tan JQ, Kerstetter JE, Turcotte MM (2021) Eco-evolutionary interaction between microbiome presence and rapid biofilm evolution
determines plant host fitness. Nat Ecol Evol 5: 670–676
Tanaka O, Cleland CF, Hillman WS (1979) Inhibition of flowering
in the long-day plant Lemna gibba G3 by Hutner’s medium and its
reversal by salicylic acid. Plant Cell Physiol 20: 839–846
Tanaka Y, Tamaki H, Tanaka K, Tozawa E, Matsuzawa H, Toyama
T, Kamagata Y, Mori K (2018) “Duckweed-microbe co-cultivation
method” for isolating a wide variety of microbes including taxonomically novel microbes. Microbes Environ 33: 402–406
Teotonio H, Chelo IM, Bradic M, Rose MR, Long AD (2009)
Experimental evolution reveals natural selection on standing genetic variation. Nat Genet 41: 251–257
Thompson CH (1898) A revision of the American Lemnaceae occurring North of Mexico. Ann Rep Miss Bot Gard 9: 21–42
Thu PTL., Huong PT, Tien VV, Ham L, Khanh T (2015)
Regeneration and transformation of gene encoding the hemagglutinin antigen of the H5N1 virus in frond of duckweed (Spirodela
polyrhiza L.). J Agric Stud 3: 48–59.
Tippery NP, Les DH, Crawford DJ (2015) Evaluation of phylogenetic
relationships in Lemnaceae using nuclear ribosomal data. Plant
Biol 17: 50–58
Tippery NP, Les DH (2020) Tiny plants with enormous potential:
phylogeny and evolution of duckweeds. In XH Cao, P Fourounjian,
W Wang, eds, The Duckweed Genomes. Springer, Berlin,
Heidelberg, pp. 19–38
Toyama T, Yu N, Kumada H, Sei K, Ike M, Fujita M (2006)
Accelerated aromatic compounds degradation in aquatic environment by use of interaction between Spirodela polyrhiza and bacteria in its rhizosphere. J Biosci Bioeng 101: 346–353.
Toyama T, Kuroda M, Ogata Y, Hachiya Y, Quach A, Tokura K,
Tanaka Y, Mori K, Morikawa M, Ike M (2017) Enhanced biomass
production of duckweeds by inoculating a plant growthpromoting bacterium, Acinetobacter calcoaceticus P23, in sterile
medium and non-sterile environmental waters. Water Sci Technol
76: 1418–1428
Trewavas AJ (1970) The turnover of nucleic acids in Lemna minor.
Plant Physiol 45: 742–751
Trewavas AJ (1972) Determination of the rates of protein synthesis
and degradation in Lemna minor. Plant Physiol 49: 40–46
Turley NE, Odell WC, Schaefer H, Everwand G, Crawley MJ,
Johnson MT (2013) Contemporary evolution of plant growth rate
following experimental removal of herbivores. Am Nat 181:
S21–S34
Urbanska WK (1980) Cytological variation within the family of
"Lemnaceae". Veröffentlichungen des Geobotanischen Institutes
der Eidg. Tech. Hochschule, Stiftung Rübel, Zürich doi:10.5169/
seals-308615
USEPA (1996) Aquatic plants toxicity test using Lemna spp. United
States Environmental Protection Agency, EPA 712-C-96-156
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Rusoff LL, Blakeney Jr. EW, CulleyJr. DD (1980) Duckweeds
(Lemnaceae family): a potential source of protein and amino acids.
J Agric Food Chem 28: 848–850
Sauter PR (1993) Cryopreservation of Lemnaceae (in German).
Veroeffentlichungen des Geobotanischen Instituts der ETH.
