The Society for Integrative and Comparative Biology
The Signature of Seeds in Resurrection Plants: A Molecular and Physiological Comparison of Desiccation Tolerance in Seeds and Vegetative Tissues1
1 Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa
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Desiccation-tolerance in vegetative tissues of angiosperms has a polyphyletic origin and could be due to 1) appropriation of the seed-specific program of gene expression that protects orthodox seeds against desiccation, and/or 2) a sustainable version of the abiotic stress response. We tested these hypotheses by comparing molecular and physiological data from the development of orthodox seeds, the response of desiccation-sensitive plants to abiotic stress, and the response of desiccation-tolerant plants to extreme water loss. Analysis of publicly-available gene expression data of 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitive Arabidopsis thaliana identified 13 LEAs and 4 anti-oxidants exclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin) are not expressed in vegetative tissues in A. thaliana, but have orthologues that are specifically activated in desiccating leaves of Xerophyta humilis. A comparison of antioxidant enzyme activity in two desiccation-sensitive species of Eragrostis with the desiccation-tolerant E. nindensis showed equivalent responses upon initial dehydration, but activity was retained at low water content in E. nindensis only. We propose that these antioxidants are housekeeping enzymes and that they are protected from damage in the desiccation-tolerant species. Sucrose is considered an important protectant against desiccation in orthodox seeds, and we show that sucrose accumulates in drying leaves of E. nindensis, but not in the desiccation-sensitive Eragrostis species. The activation of "seed-specific" desiccation protection mechanisms (sucrose accumulation and expression of LEA6 and 1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerant plants points towards acquisition of desiccation tolerance from seeds.
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INTRODUCTION |
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Desiccation tolerance (DT) is the ability of an organism to survive the loss of most (>95%) of its cellular water. It is considered to be a complex trait that is present in reproductive structures (pollen and seeds) of vascular plants. Desiccation tolerance in vegetative tissues is also relatively common in less complex plants such as bryophytes (Proctor, 1990
) and lichens (Kappen and Valadares, 1999) but rare in pteridophytes and angiosperms (Gaff, 1977, 1987; Alpert and Oliver, 2002; Porembski and Barthlott, 2000) and absent in gymnosperms (Gaff, 1980). Oliver et al. (2000) have speculated on the evolution of DT in vegetative tissue and argue that DT is the ancestral state for early land plants (e.g., mosses), which was lost early in the evolution of tracheophytes. The subsequent successful radiation of vascular plants on land was probably a consequence of the evolution of DT in seeds, in parallel to the evolution of structural and morphological modifications in vegetative tissue which allowed greater control of water status. Oliver et al. (2000) speculate that the emergence of DT in seeds was a modification of vegetative DT in early ancestors. They suggest furthermore that vegetative DT in angiosperms subsequently re-evolved independently at least eight times as an adaptation of seed DT.
DT in seeds and vegetative tissues of angiosperms is different from that in extant lower orders. In the former, it is based on induction of several relatively complex protection mechanisms during drying, with minimal reliance on repair of desiccation-induced damage during rehydration (Gaff, 1989; Vertucci and Farrant, 1995; Oliver, 1996; Oliver et al., 2000). In the lower order plants, DT is constitutive, less reliant on complex cellular protection and more on repair during rehydration (Bewley and Oliver, 1992; Oliver and Bewley, 1997; Oliver et al., 2000). Thus, although DT in seeds appears to include some mechanisms present in DT vegetative tissues of less-complex plants (reviewed in Oliver, 1996; Dickie and Pritchard, 2002), additional, more sophisticated mechanisms of protection must have evolved. We examine the possibility that DT in the vegetative tissues of angiosperms is a subsequent adaptation of these seed developmentally-regulated mechanisms.
Whereas DT is developmentally regulated in seeds with the putative mechanisms of tolerance being accumulated only at precise times after fertilisation, drought is stochastic and DT vegetative tissues must respond to environmental signals to activate protective mechanisms for the whole plant. Conceivably, environmental signals for extreme water loss activate an existing repertoire of protective seed-specific genes in vegetative tissue.
An alternative hypothesis is that DT evolved from the response of desiccation-sensitive plants to abiotic stresses such as cold, salt and drought. Desiccation-sensitive plants use an interconnected signaling network to activate a common repertoire of responses to abiotic stress (Knight and Knight, 2001). These responses appear to overlap with those described for DT plants during extreme water loss as they include the accumulation of compatible osmolytes, and the up-regulation of anti-oxidants and anti-oxidant enzymes (Knight and Knight, 2001). Under this scenario, we would predict that there should be significant overlap between genes that are induced in response to abiotic stresses and DT.
