The Plant Journal (2008) 54, 938–948 doi: 10.1111/j.1365-313X.2008.03478.x Lindernia brevidens: a novel desiccation-tolerant vascular plant, endemic to ancient tropical rainforests Jonathan R. Phillips1,†, Eberhard Fischer2,†, Miriam Baron1, Niels van den Dries1, Fabio Facchinelli1, Michael Kutzer1, Ramtin Rahmanzadeh1, Daniela Remus1 and Dorothea Bartels1,* 1 Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany, and 2 Department of Biology, Institute for Integrated Natural Sciences, University of Koblenz-Landau, Rheinau 1, D-56075 Koblenz, Germany Received 13 December 2007; revised 18 February 2008; accepted 26 February 2008; published online 24 April 2008. * For correspondence (fax +49 228 73 1697; e-mail dbartels@uni-bonn.de). † These authors contributed equally. Summary A particular adaptation to survival under limited water availability has been realized in the desiccation-tolerant resurrection plants, which tend to grow in a habitat with seasonal rainfall and long dry periods. One of the best-studied examples is Craterostigma plantagineum. Here we report an unexpected finding: Lindernia brevidens, a close relative of C. plantagineum, exhibits desiccation tolerance, even though it is endemic to the montane rainforests of Tanzania and Kenya, where it never experiences seasonal dry periods. L. brevidens has been found exclusively in two fragments of the ancient Eastern Arc Mountains, which were protected from the devastating Pleistocene droughts by the stable Indian Ocean temperature. Analysis of the microhabitat reveals that L. brevidens is found in the same habitat as hygrophilous plant species, which further indicates that the plant never dries out completely. The objective of this investigation was to address whether C. plantagineum and L. brevidens have desiccation-related pathways in common, or whether L. brevidens has acquired novel pathways. A third, closely related, desiccation-sensitive species, Lindernia subracemosa, has been included for comparison. Mechanisms that confer cellular protection during extreme water loss are well conserved between C. plantagineum and L. brevidens, including the interconversion of 2-octulose to sucrose within the two desiccation-tolerant species. Furthermore, transcriptional control regions of desiccation-related genes belonging to the late embryogenesis abundant (LEA) protein family are also highly conserved. We propose that L. brevidens is a neoendemic species that has retained desiccation tolerance through genome stability, despite tolerance being superfluous to environmental conditions. Keywords: desiccation tolerance, rainforest endemic, sucrose metabolism, late embryogenesis abundant proteins. Abbreviations: ABA, abscisic acid; LEA, late embryogenesis abundant protein. Introduction Desiccation tolerance is often observed in seeds, spores and pollen, but it is rare in the vegetative tissues of vascular plants. Of the quarter of a million species of vascular plants, approximately 300 species have been documented as having the potential to survive desiccation in the vegetative growth phase (Porembski and Barthlott, 2000). Desiccationtolerant flowering plants are known at present in 10 families of monocotyledonous and dicotyledonous angiosperms, mostly from central and southern Africa, Australia and South America, e.g. Velloziaceae, Myrothamnaceae or 938 Linderniaceae (Fischer, 1992; Gaff, 1971; Porembski and Barthlott, 2000). Phylogenetic analyses indicate that tolerance in land plants is an ancient trait (Alpert, 2006; Oliver et al., 2000; Porembski and Barthlott, 2000). It is thought that tolerance was lost in vegetative tissues in association with the evolution of internal water transport. Acquisition of tolerance in adult plants may depend mainly on changes in gene expression, as the genes necessary for tolerance in seeds or pollen are generally already present (Bartels and Salamini, 2001; Oliver et al., 2000). ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd L. brevidens: a desiccation-tolerant plant 939 Desiccation-tolerant species are often small, low-growing plants, with short internodes and compact growth, which are found as pioneers in shallow soils or on rocky outcrops (Fischer, 1992; Fischer and Theisen, 2000). Such areas experience extreme variations in moisture availability, and consequently the ability to survive desiccation and quickly re-establish normal function is a necessity. There are few exceptions to this correlation, for example Boea hygroscopica occurs in forest understoreys (Gaff, 1981). However, it is generally assumed that ‘tolerance appears to be negatively associated with occurrence in moist habitats’ (Alpert, 2006). This hypothesis is based partly on studies of seeds. Tweddle et al. (2003) discovered that angiosperm species with desiccationsensitive seeds tend to be found in warmer and wetter habitats. The