Stiftung Ruebel, Zurich, 112 Heft
Scheffer M, Szabo S, Gragnani A, Van Nes EH, Rinaldi S, Kautsky
N, Norberg J, Roijackers RM, Franken RJ (2003) Floating plant
dominance as a stable state. Proc Natl Acad Sci U S A 100:
4040–4045
Schleiden MJ (1839) Prodromus Monographiae Lemnacearum oder
Conspectus generum atque specierum. Linnaea 13: 385–392
Schlotterer C, Kofler R, Versace E, Tobler R, Franssen SU (2015)
Combining experimental evolution with next-generation sequencing: a powerful tool to study adaptation from standing genetic variation. Heredity (Edinb) 114: 431–440
Shahaf N, Rogachev I, Heinig U, Meir S, Malitsky S, Battat M,
Wyner H, Zheng S, Wehrens R, Aharoni A (2016) The
WEIZMASS spectral library for high-confidence metabolite identification. Nat Commun 7:12423
Shao J, Liu Z, Ding YQ, Wang JM, Li XF, Yang Y (2020)
Biosynthesis of the starch is improved by the supplement of nickel
(Ni2þ) in duckweed (Landoltia punctata). J Plant Res 133: 587–596
Silva GG, Green AJ, Weber V, Hoffmann P, Lovas-Kiss Á, Stenert
C, Maltchik L (2018) Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America. Biol Lett
14: p.20180703
Smarda P, Bures P, Horová L, Leitch IJ, Mucina L, Pacini E, Tichy
L, Grulich V, Rotreklová O (2014) Ecological and evolutionary significance of genomic GC content diversity in monocots. Proc Natl
Acad Sci 111: E4096–E4102
Smart CC, Trewavas AJ (1983a) Abscisic acid-induced turion formation in Spirodela polyrrhiza. I. Production and development of the
turion. Plant Cell Environ 6: 507–514
Smart CC, Trewavas AJ (1983b) Abscisic acid-induced turion formation in Spirodela polyrrhiza L. II. Ultrastructure of the turion; a stereological analysis. Plant Cell Environ 6: 515–522
Smart CC, Trewavas AJ (1984) Abscisic acid-induced turion formation in Spirodela polyrrhiza L. III. Specific changes in protein
synthesis and translatable RNA during turion development. Plant
Cell Environ 7: 121–132
Smart CC, Fleming AJ, Chaloupkova K, Hanke DE (1995) The physiological role of abscisic acid in eliciting turion morphogenesis.
Plant Physiol 108: 623–632
Smith DH, Castle JE (1960) Production of auxotrophs in a duckweed, Spirodela polyrhiza. Plant Physiol 35: 809–816
Sonta M, Rekiel A, Batorska M (2019) Use of duckweed (Lemna L.)
in sustainable livestock production and aquaculture – a review.
Ann Anim Sci 19: 257–271
Sowinski EE, Gilbert S, Lam E, Carpita NC (2019) Linkage structure
of cell-wall polysaccharides from three duckweed species.
Carbohydr Polym 223: 115119
Sree KS, Maheshwari SC, Boka K, Khurana JP, Keresztes A,
Appenroth KJ (2015a) The duckweed Wolffia microscopica: a
unique aquatic monocot. Flora 210: 31–39
Sree KS, Sudakaran S, Appenroth KJ (2015b) How fast can angiosperm grow? Species and clonal diversity of growth rates in the genus Wolffia (Lemnaceae). Acta Physiol Plant 37: 204
Sree KS, Adelmann K, Garcia C, Lam E, Appenroth KJ (2015c)
Natural variance in salt tolerance and induction of starch accumulation in duckweeds. Planta 241: 1395–1404
Sree KS, Dahse HM, Chandran JN, Schneider B, Jahreis G,
Appenroth KJ (2019) Duckweed for human nutrition: no cytotoxic
and no anti-proliferative effects on human cell lines. Plant Foods
Hum Nutr 74: 223–224
Stiekema WJ, Wimpee CF, Tobin EM (1983) Nucleotide sequence
encoding the precursor of the small subunit of ribulose
THE PLANT CELL 2021: 33: 3207–3234
3234
| THE PLANT CELL 2021: 33: 3207–3234
Xu XJ, Sun JQ, Nie Y, Wu XL (2015) Spirodela polyrhiza stimulates
the growth of its endophytes but differentially increases their
fenpropathrin-degradation capabilities. Chemosphere 125: 33–40
Xu H, Yu C, Xia X, Li M, Li H, Wang Y, Wang S, Wang C, Ma Y,
Zhou G (2018) Comparative transcriptome analysis of duckweed
(Landoltia punctata) in response to cadmium provides insights
into molecular mechanisms underlying hyperaccumulation.