The stresses associated with extreme water loss include the mechanical stress associated with turgor loss, oxidative stress from free radical-mediated processes and the destabilization or loss of macromolecular integrity (reviewed by Vertucci and Farrant, 1995; Oliver and Bewley, 1997; Walters et al., 2002). Protection mechanisms associated with prevention of damage from these stresses have been extensively reviewed and the maintenance of subcellular integrity has been widely attributed to the accumulation of stress-associated proteins, non-reducing sugars and free radical scavenging systems (for example Farrant, 2000; Scott, 2000; Oliver et al., 2000; Vicre et al., 2004).
Late Embryo Abundant (LEA) proteins accumulate during the onset of DT in orthodox seeds and have been shown to occur in response to the drying of DT vegetative tissues (Bray, 1997; Ingram and Bartels, 1996). Expression of LEAs has also been reported to be up-regulated during abiotic stress such as cold, drought and osmotic stress (Wise and Tunnacliffe, 2004). Several LEA mRNA transcripts and proteins have been identified as being up-regulated in leaves of the resurrection plants Craterostigma plantagineum (4) and Xerophyta viscosa (2) during a cycle of desiccation (Piatkowski et al., 1990; Mundree and Farrant, 2000; Ndima et al., 2001). The largest set of LEAs (16) from a single resurrection plant was recently identified in a mini-microarray screen of 400 cDNAs from Xerophyta humilis (Collett et al., 2004). LEAs are low complexity proteins which have been classified into several unrelated groups on the basis of conservation of peptide motifs (or Pfam domains) (Close, 1997; Wise, 2003). It has not been possible to assign structures to LEAs, or experimentally determine their exact cellular role because they are unfolded in the hydrated state. Based on evidence of their abundant expression, and rich hydrophilic amino acid content, it has been proposed that LEAs maintain subcellular integrity by protecting cellular structures from the effects of water loss by either acting as a hydration buffer, by sequestering ions, by direct protection of other proteins or membranes, or by renaturing unfolded proteins (Bray, 1991). Recently, it was shown that two LEAs can prevent protein aggregation during water stress (Goyal et al., 2005) and the ability of plant LEAs to confer increased tolerance to water deficit stress on yeast and other plants (for example, Swire-Clark and Marcotte [1999], Xu et al. [1996] and Sivamani et al. [2000]) also suggests LEAs play an important role in protecting tissues from the effects of water loss.
Sucrose is the only sugar commonly accumulated in DT tissues of seeds and resurrection plants (Scott, 2000; Ghasempour et al., 1998). Like LEAs, it is commonly acknowledged to play an important and varied role in DT, although the exact nature of this in vivo has yet to be demonstrated. The general protective roles ascribed to sucrose are as water replacement molecules and a facilitator, together with proteins, of glass formation (Leopold et al., 1994; Vertucci and Farrant, 1995; Crowe et al., 1998). It has also been suggested that the formation of sucrose reduces the monosaccharide pool, which in turn reduces the chances of damaging Maillard-type reactions occurring and puts a stasis on respiratory metabolism, both of which reduce free radical formation (Vertucci and Farrant, 1995).
Free radical scavenging systems are ubiquitous in plants and include well known antioxidants such as ascorbate, glutathione and tocopherol, and enzymes such as the peroxidases (ascorbate peroxidase [AP], glutathione peroxidase, thioredoxin peroxidase, catalase), glutathione reductase (GR) and superoxide dismutase (SOD) inter alia. Other anti-oxidant enzymes such as 1-cys peroxiredoxin have been identified in the seeds of desiccation-sensitive angiosperms (Aalen, 1999) and recently in the vegetative tissues of the resurrection plant X. viscosa (Mowla et al., 2002).
We have focused on the above-mentioned mechanisms of DT to test the hypothesis that DT in vegetative tissue in resurrection plants is an adaptation of DT in seeds. We have compared some of the putative mechanisms of tolerance in the seeds of desiccation-sensitive angiosperms with those found to occur during desiccation of resurrection plants (viz. the accumulation of LEA proteins, anti-oxidants and sucrose) using a combination of molecular and physiological studies. In this study we aim to distinguish between seed-specific and abiotic stress-specific LEA and anti-oxidant enzyme gene expression in Arabidopsis with a view to showing that some of the "seed-specific" genes are expressed in the vegetative tissue of resurrection plants such as X. humilis during desiccation. We also compare antioxidant activity and sucrose accumulation in the seeds and vegetative tissue of miscellaneous desiccation-sensitive and desiccation-tolerant plants to test our hypotheses.
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MATERIALS AND METHODS |
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Plant material
Resurrection plants were collected and maintained in a glasshouse at the University of Cape Town as previously reported (Sherwin and Farrant, 1996
; Dace et al., 1998; Vander Willigen et al., 2001). Plants of Eragrostis curvula and E. teff were grown from seeds sown in seedling flats (1:1 potting soil:river sand mix) and maintained in the glasshouse for three months. Prior to experimentation, fully hydrated plants were transferred to a controlled environment chamber where they were acclimated to conditions of 50–65% relative humidity, 14 hr light (500 µmol photons·m–2·s–1; 25°C): 10 hr dark (18°C). After two weeks plants were used for dehydration/rehydration experiments. Plants were naturally dried by withholding water until the plants had reached an air-dry state (<5% relative water content [RWC]). Leaves were sampled at regular intervals during dehydration for procedures described below. They were held in the dry state for a minimum of two weeks prior to rehydration, which was by soil watering. Seeds of E. nindensis were obtained from the Agricultural Research Council (Pretoria, South Africa) and E. curvula seeds were purchased from Silverhill Seeds (Cape Town, South Africa). E. teff seeds were a kind donation from Mr Z. Ginbot.