molecular basis of desiccation tolerance in dicotyledonous angiosperms has been investigated extensively using Craterostigma plantagineum Hochst. as a model system (Bartels and Salamini, 2001). The acquisition of desiccation tolerance in C. plantagineum requires the induction of a co-ordinated programme of genetic and biochemical processes during drying. The most prominent metabolic changes that take place during drying are the de novo synthesis of proteins and sugars, which are postulated to form the basis of protective mechanisms that limit damage to cellular constituents. Late embryogenesis abundant (LEA) proteins are an abundant group of proteins that play a major role in desiccation tolerance (Wise, 2003). The accumulation of non-reducing disaccharides appears to be correlated with the acquisition of desiccation tolerance, for example, 2-octulose stored in the hydrated leaves of C. plantagineum is converted to sucrose during drying, and comprises about 40% of the dry weight (Bianchi et al., 1991) in the desiccated state. Recently, Rahmanzadeh et al. (2005) demonstrated the monophyly of the lineage that includes the genera Craterostigma and Lindernia. As a consequence of this study, the ability of species within the Linderniaceae to survive desiccation was reviewed. Besides all species of Craterostigma, several Lindernia species from rock outcrops have been shown to be desiccation tolerant; however, the majority of Lindernia species such as Lindernia rotundata are desiccation sensitive (Fischer, 1992, 1995; Seine et al., 1995). Here, we report that Lindernia brevidens Skan is also a desiccation-tolerant species. Desiccation tolerance is demonstrated at the cellular level by analysing cell architecture and membrane integrity, in addition to the presence of metabolic and molecular signatures. This is a surprising discovery because L. brevidens is endemic to the montane rainforests of coastal Africa, a niche that does not experience drought. Given that our results suggest that the mechanisms that confer cellular protection during extreme water loss appear well conserved, we propose that L. brevidens is a neoendemic species that has remarkably retained desiccation tolerance through genome stability. Results Desiccation-tolerant species within the Linderniaceae The objective of these studies was to determine the physiological and molecular requirements of desiccation tolerance. Members of the Craterostigma genus are known to be desiccation tolerant (Fischer, 1992). Studies of phylogenetic relationships showed that desiccation-tolerant Craterostigma species are contained in one branch. Other species that co-localize in the same branch as Craterostigma are L. brevidens, Lindernia subracemosa and Torenia vagans (Figure 1). Rahmanzadeh et al. (2005) reported that T. vagans and L. subracemosa are not desiccation tolerant; however, to date no information was known about L. brevidens. Lindernia brevidens is desiccation tolerant The ability of L. brevidens to survive desiccation was tested, and an example of a plant specimen undergoing dehydration/rehydration is shown in Figure 2(a–c). Water was withheld from L. brevidens plants and the vegetative tissues slowly dried, typically over a period of 12–21 days, at which point the tissues were desiccated. Desiccation is defined as <0.1 g of water per 1 g of dry weight (dw), as described by Figure 1. Phylogenetic relationships between selected members of the Linderniaceae, as inferred by maximum parsimony analyses of the chloroplast maturase (matK) gene. Bootstrap percentages are indicated above the branches. The figure has been derived from studies first published by Rahmanzadeh et al., 2005; here, two more species Lindernia brevidens and Craterostigma lanceolatum were included. The desiccation-tolerant species are marked with an asterisk. ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 940 Jonathan R. Phillips et al. (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Figure 2. Lindernia brevidens is desiccation tolerant. The effect of extreme dehydration on L. brevidens (a), is shown in (b). Upon rehydration, the plants recover and resume normal physiological activity (c). Scale bar: 5 cm. Hydrated and rehydrated leaves appear structurally similar. (d–i) Scanning electron micrographs of hydrated, desiccated and rehydrated L. brevidens leaves. Surface view of the epidermis of hydrated (d), desiccated (e) and rehydrated (f) leaves. Note the presence of glands on the leaves. Transverse sections of hydrated (g), desiccated (h) and rehydrated (i) leaves. Scale bar: 40 lm. The water status during the dehydration and rehydration of L. brevidens is shown in a drying curve (j). The water content is expressed on a dry-weight (dw) basis. Plants were dehydrated for 12 days and then rehydrated for 24 h. The values are shown as mean values with standard deviation