Chemosphere 190: 154–165
Xu S, Stapley J, Gablenz S, Boyer J, Appenroth KJ, Sree KS,
Gershenzon J, Widmer A, Huber M (2019) Low genetic variation
is associated with low mutation rate in the giant duckweed. Nat
Commun 10: 1243
Yamaga F, Washio K, Morikawa M (2010) Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from
the rhizosphere of duckweed Lemna aoukikusa. Environ Sci
Technol 44: 6470–6474
Yamakawa Y, Jog R, Morikawa M (2018) Effects of co-inoculation
of two different plant growth-promoting bacteria on duckweed.
Plant Growth Regul 86: 287–296
Yang L, Han Y, Wu D, Yong W, Liu M, Wang S, Liu W, Lu M, Wei
Y, Sun J (2017) Salt and cadmium stress tolerance caused by overexpression of the Glycine max Naþ/Hþ Antiporter (GmNHX1)
gene in duckweed (Lemna turionifera 5511). Aquat Toxicol 192:
127–135
Yang J, Li G, Hu S, Bishopp A, Heenatigala PPM., Kumar S, Duan
P, Yao L, Hou H (2018a) A protocol for efficient callus induction
and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens. Aquat Bot 151: 80–86
Yang GL, Fang Y, Xu YL, Tan L, Li Q, Liu Y, Lai F, Jin YL, Du AP,
He KZ, Ma XR, Zhao H (2018b) Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor. Plant Mol
Biol 98: 319–331
Yu C, Zhao X, Qi G, Bai Z, Wang Y, Wang S, Ma Y, Liu Q, Hu R,
Zhou G (2017) Integrated analysis of transcriptome and metabolites
reveals an essential role of metabolic flux in starch accumulation under nitrogen starvation in duckweed. Biotechnol Biofuels 10: 1–14
Zeller MA, Hunt R, Sharma S (2013) Sustainable bioderived polymeric materials and thermoplastic blends made from floating
aquatic macrophytes such as ‘‘duckweed’’. J Appl Polym Sci 127:
375–386
Zhang Y, An D, Li C, Zhao Z, Wang W (2020) The complete chloroplast genome of greater duckweed (Spirodela polyrhiza 7498) using
PacBio long reads: insights into the chloroplast evolution and transcription regulation. BMC Genomics 21: 76
Zhao Y, Fang Y, Jin Y, Huang J, Ma X, He K, He Z, Wang F, Zhao
H (2015) Microbial community and removal of nitrogen via the
addition of a carrier in a pilot-scale duckweed-based wastewater
treatment system. Bioresour Technol 179: 549–558
Ziegler P, Adelmann K, Zimmer S, Schmidt C, Appenroth KJ
(2015) Relative in vitro growth rates of duckweeds (Lemnaceae)—the
most rapidly growing higher plants. Plant Biol 17: 33–41
Ziegler P, Sree KS, Appenroth KJ (2016) Duckweed for water remediation and toxicity testing. Toxicol Environ Chem 98: 1127–1154
Ziegler P, Sree KS, Appenroth KJ (2018) Duckweed biomarkers for
identifying toxic water contaminants? Environ Sci Pollut Res 26:
14797–14822
Züst T, Heichinger C, Grossniklaus U, Harrington R, Kliebenstein
DJ, Turnbull LA (2012) Natural enemies drive geographic variation
in plant defenses. Science 338: 116–119
Downloaded from https://academic.oup.com/plcell/article/33/10/3207/6323365 by guest on 14 January 2023
Van Bel M, Diels T, Vancaester E, Kreft L, Botzki A, Van de Peer
Y, Coppens F, Vandepoele K (2018) P LAZA 4.0: an integrative resource for functional, evolutionary and comparative plant genomics. Nucleic acids research. 46: D1190–D1196