Molecular studies and bio-informatics
Sequencing and classification of X. humilis LEAs
The 5' ends of 16 cDNAs described as LEAs, dehydrins, putative LEAs or LEA-like proteins on the basis of blastx analysis (Collett et al., 2004) were sequenced on a MegaBACE 500 sequencer (Amersham Biosciences, Little Chalfont, UK) using the DYEnamic ET Dye terminator Cycle sequencing Kit for MegaBACE (Applied Biosystems). Sequences were analysed by InterProScan (EBI) to identity conserved Pfam domains and to assign the LEAS, putative LEAs, LEA-like proteins, and dehydrins to InterPro (IPR) Superfamilies (Zdobnov and Apweiler, 2001).
Classification of LEAs from other resurrection plants
Sequences of LEAs that have been reported from other resurrection plants were obtained from GenBank, analysed by Interproscan, and assigned to IPR Superfamilies.
Northern blot analysis
The relative abundance of LEA mRNA transcripts during a cycle of desiccation and rehydration was examined by northern blot analysis of total RNA samples extracted from X. humilis leaves at early and late stages of desiccation and rehydration, as described by Collett et al. (2004).
Microarray analysis
The AGI gene locus number for all 49 Arabidopsis LEA and LEA-like proteins grouped under the different LEA superfamilies (IPRs) were downloaded from the Interproscan website (Table 1). These superfamilies included the LEA groups defined by Wise (2003) and IPR00839, since several of the genes in this Superfamily are listed as encoding LEA-like proteins (e.g., AAD10377
[GenBank]
hydrophobic LEA-like protein, Oryza sativa).
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Scaled microarray expression data during seed development and abiotic stress treatments corresponding to the Arabidopsis locus IDs listed in Table 1, and for 71 anti-oxidants listed by Mittler et al. (2004)
was downloaded from the Genevestigator database (www.genevestigator.ethz.ch) (Zimmerman et al., 2004) (Experiments 90, 120, 121, 122, 124 and 124). These experiments are part of AtGenExpress, a multinational effort to profile the transcriptome of Arabidopsis (http://web.uni-frankfurt.de/fb15/botanik/mcb/AFGN/atgenex.htm). 35 out of the 49 Arabidopsis LEAs were present on the Affymetrix 25K chip used in these experiments. We checked that the distribution of signal intensities for each of the full datasets was similar to validate our cross-comparison between the seed development and abiotic stress series. Average signal values were calculated as the mean from the repeats for each data point in the silique/seed and the abiotic stress series. A general signal value for each gene in control plants was determined by taking the mean of the control data points for green shoot and root respectively from the abiotic stress series. For each gene, each average signal intensity in the silique and seed development series and in the abiotic stress experiments, as well as the general control values were expressed as a percentage of the maximum value across all the experiments. Genes were manually grouped into the following clusters based on their profiles of expression; A) Seed specific genes: maximum expression in seed, less than 30% of the maximum expression in any of the abiotic stress treatments, B) maximum expression in seed and more than 30% of the maximum expression in any of the abiotic stress treatments, C) maximum expression in one of the abiotic stress treatments as well as more than 30% of the maximum expression during seed development, D) Stress specific genes: maximum expression in one of the abiotic stress treatments and less than 30% of the maximum expression during seed development and E) Housekeeping genes: expression in the control plants is more than 40% of the maximum expression value of either the seed development or abiotic stress treatment series. The average expression in controls was less than 30% of the maximum expression value in groups A–D.
Relative water content (RWC) determination
The RWC was calculated as the water content divided by the water content estimated at full turgor. The means of the water content of leaves at full turgor were recorded for each species using more than 20 representative leaf samples from plants that had been fully hydrated overnight in plastic bags. Water contents were gravimetrically determined by oven drying at 70°C for 48 hr.
Antioxidant enzyme activity
Leaf tissues and seeds were extracted and analysed for AP, GR and SOD analysis as described previously (Farrant et al., 2003, 2004). Three 250 mg replicates of leaf tissue were ground in liquid nitrogen and extracted in 3 ml of cold buffer (0.1 M phosphate buffer pH 7.8; 2 mM dithiothreitol [DTT]; 0.1 mM EDTA; 1.25 mM PEG 4000; 0.1 g insoluble polyvinylpyrrolidone [PVP]). The extract was centrifuged at 16,000 g for 15 min and the supernatant was desalted on a Sephadex G-25 PD10 column (Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 7.8). Samples were eluted with phosphate buffer (0.1 M, pH 7.8) and solutions collected for analysis of enzyme activities. AP was measured as described in Wang, et al. (1991), GR by the method of Esterbauer and Grill (1978) and SOD was measured using the method of Giannopolitis and Ries (1971) and modified by Bailly et al. (1996).