for n = 3 replications. Wood and Jenks (2007). In Figure 2(a–c), the original relative water content (RWC) of the hydrated leaves was 72% (corresponding to approximately 4 g water g)1 dw) and was reduced to approximately 1% (corresponding to approximately 0.09 g water g)1 dw) in the desiccated state. A typi- cal drying curve that illustrates the rate of water loss during drying, and the water content during rehydration, is shown in Figure 2(j). Hydrated leaves were of a uniform green colour. Leaves became folded upon dehydration; however, the green colouration was retained, which is indicative of ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 L. brevidens: a desiccation-tolerant plant 941 homiochlorophyllous (chlorophyll-retaining) desiccationtolerant plant species (Bartels and Salamini, 2001). Rehydrated leaves were largely indistinguishable from hydrated leaves, although the leaf tips were sometimes brown. The leaf and root structures do not present any xerophytic structures. Water loss resulted in the significant shrinkage of the epidermal, palisade and mesophyll cells of the leaves, which was accompanied by extensive cell-wall folding. Upon rehydration, the leaves returned to normal cellular morphology. Scanning electron microscopy of hydrated and rehydrated leaf tissues shows that desiccation-induced cellular damage was minimal. Surface views of the epidermis from hydrated, desiccated and rehydrated leaf tissues are shown in Figure 2(d–f). In contrast to the hydrated and rehydrated samples, the epidermis of the dried leaf was highly folded. Glands were apparent in the epidermis, which became trapped in the epidermal folds of the dried leaves. Transverse sections through the hydrated, desiccated and rehydrated leaves are shown in Figure 2(g–i). The scanning electron micrographs indicate that, in the case of L. brevidens, there was broadly no difference in the cellular architecture of the hydrated and rehydrated leaf specimens, which indicates that vegetative tissue had recovered intact from desiccation. As a comparator, water was also withheld from L. subracemosa plants under identical conditions (Figure 3a–c). Hydrated leaves were of a uniform green colour; however, unlike L. brevidens, the leaves became brown and necrotic upon dehydration. Upon rehydration, the leaves remained brown and did not recover. The leaf structure of L. subracemosa was broadly similar to that observed for L. brevidens, including the presence of glands (Figure 3d). Again, water loss gave rise to significant cell shrinkage, and extensive cellwall folding was also observed (Figure 3d–f). L. subracemosa leaves did not resume normal cellular morphology upon rehydration, as illustrated by the compressed structure shown in Figure 3(f) (surface)/(i) (transverse) compared with Figure 3(d) (surface) (g))1 (transverse). Irreversible desicca- (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 3. Lindernia subracemosa is desiccation sensitive. The effect of extreme dehydration on L. subracemosa (a), is shown in (b). Upon rehydration, the plants recover and resume normal physiological activity (c). Scale bar: 5 cm. Hydrated and rehydrated leaves appear structurally similar. (d–i) Scanning electron micrographs of hydrated, desiccated and rehydrated L. subracemosa leaves. Surface view of the epidermis of hydrated (d), desiccated (e) and rehydrated (f) leaves. Note the presence of glands on the leaves. Transverse sections of hydrated (g), desiccated (h) and rehydrated (i) leaves. Scale bar: 40 lm. ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 942 Jonathan R. Phillips et al. soluble sugars in photosynthetically active leaves. Upon dehydration, the 2-octulose levels decline and conversely sucrose accumulates; the reverse is observed during rehydration (Bianchi et al., 1991). To test whether a similar transition occurs in L. brevidens, soluble sugars were extracted from lyophilized leaf material. Table 1 shows that like C. plantagineum, 2-octulose was present in hydrated L. brevidens leaves, and that upon dehydration, sucrose accumulated. In contrast, 2-octulose occurs only at very low levels in the hydrated or desiccated L. subracemosa leaves, and no sucrose accumulation was observed upon dehydration. In other desiccation-sensitive plants, such as L. rotundata and Crepidorhopalon whytei, no 2-octulose was detected. Figure 4. Membrane integrity analysis. Desiccated leaves from Lindernia brevidens (s), Craterostigma plantagineum (.) and Lindernia subracemosa (•) were assayed for membrane permeability via ion-leakage assays. Values are representative of three independent experiments, and the standard deviation is shown. tion-induced cellular damage was evident by the collapsed structure of the leaf cells following rehydration (Figure 3d,g). To confirm that L. brevidens is desiccation tolerant, electrolyte leakage assays