Van Der Heide TJ, Roijackers RM, Peeters ET, Van Nes EH (2006)
Experiments with duckweed–moth systems suggest that global
warming may reduce rather than promote herbivory. Freshw Biol
51: 110–116
Van Hoeck A, Horemans N, Monsieurs P, Cao HX, Vandenhove H,
Blust R (2015) The first draft genome of the aquatic model plant
Lemna minor opens the route for future stress physiology research
and biotechnological applications. Biotechnol Biofuels 8: 188
Vorholt JA, Vogel C, Carlström CI, Müller DB (2017). Establishing
causality: opportunities of synthetic communities for plant microbiome research. Cell Host Microbe 22: 142–155
Vunsh R, Heinig U, Malitsky S, Aharoni A, Avidov A, Lerner A,
Edelman M (2015) Manipulating duckweed through genome duplication. Plant Biol 17: 115–119
Wang WQ, Wu YR, Yan YH, Ermakova M, Kerstetter R, Messing J
(2010) DNA barcoding of the Lemnaceae, a family of aquatic
monocots. BMC Plant Biol 10: 205
Wang W, Kerstetter RA, Michael TP (2011) Evolution of genome
size in Duckweeds (Lemnaceae). J Bot 2011: 1–9
Wang W, Messing J (2011) High-throughput sequencing of three
Lemnoideae (duckweeds) chloroplast genomes from total DNA.
PLoS One 6: e24670
Wang W, Wu Y, Messing J (2012) The mitochondrial genome of an
aquatic plant, Spirodela polyrhiza. PLoS One 7: p.e46747.
Wang W, Haberer G, Gundlach H, Glaber C, Nussbaumer TC, Luo
MC, Lomsadze A, Borodovsky M, Kerstetter RA, Shanklin J,
et al. (2014) The Spirodela polyrhiza genome reveals insights into
its neotenous reduction fast growth and aquatic lifestyle. Nat
Commun 5: 3311
Wang B, Chu J, Yu T, Xu Q, Sun X, Yuan J, Xiong G, Wang G,
Wang Y, Li J (2015) Tryptophan-independent auxin biosynthesis
contributes to early embryogenesis in Arabidopsis. Proc Natl Acad
Sci U S A 112: 4821–4826
Wang P, Zhao FJ, Kopittke PM (2019) Engineering crops without
genome integration using nanotechnology. Trends Plant Sci 24:
574–577
Ward DB, Hall DW (2010) Keys to the flora of Florida–25,
Lemnaceae. Phytologia 92: 241–248
Watanabe S, Fucı́ková K, Lewis LA, Lewis PO (2016) Hiding in plain
sight: Koshicola spirodelophila gen. et sp. nov. (Chaetopeltidales,
Chlorophyceae), a novel green alga associated with the aquatic angiosperm Spirodela polyrhiza. Am J Bot 103: 865–875
White SL, Wise RR (1998) Anatomy and ultrastructure of Wolffia
columbiana and Wolffia borealis, two nonvascular aquatic angiosperms. Int J Plant Sci 159: 297–304
Xie WY, Su JQ, Zhu YG (2014) Arsenite oxidation by the phyllosphere bacterial community associated with Wolffia australiana.
Environ Sci Technol 48: 9668–9674
Xie WY, Su JQ, Zhu YG (2015) Phyllosphere bacterial community of
floating macrophytes in paddy soil environments as revealed by
Illumina high-throughput sequencing. Appl Environ Microbiol 81:
522–532
Xu JL, Cheng JJ, Stomp AM (2012) Growing Spirodela polyrrhiza in
swine wastewater for the production of animal feed and fuel ethanol: a pilot study. Clean Soil Air Water 40: 760–765
K. Acosta et al.