Sucrose determination
Three 100 mg replicates of leaf tissue and seeds were ground in liquid nitrogen and samples extracted in cold 100 mM NaOH (50% v/v ethanol/water). Chloroform (15% v/v) was added and the samples incubated on ice for 10 min. Thereafter the pH was adjusted to 7.5 with 100 mM HEPES in 100 mM glacial acetic acid. After centrifugation for 20 min at 4°C at 28,000 g, the supernatant was removed. A repeat extraction was performed on the pellets. The supernatants were pooled and then centrifuged. Sucrose in the supernatant was calculated from the spectrophotometric measurement of NADPH production using a D-glucose/D-fructose sugar assay kit (Boehringer Mannheim, Germany).
Measurement of photosynthesis
Light-saturated net photosynthesis (A) of leaves of E. curvula and E. nindensis during desiccation was measured using a Ciras-1 infrared gas analyzer with a Parkinson's Leaf Cuvette and in built illumination unit (PP Systems, Hertfordshire, UK) operated in differential mode at an ambient CO2 concentration of 350 ppm and 22°C (50% RH). The parameter A was calculated according to the equations of von Caemmerer and Farquhar (1981) and the data were expressed as a percentage of A measured in control hydrated tissues. Measurements were taken on 5 individual plants and repeated during at least two cycles of drying.
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RESULTS |
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Clustering expression profiles of Arabidopsis LEA mRNAs during seed development and in response to abiotic stress
LEAs were identified as being up-regulated in leaves in the resurrection plant X. humilis following desiccation (Collett et al., 2004
). We posed the question: Are any of the LEA groups restricted to a profile of seed-specific or abiotic-stress specific gene expression in a desiccation-sensitive plant? To this end, we first analysed the expression patterns of LEAs from a desiccation-sensitive plant during seed development and abiotic stress by mining publicly-available Arabidopsis genome and microarray data. All the AGI gene locus IDs that fall into 10 different LEA superfamilies, defined on InterProScan, were identified (Table 1). Out of 49 Arabidopsis LEAs, 35 had specific probes on the 25K Affymetrix gene chip and the expression of these 35 was compared between silique and seed development and during abiotic stress treatments (cold, osmotic, salt and drought stress).
We aimed to distinguish between seed-specific and stress-specific LEAs on the basis of expression profiles in the selected AtGenExpress dataset. LEA expression profiles grouped into different clusters illustrated in Figure 1 and summarized in Table 1. According to this analysis, we were able to identify LEA groups that fell only into Cluster A (i.e., seed specific), but no LEA groups that were restricted to Cluster D (i.e., abiotic stress specific). The Group 1 LEAs (IPR000389) and a Group 9 LEA (PD68804) were identified as being seed-specific (i.e., cluster A) as there was very low expression (<10% of seed expression) in roots or leaves during abiotic stress treatments. Although only 3 out of the 6 Group 6 LEAs (Table 1) were represented on the Affymetrix 25K genechip, these were also only expressed during silique development and for the latter stages of seed maturation. The maximum absolute level of expression of the silique-specific At5g27980 was very low in comparison to the other expression profiles reported in this study (<0.5% of most abundant LEA, i.e., At5g664400/LEA2, cluster B). Group 7 LEAs (IPR004926), were expressed under stress conditions (cluster C and D) or as housekeeping genes in roots (cluster E). The one LEA from Group 8 (IPR004864) that was represented in these experiments was expressed at high levels in both maturing seed, and in response to abiotic stress (Table 1, Fig. 1). Some members within LEA groups 2, 3, 4, 10 were seed-specific, whilst others were activated in response to abiotic stress treatment and seed maturation, or were housekeeping genes.
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What is remarkable is the diversity of LEAs expressed during seed maturation; 74% of the LEAs are expressed during seed development with 19/35 LEAs being expressed at their maximum in seed development and 7/35 LEAs being expressed at >30% maximum levels in seeds. In contrast, 46% of the LEAs were expressed in response to abiotic stress, with 10/ 35 being expressed at maximum level in either leaves or roots and 6/35 LEAs being expressed at >30% level. Expression of homologues of Arabidopsis LEAs from Groups 1, 6 and 9 in desiccating vegetative tissue of resurrection plants, could be considered an indicator that vegetative DT is due to appropriation of seed DT. This could arise from a change in regulation of genes that in desiccation-sensitive plants, such as Arabidopsis, are only expressed during the desiccation phase of seed maturation.