were performed (Figure 4). The membrane integrity of desiccated leaves (approximately 1% RWC 0.1 g water g)1 dw) from L. brevidens and C. plantagineum was assessed as a percentage of the maximum electrolyte leakage. Broadly, no difference in electrolyte leakage was observed, indicating that both species are desiccation tolerant. As a reference, desiccated leaves from L. subracemosa were also assayed, and a higher degree of membrane permeability was observed, which was consistent with a desiccationsensitive species. Sucrose/2-octulose transition occurs in response to desiccation In C. plantagineum, the unusual C8 sugar 2-octulose is present in large quantities, constituting up to 90% of the Desiccation-responsive LEA gene expression profiles are conserved The acquisition of desiccation tolerance in C. plantagineum involves the expression of a diverse set of genes (Bartels and Salamini, 2001). Analysis of the expressed sequence tags (ESTs) from desiccated L. brevidens leaves revealed the presence of homologues to known desiccation-related genes, such as aldehyde dehydrogenase (Kirch et al., 2001), sucrose synthase (Kleines et al., 1999), glyceraldehyde3-phosphate dehydrogenase (Velasco et al., 1994) or early light-inducible proteins (Bartels et al., 1992). Likewise, homologous LEA genes that are induced during desiccation of C. plantagineum (Bartels et al., 1990) were found to be abundantly expressed in desiccated L. brevidens leaves. This is illustrated by genes encoding Lb LEA2_6–19 (group 2) and Lb LEA14_27–45 (group 14) proteins that possessed 66% and 93% identity to Cp LEA2_6–19 and Cp LEA14_27–45 respectively (Figure S1). The expression profiles of Lb LEA2_6–19 and Lb LEA14_27–45 genes in leaves undergoing water loss and subsequent rehydration are shown in Figure 5. The expression profiles of each gene were found to be closely correlated to the water content of the leaves, with subtle differences in the temporal expression profile, and were strikingly similar to those obtained in similar experiments with the cognate clones from C. plantagineum Table 1 Octulose and sucrose contents in fully hydrated and desiccated leaves Hydrated Craterostigma plantagineum Lindernia brevidens Lindernia subracemosa Lindernia rotundata Crepidorhopalon whytei Desiccated 2-Octulose Sucrose 2-Octulose Sucrose 41.91  2.59 22.04  1.82 4.00  0.00 0.00  0.00 0.00  0.00 0.5  0.36 1.93  0.32 0.81  0.32 0.01  0.00 0.00  0.00 0.09  0.04 3.12  0.51 1.43  0.14 0.00  0.00 0.00  0.00 43.74  7.41 16.18  0.6 0.18  0.06 0.56  0.00 0.20  0.00 Soluble carbohydrates were determined in hydrated and totally dehydrated (desiccated) leaves of L. brevidens, L. subracemosa, C. plantagineum, L. rotundata and C. whytei. The values are given as mg 100 mg)1 dry mass. The numbers represent the mean values  SD of four repetitions. ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 L. brevidens: a desiccation-tolerant plant 943 (a) (b) (c) Figure 5. Expression profiling of conserved desiccation-associated genes in Lindernia brevidens and Craterostigma plantagineum. Morphology of the L. brevidens leaves at different stages of dehydration/ rehydration: dehydration, (1) 9.16 g water g)1 fry weight (dw), (2) 7.85 g water g)1 dw, (3) 2.45 g water g)1 dw, (4) 0.07 g water g)1 dw; and rehydration, (5) 3.35 g water g)1 dw (6) 9.82 g water g)1 dw. Total RNAs were extracted from all samples. RNA (10 lg) was analysed by gel-blot hybridization with 32P-labelled cDNA probes corresponding to the indicated genes. (Bartels et al., 1990). The expression profiles of LEA group 3 (3–06), LEA-like (11–24) and desiccation-related protein (13– 62) genes were also investigated, and similar desiccationresponsive profiles were observed (Figure 5). Microsynteny between the genomes of Craterostigma plantagineum and Lindernia brevidens: sequence conservation between the ABA-responsive regions of LEA gene promoters Our results indicated that protein coding regions are highly conserved between the desiccation-related genes of L. brevidens and C. plantagineum genes. We examined the level of sequence conservation that exists in the transcriptional regulatory region of two desiccation-responsive Lb LEA2_6–19 and Lb LEA14_27–45 genes (Figures 6a,b and S1). Genomic DNA fragments were isolated from L. brevidens that corresponded to the 5¢ upstream regions of the LEA group 14 and LEA group 2 genes, respectively (Figure 6a,b). In the case of the LEA group 14 promoter, a fragment of the upstream xyloglucan endotransglycosylase gene is included in the alignment to demonstrate that the gene order is identical. Sequence analysis revealed a high level of nucleotide homology between the LEA promoter region from Figure 6. The promoter regions of desiccation-related late embryogenesis abundant (LEA) genes from Lindernia brevidens and Craterostigma plantagineum are structurally similar