Sequence analysis of sixteen X. humilis LEAs
To see whether any of the sixteen desiccation up-regulated LEA ESTs identified in the leaves of X. humilis (Collett et al., 2004) belonged to the unique seed-specific LEAs we identified in Arabidopsis (i.e., groups 1, 6 or 9), we sequenced the 5' end of each X. humilis cDNA. The closest homologue for each X. humilis putative LEA was identified by a BlastX search and the nucleotide sequences for each LEA were analysed by InterProScan, and grouped into LEA superfamilies on the basis of their IPR Superfamily classification (Table 2) (Zdobnov and Apweiler, 2001). When InterProScan failed to identify PFAM domains in the X. humilis sequence information, LEAs were grouped on the basis of the InterProScan of their closest homologues identified by BlastX analysis (Table 2).
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With the exception of the Group 1 LEAs (IPR000389) and Group 9 LEAs (PD688044), genes belonging to all the other LEA groups were identified in the 16 LEAs isolated from 400 sequenced X. humilis cDNAs (Table 2). In Arabidopsis, 10 genes are classified in the LEA-2 (IPR000167) and 14 genes are classified in the LEA-3 (IPR004238) superfamilies (Table 1). These two superfamilies were also well-represented in the X. humilis cDNAs (Table 2). We also analysed LEAs reported as up-regulated during desiccation in other resurrection plants, namely Craterostigma plantigineum, X. viscosa and the moss, Tortula ruralis These LEAs can be classified into groups 2, 3, 8 and 10 (Table 3). Again, Groups 2 and 3 are predominant. Only a small number of Arabidopsis genes are classified in the Group 1 and Group 9 superfamilies, and the lack of representation of genes in these groups from resurrection plants could be due to the fact that the EST datasets analysed to date do not constitute the full LEA complement.
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The LEA6 cDNA (HC248) was the only LEA in the X. humilis geneset that clearly corresponded to a LEA that was expressed only in maturing seed in Arabidopsis. No conclusions could be drawn on the roles that the other X. humilis LEAs play in the evolution of desiccation tolerance in vegetative tissues, since they belong to superfamily groups which include both seed-specific members as well as members which are expressed in both seed and abiotic stress in Arabidopsis (Fig. 1).
Northern blot analysis of X. humilis LEAs
Many of the Group 2 and Group 3 LEAs are up-regulated in response to abiotic stress treatments in Arabidopsis (Fig. 1). We tested the hypothesis that X. humilis Group 2 and Group 3 LEAs corresponding to the stress LEAs from desiccation-sensitive plants, might be up-regulated during the early stages of dehydration (i.e., >65% RWC), and that a second class of LEAs, which correspond to those expressed during the desiccation phase of seed development, are expressed during the later stages of desiccation (<65% RWC). Northern blot analysis of 13 of the 16 LEAs (Fig. 2) clearly showed that the X. humilis LEAs investigated all have similar profiles of expression. They are all significantly up-regulated only in the later stages of desiccation (i.e., <65% RWC) in leaves, and most LEA mRNA transcripts are stably stored in dry leaves (<6% RWC).
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Clustering Arabidopsis anti-oxidant mRNA expression profiles during seed development and in response to abiotic stress
We extended our analysis of the selected Arabidopsis AtGenExpress microarray dataset, posing the question: can we identify any anti-oxidant genes that are expressed exclusively during the desiccation phase of seed maturation in this desiccation-sensitive plant, and are up-regulated during desiccation in the vegetative tissue of desiccation tolerant plants? Microarray data from the seed development and abiotic stress series corresponding to AGI gene locus IDs for 68/71 different anti-oxidants listed by Mittler et al. (2004)
which were present on the Affymetrix 25K genechip, was analysed. Genes were classified into the same seed development and abiotic stress clusters that were used for the LEA analysis. In contrast to the LEA genes, 72% of the Arabidopsis antioxidant genes were expressed at high levels in control plants (>40% maximum seed or stress expression) and were therefore classified as housekeeping genes (cluster E) (Table 4). Very few (4/71) of the anti-oxidants were seed-specific. However, At1g48130 (1-cys peroxiredoxin) gave the highest intensity reading of all the antioxidants assayed in this study, with less than 0.1% expression in vegetative tissue. Homologues of 1-cys peroxiredoxins have been identified in both X. viscosa (Mowla et al., 2002) and X. humilis (Collett et al., 2004). This gene is thus another example of a seed-specific gene from desiccation-sensitive plants that is expressed at high levels in vegetative tissues during desiccation in desiccation tolerant plants.
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Physiological studies
To further address the main question of whether DT mechanisms in vegetative tissue of desiccation-tolerant plants is an adaptation of DT mechanisms in seeds, we examined two physiological responses of DT in seeds, namely, anti-oxidant activity and sucrose accumulation.
Anti-oxidants
The percent change in AP, GR and SOD enzyme activity, taken from published data on desiccation-tolerant (resurrection plants and orthodox seeds) and -sensitive tissues upon dehydration from full turgor is shown in Figure 3. Discrepancies in reporting (for example many studies, particularly those on vegetative tissues, do not report tissue water content but only drying time) has limited the number of species that could be compared. The data in Figure 3 show that there is no consistent trend in AP activity among tolerant tissues, nor between tolerant and sensitive types. Among the resurrection plants only the Xerophyta spp., and among orthodox seeds only Acer platanoides, show an overall increase in AP activity on drying. In other tolerant tissue, with the exception of wheat seeds, AP activity is down regulated. Amongst sensitive tissues a small increase occurred on drying of wheat seedlings, the remainder also showing down-regulation of AP activity. A decrease in AP activity during seed desiccation appears common and Bailly (2004) has suggested that the ascorbate system is probably not involved in DT. GR activity, on the other hand, is elevated in dry relative to hydrated tissues in all tolerant species, but also in all but one (Quercus robur) sensitive type (Fig. 3). This suggests that GR is a general stress responsive enzyme (and may be considered a "housekeeping" protectant) rather than being specific to DT. Studies aimed at understanding the putative role(s) of GR and the glutathione system give no clear trend in relation to DT. For example GR activity increased during maturation drying of French bean seeds (Bailly et al., 2001) but declined in the case of wheat seeds (De Gara et al., 2003) and remained unchanged in sunflower seeds (Bailly et al., 2003). Reviews on vegetative tissues and recalcitrant seeds similarly indicate differences among species in response to water deficit (Kermode and Finch-Savage, 2002; Farrant, 2000; Kranner and Grill, 1997; Navari-Izzo et al., 1997; Kranner, 2002). SOD activity (Fig. 3) is elevated in all desiccation-tolerant tissues surveyed but only in one sensitive species (Digitaria sanguinalis leaves). SOD activity was down-regulated in all other desiccation-sensitive types. While this might suggest a role unique to DT, reviews of the literature suggest that the SOD enzymes are probably also housekeeping and also play a role in response to various degrees of stress (Pammenter and Berjak, 1999; Bailly, 2004).
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The data assembled in Figure 3 is a comparison between hydrated and dry states and gives no information on changes in enzyme activity during the process of drying (the data are not reported in most studies). It is possible that there was a change in enzyme activity in response to initial drying. Our study on dehydration of vegetative tissue of three Eragrostis spp. with differing degrees of tolerance to water deficit shows that anti-oxidant enzymes were active in hydrated tissues of all species (Fig. 4), suggesting a housekeeping role (as predicted by the analysis above). There was an initial increase in activity of AP, GR and SOD in all the species at 70% RWC. In the desiccation-sensitive E. teff and E. curvula enzyme activity ceased when the plants were dried below their critical water contents (Balsamo et al., 2005
) of 50 and 40%, RWC respectively. In contrast, the activities of all three enzymes remained elevated at lower RWC in the DT E. nindensis compared to the desiccation-sensitive species. Mature air-dry orthodox seeds of both E. nindensis and E. teff retained significant anti-oxidant enzyme activity (Fig. 5). Since the enzyme assays are done in vitro this reflects only their potential for activity and it is unlikely that they are active in situ at 5% RWC. Nonetheless, the ability to remain active when extracted from dry tissue is evidence that the enzymes had been sufficiently protected in the dry state. The lack of enzyme activity in dry tissues of E. teff and E. curvula is likely to be due to their desiccation-induced denaturation (rather than down-regulation) since the plants did not survive drying below 50% RWC (data not shown). Analysis of anti-oxidant enzyme activity therefore corroborates the conclusions from the Arabidopsis antioxidant expression profiles, in that anti-oxidant activity does not appear to be a mechanism specific to DT.
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) and desiccation-sensitive grasses E. teff (
) and E. curvula (
). E. nindensis survives drying to 5% RWC; E. teff and E. curvula die below 50% and 40% RWC respectively. Values shown are means of nine replicates (three separate extracts, three internal replicates). Vertical bars denote standard deviation
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Sucrose
Our survey of changes in sucrose content in response to drying in tissues of desiccation-tolerant and -sensitive species (Fig. 6) shows that this sugar does indeed increase, to varying extents, on drying in angiosperm resurrection plants and orthodox seeds. There was also a general increased level in response to drying in those sensitive tissues from which we could obtain drying course data. However, all but one (wheat seedling) of the reports on sensitive tissues deal with recalcitrant seeds (not desiccation-tolerant) and the accumulation of sucrose may well be as storage reserve rather than pertaining to water deficit during maturation. At least one species (Avicennia marina) does not dry during the terminal stages of development and yet considerable levels of sucrose accumulate (Farrant et al., 1992
). The work on wheat coleoptiles indicated that these tissues were newly germinated and still relatively plastic with respect to re-induction of DT (Farrant et al., 2004) and thus sucrose accumulation in that system might well be related to DT.