and functionally equivalent. (a, b) Comparison between the desiccation and ABA-inducible LEA2_6–19 and LEA14_27–45 promoter regions from L. brevidens and C. plantagineum. The regions that are necessary and sufficient for dehydration/ABA responsiveness are indicated by the white line. A xyloglucan endotransglycosylase gene is located upstream of the LEA14_27–45 gene (the orange box). The conserved dehydration/ABA-related cis-elements are annotated. Blue boxes indicate ABA-responsive elements (ABREs; Guiltinan et al., 1990; Hattori et al., 2002; Mundy and Chua, 1988; Simpson et al., 2003). Red boxes indicate dehydration-responsive elements (DREs; Dubouzet et al., 2003). Green boxes indicate MYB1 and MYB2 recognition sites found in the promoter of the dehydrationresponsive gene rd22 (Abe et al., 2003). Gold boxes indicate Dc3 promoterbinding factor-1 and -2 binding core sequences, bound by bZIP transcription factors such as DPBF-1 and -2, and ABI5 (Finkelstein and Lynch, 2000; Kim et al., 1997). Pink boxes indicate the binding site for MYC (rd22BP1) in the promoter of the dehydration-responsive gene, rd22 (Abe et al., 1997). Purple boxes indicate the R18-SAP domain transcription factor binding sites (Hilbricht et al., 2002). (c) Transient expression of the Lb LEA2_6–19 (GenBank: EF429272) GUS gene in L. brevidens leaves treated with water, 100 lM ABA or 0.8 M mannitol (dehydration). Two deletion constructs ()463 and )203) are shown that contain the minimal sequences necessary to respond to ABA/dehydration. Values represent the percentage level of promoter activity compared with the CaMV35S promoter, and the standard deviation is calculated from four independent experiments. C. plantagineum and the equivalent L. brevidens regions. The 448 and 282-bp promoter regions for Cp LEA2_6–19 and Cp LEA14_27–45, which were described by Michel et al. (1993, 1994) as being necessary to mediate ABA-responsive gene expression in C. plantagineum cells, were 56% and 64% identical, respectively (Figures 6b,d and S1). Specific types of cis-acting elements are required for dehydrationresponsive, coordinated gene expression. These elements were retrieved from the Plant Cis-acting Regulatory DNA Element database (Higo et al., 1999), and were mapped to the LEA2 and LEA14 promoter regions (Figure 6a,b). Wellcharacterized promoter elements such as ABREs (ABAresponsive elements) and DREs (dehydration-responsive elements) were conserved, with respect to sequence identity and spacing, within the promoter, and the order of the cis-elements was strikingly similar. ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 944 Jonathan R. Phillips et al. The conserved structure of the LEA genes was reflected in the functional equivalence between the L. brevidens and the C. plantagineum promoter regions (Figure 6c; Michel et al., 1993, 1994). The Lb LEA2_6–19 promoter was shown to be both ABA and dehydration inducible, with the ABA-responsive region localized between )464 and )203 bp. ABAindependent promoter activity was retained in the )203-bp fragment. Similarly, the Lb LEA14_27–45 promoter was also shown to be desiccation and ABA responsive (SmithEspinoza et al., 2007). Two related desiccation-tolerant plants colonize radically different ecological niches The monophyly of the Linderniaceae lineage encompassing the genera Craterostigma, Lindernia, Artanema, Torenia and Crepidorhopalon has already been described (Rahmanzadeh et al., 2005). Floral morphology fully agrees with the Linderniaceae clade. This is typified by the corollas of (a) (b) (c) C. plantagineum and Lindernia, which are both similar in shape and colour, although they differ in size (i.e. C. plantagineum, 14-mm long; L. brevidens, 8-mm long; Figure 7a,b). All genera resolved in the Linderniaceae clade share the same type of stamens in which the abaxial filaments are geniculate, zig-zag shaped or spurred (Fischer, 1992). Mostly these geniculations have a knob- or clubshaped outgrowth, and are covered with blue to yellow glandular hairs, mimicking an anther with pollen. There are differences in the growth habit: C. plantagineum has a flattened leafy rosette structure resulting from the relatively short internode length, whereas L. brevidens is between 10 and 15-cm tall with simple or branched stems. Craterostigma plantagineum colonizes areas with restricted water availability, such as rocky outcrops at low to moderate elevations below 2000 m a.s.l. in a relatively broad range of locations from Niger to Ethiopia, Sudan, East Africa extending to South Africa, Arabia and India (Figure 7c; Fischer, 1992). In contrast, L. brevidens is only reported from the Taita Hills and the Western Usambara Mountains (Fischer, 1992; Hemsley and Skan, 1906) (Figure 7c; Appendix S1). The species occurs in open places within the forest or along footpaths in the montane rainforest, where