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Our study on Eragrostis spp. provides additional evidence that sucrose accumulation is a mechanism linked to DT. In vegetative tissues, sucrose accumulates only in the desiccation-tolerant species E. nindensis in response to drying (Fig. 7). In contrast, we find that the mature orthodox seeds of both E. nindensis and E. teff accumulate high sucrose levels of 88 (±12) and 145 (±16) µmol·mg dw–1 respectively.
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) (sucrose only). E. nindensis survives drying to 5% RWC; E. teff and E. curvula die below 50% and 40% RWC respectively. The values shown are means of three separate extractions. Vertical bars denote standard deviationThe accumulation of sucrose in E. nindensis (Fig. 7) is not likely to be due to photosynthesis, as this is shut down early in the drying time course before maximal sucrose accrual. This is true too of other resurrection plants studied to date (Farrant, 2000
; Mundree et al., 2002; Whittaker et al., 2001, 2004) and sucrose accumulation is proposed to be from mobilization of other storage oligo- and/or polysaccharides such as octulose, stachyose and starch (Schwall et al., 1995; Norwood et al., 2000, 2003). In seeds, sucrose is imported from the parent plant. Although photosynthesis continues to lower water contents in E. curvula, sucrose is not accumulated in this species (Fig. 7) and this species apparently does not have the ability to up-regulate alternative means of sucrose accumulation upon water deficit stress. Sucrose accumulation is therefore common to both seed development and DT in vegetative tissues, and we suggest that sucrose accumulation for protection against desiccation damage is not associated with photosynthesis but involves up-regulation of alternative pathways, with signals that might be common to those present during orthodox seed development. |
DISCUSSION |
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This study offers an approach to exploring the origins of DT in angisosperms and resurrection plants. Two possibilities were entertained which are not necessarily mutually exclusive: DT in resurrection plants was acquired via DT of seeds and/or via effective adaptation of abiotic stress responses. We have looked at both molecular and physiological/biochemical responses to desiccation and abiotic stress to ask whether responses commonly associated with DT in vegetative tissues are also active in desiccating seeds and/or during abiotic stress.
LEAs
First we systematically compared the expression levels of 35 LEAs represented on an Arabidopsis Affymetrix 25K gene chip during seed development, and under a standardized set of conditions for abiotic stress treatments. A large diversity of LEAs were expressed during seed development in comparison to the response of vegetative tissue to abiotic stress. Whereas there is no particular class of LEAs that is uniquely expressed during abiotic stress, genes belonging to LEA-1, -6 and -9 superfamilies are only significantly expressed during seed development. These LEAs may thus be uniquely associated with defense against severe water loss such as would occur in desiccation-tolerant angiosperms/resurrection plants or orthodox seeds.
Many of the abiotic stress-responsive LEAs identified in the expression dataset have also been identified in other Arabidopsis microarray studies on abiotic stress. These include At1g01470 (LEA-8), At1g20440 (LEA-2), At1g52690 (LEA-3), At5g06760 (LEA-4) and At5g66400 (LEA-2) (Bray, 2004). Our comparison with expression data for seed development shows that only At1g20440 (LEA-2) is specific to the abiotic stress response. Notably, no LEA-1, -6, -7, -9 or -10 genes were identified as stress up-regulated in these studies.
We have shown that the expression of at least 16 different LEA genes, representing the LEA-2, -3 -4, -6 -7, -8 and -10 superfamilies, is activated during desiccation in X. humilis leaves (Collett et al., 2004). LEA-6 was identified as a ‘seed-specific’ group in Arabidopsis. Northern blot analysis has shown that the LEAs investigated are specifically activated during the late stages of desiccation, and not during the early stages of water loss, suggesting that they are a uniquely desiccation-specific set of LEAs. We speculate that the simultaneous activation of such a large complement of LEAs under conditions of water loss could point towards the formation of an interacting network necessary for the stabilization of membranes and the protection of proteins. An alternative explanation could be that different LEAs are specifically targeted to different organelles or cellular structures, where they play a local role in protecting proteins, nucleic acids and membranes from the effects of water loss. LEAs have been reported to be expressed at high levels in several other desiccation tolerant plants including the bryophyte T. ruralis, and the angiosperms C. plantagineum and X. viscosa (Table 3). These LEAs all represent the LEA-2, LEA-3 LEA-8 and LEA-10 superfamilies. Six different LEA-3 ESTs were amongst the most abundantly represented mRNA transcripts in a T. ruralis rehydration cDNA library (Oliver et al., 2004) and a LEA-3 gene has been shown to expressed in the anhydrobiotic nematode Aphelenchus avenae (Browne et al., 2002). The LEA-3 superfamily may thus represent very ancient proteins that play important roles during desiccation.