precipitation is usually above 1500 mm per year. It has also been observed near the edge of a small pond. Accompanying species are hygrophilous Acanthaceae, e.g. Hypoestes spp., ferns (Doryopteris concolor) and forest grasses (Oplismenus hirtellus). Its closest relative is the desiccation-sensitive (Figure 3) perennial L. subracemosa De Wild., an afromontane taxon known from similar habitats in montane forests from Ethiopia to Eastern Congo and western Tanzania. Discussion Figure 7. Morphology and distribution of Lindernia brevidens and Craterostigma plantagineum. (a) C. plantagineum and (b) L. brevidens share similar floral morphology. (c) L. brevidens (yellow dots) is restricted to the rainforests of the Usambara Mts and the Taita hills, whereas C. plantagineum (red dots) has colonized areas with limited seasonal water availability from Niger to Sudan, Ethiopia and East Africa, extending to Angola, Namibia and South Africa. Lindernia brevidens is a novel example of a desiccationtolerant angiosperm associated with a tropical rainforest. The area of tropical rainforest where L. brevidens is found is highly fragmented, with discrete areas of localized rainfall surrounded by comparatively arid woodland. Climatic changes throughout the Pleistocene period are thought to have caused major extinctions by substantially reducing the area of African rainforest. In contrast, the forest patches of the Eastern Arc Mountains have existed for approximately 30 million years because of the stability of the Indian Ocean currents that bring moisture to the tropical East African coast (Lovett and Wasser, 1993). The age and fragmented nature of the Eastern Arc forests have combined to produce high levels of endemism and diversity. It is estimated that 25–30% of the 2000 vascular plant species are endemic to the area (Lovett and Wasser, 1993). A refugial model fits with the unusually high level of biodiversity in the montane rainforests of eastern Africa. Endemic species may have speciated allopatrically, given that they must have been genetically isolated from the ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 L. brevidens: a desiccation-tolerant plant 945 neighbouring species. Pockets of forest have survived in the wettest locations, which form a refuge for all the forest flora and fauna. Cut off from the populations of other forest organisms in other refugia, the fragmented populations underwent speciation. Eastern Arc endemics can be divided into paleoendemics and neoendemics. Paleoendemics are taxa that have survived in a limited portion of their past territory, and rarely have close relations in adjacent regions. Neoendemics are taxa in rapidly evolving species complexes, which often have their most closely related taxa in the same or nearby regions (Rodgers and Homewood, 1982). Based on the refugial model, it is likely that an ancestor of L. brevidens was desiccation tolerant. Both C. plantagineum and L. brevidens are probably highly derived within the Linderniaceae. L. brevidens evolved in rainforest areas; however, the species did not lose the ability to adapt to extreme dehydration. Molecular analysis of L. brevidens suggests that the structure and expression profiles of desiccation-related genes from C. plantagineum are well conserved. The highest homology was found in the exonic regions; however, a relatively high conservation score (56– 64%) was also observed in functionally important promoter regions, with respect to ABA/dehydration responsiveness (see Figure S1). Comparison between promoter regions of different dehydration-responsive C. plantagineum genes did not reveal significant homology, nor could a common set of cis-acting elements be determined specifically for resurrection plants (Bartels et al., 2006). Only limited experimental data relating to functional promoter comparisons between different plant species are available; however, studies with animal systems have shown that a similar degree of promoter conservation is obtained when comparing some human–rodent promoter pairs (Xuan et al., 2005). The accumulation of sucrose from octulose during drying was another tolerance diagnostic indicator shared by both species. Octulose is rarely found in plants, and appears to act as a temporary storage carbohydrate in leaves during photosynthesis (Bianchi et al., 1991; Norwood et al., 2000). In tandem with LEA proteins, sucrose probably stabilizes drying cells both by direct interaction with macromolecules and membranes, and by reversibly immobilizing cytoplasm through glass formation (Buitink and Leprince, 2004; Wolkers et al., 2001). Given that the mechanisms that confer cellular protection during extreme water loss appear well conserved, we propose that L. brevidens is a neoendemic species that has retained desiccation tolerance through genome stability. The question is why L. brevidens did not lose the ability to adapt to extreme dehydration. A possible explanation for the absence of desiccation-tolerant species in moist habitats is competitive exclusion by desiccation-sensitive species, such that selection against tolerance occurs when water availability is high (Alpert, 2006). Desiccation sensitivity in species from moist habitats could therefore result from a lack of selection to maintain tolerance or from selection against it, if there is a trade-off between tolerance and another trait, such as growth or reproduction. Desiccation tolerance is considered to be a primitive trait that is negatively correlated with rapid growth (Alpert, 2006; Oliver et al., 2005); however, we assume that no selection pressure for rapid growth occurred in the ecological niche inhabited by L. brevidens. Alternatively, desiccation tolerance may be linked to another, as yet unknown trait that is important for survival in the rainforest. In this way, the ability to survive desiccation remains, but the mechanisms that protect the vegetative tissues are never induced in nature. Experimental procedures Plant material Lindernia brevidens, L. subracemosa and L. rotundata (Pilger) Eb. Fisch. plants were collected by one of the authors (EF) from the Taita Hills, Kenya (L. brevidens and L. subracemosa) and Rwanda (L. rotundata). Voucher specimens were deposited at Koblenz University (KOBL). A list of all collections of L. brevidens is provided (see Appendix S1). C. plantagineum plants were collected and grown as described by Bartels et al. (1990). L. brevidens seeds were germinated directly in potting compost and maintained in a climate chamber at day/night temperatures of 22 and 18C, respectively. Plants were grown under a 16-h day/8-h night regime, with a light intensity of 80 lE m)2 sec)1. Light intensities greater than 80 lE m)2 sec)1 resulted in stress, as indicated by the appearance of the red pigmentation caused by anthocyanin accumulation. Plants were gradually dried in pots over a period of 12–21 days, and were re-watered by submerging the leaves under water. Relative water content measurements were made according to the method described by Bernacchia et al. (1996). Water content was also measured on a dw basis using the formula WC (g water g)1 dw) = (fresh weight – dry weight)/dry weight. Phylogenetic analysis Phylogenetic relationships were calculated as described by Rahmanzadeh (2002) and Rahmanzadeh et al. (2005). Here, the part of the analysis is shown that is relevant for the work reported. The analysis includes the designation of the desiccation-tolerant and desiccation-sensitive species. The construction of the tree is based on PCR amplification of the trnK intron, which was amplified in two overlapping fragments using primers and sequence analysis according to Müller et al. (2004). Parsimony analyses were performed with PAUP version 4.0b10 (Swofford, 1998). Bootstrapping was used to address confidence in the analysis. Swapping of up to 100 trees was performed, and resulted in 95% confidence intervals of << 2% for bootstrap proportions of ‡ 90% provided sufficient accuracy for the conclusions. Nodes with less than 50% confidence were regarded as unsupported. Scanning electron microscopy Hydrated and rehydrated samples were fixed in 2% (v/v) glutaraldehyde in phosphate-buffered saline (PBS; pH 7.2), and were dehydrated through a graded ethanol series (from 10% to 100%) diluted in PBS prior to critical-point drying with a critical-point device (CPD 020; ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 938–948 946 Jonathan R. Phillips et al. Balzers, http://www.oerlikon.com/balzers) according to the method of Svitkina et al. (1984). Desiccated leaf samples were observed directly. All leaf specimens were gold coated with a sputter-coater (SCD 040, Balzers) and viewed using an LEO 1450 scanning electron microscope (Carl Zeiss Jena, http://www.zeiss.com). Electrolyte leakage Ion leakage of desiccated leaf samples was measured as the change in conductivity during a 60-min period. Maximum electrolyte leakage, corresponding to 100%, was measured after heating the leaves at 80C for 30 min. Nucleic acid analysis The procedures used for RNA extraction and gel-blot analysis were as described by Bartels et al. (1990). A 10-lg sample of total RNA was separated on a 1.2% denaturing agarose gel. Equal loading of RNA blots was confirmed using the rDNA probe, pTA71 (data not shown) (Gerlach and Bedbrook, 1979). cDNA clones were obtained from a Uni-ZAP XR library (Stratagene, http://www.stratagene. com) prepared from desiccated (RWC < 5%) L. brevidens leaves. DNA analysis (plasmid extraction, sequencing) was performed according to standard techniques (Sambrook et al., 1989). 