The nomenclature used in this study to describe the LEA superfamilies corresponds to the convention described by Wise (2003), based on differences in peptide composition (see Wise and Tunnacliffe, 2004). We have included a LEA-10 group to accommodate an unclassified LEA-like protein in the Pfam database. It remains to be seen whether different functions can be assigned to the different LEA superfamilies. We used the terms ‘LEA’ and ‘LEA-like’ to identify superfamilies which represent this large class of proteins in the Pfam database. It is important to bear in mind that the annotation of sequences as LEAs tends to be arbitrary, originally assigned according to the abundance of a transcript during late embryogenesis. A unifying feature of this group of proteins is their large number of homologous repetitive hydrophilic peptide motifs and their high percentage of glycine residues. Recent reports on functional analysis of recombinant proteins of AvLEA1, a Group 3 LEA protein from the anhydrobiotic nematode A. avenae, and Em, a group I LEA protein from wheat (Goyal et al., 2005) have shown that both these LEAs show anti-aggregation properties and protected enzyme activity under conditions of water loss. These properties were synergistically enhanced in the presence of sucrose.
Antioxidants
In contrast to the LEAs, mRNA transcripts for most of the anti-oxidant enzymes were abundant in Arabidopsis under control conditions. Very few anti-oxidants were seed-specific but notably, one of these, a 1-cys-peroxiredoxin, has been previously shown to be abundantly expressed during desiccation in the moss T. ruralis and in the leaves of X. humilis and X. viscosa.
Overall, the physiological data on antioxidant enzymes such as AP, GR and SOD suggest that these are ‘housekeeping’ protectants, responsive in the case of most abiotic stresses. We propose that they while they are part of the protection systems in desiccation-tolerant tissues, they are not unique to them and thus are not useful in the evaluation of evolution of DT. Only in true desiccation-tolerant tissues can the activity remain elevated, but this is likely to be a consequence of mechanisms that protect the anti-oxidant enzymes, rather than a unique DT mechanism.
However, there are some antioxidants that appear to be novel to DT, expressed only in maturation drying of orthodox seeds and desiccation of resurrection plants. For example, a 1-Cys peroxiredoxin has been reported to be seed specific (Aalen, 1999; Haslekas et al., 1998) but is induced on drying of the resurrection plant X. viscosa (Mowla et al., 2002). Interestingly, a 1-Cys peroxiredoxin is also expressed during rehydration of the desiccation-tolerant moss T. ruralis (Oliver, 1996) and thus might be indicative of the evolutionary process of these antioxidants in DT.
Sucrose
Sucrose appears to be universally present in mature orthodox seeds (Chen and Burris, 1990; Vertucci and Farrant, 1995) and increases in response to desiccation in all angiosperm resurrection plants studied to date (Ghasempour et al., 1998; Scott, 2000; Whittaker et al., 2001, 2004). Although the precise role of sucrose in DT is still unclear, it is generally agreed that the accumulation of this metabolite is important for protection against desiccation damage (Koster and Leopold, 1988; Scott, 2000; Walters et al., 2002). Our data show that sucrose accumulates in vegetative tissues of only the desiccation-tolerant species of Eragrostis and, like other resurrection plants studied to date (Farrant, 2000; Mundree et al., 2002; Whittaker et al., 2001, 2004), appears unrelated to photosynthesis. Thus while sucrose itself is a common metabolite, its method of accumulation under stress conditions might be an important factor in DT. Breakdown of oligo- and polysaccharides and their mobilization and resynthesis into sucrose within tissues that become protected against desiccation damage is a common phenomenon in seeds and resurrection plants. It is not known whether the mechanisms and signals for this accumulation are similar in seeds and resurrection plants but it is clear that they are absent from vegetative tissues of desiccation-sensitive species E. teff and E. curvula.
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IN SUMMARY |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIN SUMMARYReferences |
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Some of the LEAs and the majority of anti-oxidants analysed in the present study appear to be "housekeeping" or are activated under conditions of abiotic stress (cold, osmotic, salt or drought) in desiccation-sensitive and -tolerant tissues. We propose that these are active and protect only at higher water contents (>65% RWC), but that for true desiccation-tolerance a further repertoire of protectants are necessary and that these are activated below 65% RWC in desiccation-tolerant tissues. The activation of a number of ‘seed-specific’ desiccation protection mechanisms, such as sucrose accumulation and expression of a LEA-6 and a 1-cys-peroxiredoxin gene, in the vegetative tissues of desiccation-tolerant plants points towards acquisition of DT from seeds.
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ACKNOWLEDGMENTS |
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We thank Cathal Seoighe and Brigitte Hamman for assistance in compiling the data for the study and Bronwen Aken and Zek Ginbot for some of the antioxidant and sucrose data generated in the Eragrostis study. This research was supported by funding from the University of Cape Town, the National Research Foundation, South Africa and the National Bioinformatics Network, South Africa.
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FOOTNOTES |
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1 From the Symposium Drying Without Dying: The Comparative Mechanisms and Evolution of Desiccation Tolerance in Animals, Microbes, and Plants presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2005, at San Diego, California.
2 E-mail: Farrant{at}science.uct.ac.za
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