32 P-labelled cDNA insert probes were obtained by random priming using the Fermentas (http://www.fermentas.com) labelling kit. Hybridization probes were generated from Lb LEA2_6–19, Lb LEA14_27–45, Lb LEA3_3–06, Lb LEA-like_11–24, Lb desiccation related protein_13–62 cDNA clones (accession numbers EF429273, EF364563, EU478590, EU478591 and EU478592, respectively). The gene nomenclature includes information regarding the species origin (i.e. L. brevidens) followed by the gene function and the homologous gene sequence from C. plantagineum. Promoter analysis Genomic fragments were obtained using a GenomeWalker kit (Clontech, http://www.clontech.com). Oligonucleotide primers are available upon request. The Lb LEA2_6–19 and Lb LEA14_27–45 promoter sequences are deposited in GenBank (EF429272 and EF364562, respectively). Biolistic transient transformation was performed according to Ditzer and Bartels (2006). Lb LEA2_6–19 promoter GUS constructs were co-bombarded with a CaMV35S promoter GFP construct (pGJ280, kindly provided by Dr G. Jach, Max-Planck-Institute, http://www.mpg.de). The relative expression levels were calculated using the method of Schenk et al. (1998). Gold particles (1.6 lm in diameter) were used as microcarriers for the bombardment, and were prepared as follows: 60 mg of gold particles was vortexed twice for 2 min in 1 ml of 100% (v/v) ethanol. The suspension was centrifuged for 1 min at 10 000 g, and the gold pellet was washed in 1 ml ddH2O and resuspended in 1 ml of 50% (v/v) glycerol. Aliquots (50 ll) were used for the following coating procedure: 5 lg Lb LEA2_6–19 promoter GUS reporter plasmid-DNA, 5 lg GFP reporter plasmidDNA (pGJ280), 50 ll of 2.5 M CaCl2 and 20 ll of 100 mM freshly prepared spermidine (Sigma, Germany) were added to an aliquot of the gold suspension while vortexing for 3 min at maximum speed. The suspension was briefly centrifuged and the gold was washed with 250 ll of 100% (v/v) ethanol, sedimented again and finally mixed with 80 ll of 100% (v/v) ethanol. For each bombardment, a 20-ll aliquot of the gold suspension was spread over a macrocarrier. The bombardment procedure followed the PDS-1000/He manufac- turer’s instructions (BioRad, http://www.bio-rad.com). The upper surface of the L. brevidens leaves were bombarded with a pressure of 9.3 kPa and a distance of 6 cm with a subpressure of 91 kPa. After the bombardment, the leaves were incubated in either 0.8 M mannitol, 100 lM ABA or water for 48 h at 22C. After 48 h, the number of cells that express GFP was determined using a confocal laser scanning microscope (e-C1 confocal microscope system; Nikon, http://www.nikon.com). The leaves were subsequently stained to determine the number of cells that express b-glucuronidase [for 16 h at 37C in 3 mM 5-bromo-4-chloro-3-indolyl-b-Dglucuronic acid, 50 mM NaH2PO4, pH 7.0, 0.1% (v/v) Triton X-100 and 8 mM b-mercaptoethanol]. The GUS staining solution was vacuum infiltrated into bombarded leaves. Plant pigments were extracted with several changes of ethanol at 80C and were stored in 10% (v/v) glycerol. The GUS spots/number of GFP-producing cells after co-bombardment of leaves was then determined. The pGJ280 vector was used as an internal standard. The experiments were replicated four times, and the values for each Lb LEA2_6–19 promoter GUS reporter plasmid assayed were normalized against values for the internal standard. The values (%) are each given as a mean activity with a standard deviation. Carbohydrate analysis Carbohydrate extraction and quantification was performed according to the method described by Bianchi et al. (1991). Freeze-dried leaf material was ground to a fine powder and extracted twice with 80% (v/v) methanol, followed by chloroform. The solvents were evaporated, followed by three extractions against ethyl acetate. The solution was again lyophilized and dissolved in 200 ll of water, and applied to a Dionex HPLC system (http://www.dionex.com) using a CarboPacPA 100 column. Fractions were quantified by GC and GC mass spectrometry. Fractions for GC were dried at 60C under N2. Pyridine (20 ll) and TMS (trimethylsilyl-trifluoroacetamide; 20 ll) were added, and the sample was diluted with chloroform to reach a mass of between 1 and 20 ng. Xylitol (5 ng) was used as an internal standard. The sugar derivatives were separated on a DB1 column (J&W Scientific, now Agilent Technologies, http://www.agilent.com). Qualitative GC/MS analysis was performed with a gas chromatograph 6890 N, detector 5973 MSDetection HP (Agilent Technologies); quantitative analysis was performed with GC/flame ionization detector (5890 Series II Plus, HP (Agilent Technologies). Acknowledgements We thank Dr B. Buchen and H.-J. Ensikat for help with microscopy, C. Buchholz and S. Smolny for technical support, S. Tückmantel for reprobing RNA filters, T. Borsch and K. Müller for help with phylogenetic studies and Dr A. Richter (Vienna, Austria) for initial sugar measurements. 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