The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

Authors


Correspondence: A.D. Neale E-mail: Neale@sci.monash.edu.au

ABSTRACT

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought-stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation-tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine- and proline-rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast-intrinsic protein (TIP) and an early light-inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation-tolerant and desiccation-sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought-responsive genes to be selectively expressed at successive levels of water loss.

INTRODUCTION

Land plants have evolved a variety of mechanisms to enable them to grow and reproduce in environments where water is in short supply. Resurrection plants are a group of desiccation-tolerant plants that have adapted to environments where the rainfall is extremely sporadic. These plants are capable of withstanding severe drought stress, retaining less than 5% of their total water and then reviving completely and recommencing normal metabolism and growth within approximately 24 h of watering ( Gaff 1989). In the majority of higher plants only the mature seed and the pollen grain have this characteristic, and some of the drought-responsive genes isolated from the vegetative tissue of resurrection plants ( Bartels & Nelson 1994; Blomstedt et al. 1998a , b; O’Mahony & Oliver 1999) are also believed to be associated with desiccation tolerance in the embryos of many angiosperms ( Dure 1993; Bray 1993; Bartels & Nelson 1994).

Several regulatory proteins which are thought to mediate gene expression in response to drought or cold treatment via abscisic acid (ABA)-dependent pathways and ABA-independent pathways ( Jonak et al. 1996 ; Shinozaki et al. 1998 ) have been identified in desiccation-sensitive plant species such as Arabidopsis and alfalfa, and include protein kinases, protein phosphatases and transcription factors ( Jonak et al. 1996 ; Meskiene et al. 1998b ; Shinozaki et al. 1998 ).

ABA plays a key role in the induction of desiccation tolerance in the monocotyledonous resurrection plant Borya constricta ( Gaff & Loveys 1984) and in the dicotyledonous resurrection plant Craterostigma plantagineum ( Ingram & Bartels 1996). A C. plantagineum gene of unknown function (CDT-1), isolated by Furini et al. (1997) , is thought to act downstream of ABA and can induce desiccation tolerance when over-expressed in callus tissue. In addition, drought-responsive genes encoding a root-specific myb-like product (cpm7) and two members of the homeodomain–leucine zipper (HD-Zip) family have been isolated from C. plantagineum, and identification of the target genes for these DNA-binding proteins will elucidate their role in drought tolerance in this resurrection dicot ( Frank et al. 1998 ; Iturriaga et al. 1996 ).

We have selected Sporobolus stapfianus as a model system for examining the molecular mechanisms of desiccation tolerance in a monocotyledonous representative of the resurrection plants, with the expectation that the findings will have relevance for manipulating drought resistance in many crop and pasture species. The physiological features of the tolerance of this plant to drought have been studied extensively (reviewed in Gaff 1997; Oliver 1996). Desiccation tolerance in the leaf tissue of S. stapfianus is thought to be induced via an ABA-independent process as the plant dries down to 60% relative water content (RWC). Leaves detached from the plant after this stage of drying retain viability, whereas leaves detached before this stage are not viable on further drying ( Gaff & Loveys 1984). Analyses of proteins extracted at various stages of desiccation from the tolerant S. stapfianus and a desiccation-sensitive member of the Sporobolus genus, S. pyramidalis, indicate that there are two phases of novel protein synthesis in S. stapfianus associated with the development of desiccation tolerance. The first phase occurs at mild to moderate drought stress (85–50% RWC) and the second at extreme drought stress at around 40% RWC ( Kuang et al. 1995 ).

Genes whose transcripts are present at very low levels during the early stages of dehydration and whose expression profile alters during severe drought stress may play a significant role in the ability of S. stapfianus to survive severe desiccation. To isolate these types of genes, a technique was used which selects ‘cold plaque’ clones ( Hodge et al. 1992 ). These ‘cold plaques’ were distinguished by their inability to be visualized during differential hybridization with cDNA probes synthesized from mRNA of either fully hydrated or moderately desiccated leaf material of S. stapfianus. This suggests that the cold plaques represent gene transcripts which are not present in the probe population at high enough levels to allow detection and therefore may be constitutively present at low levels in the cell or may require specific conditions to be induced. Analysis of a large number of cold plaques present in a library prepared from a range of desiccated S. stapfianus tissue resulted in the identification of several low-abundance transcripts expressed specifically during severe drought stress.

MATERIALS AND METHODS

Plant material

Plants of the desiccation-tolerant species S. stapfianus Gandoger and the related desiccation-sensitive S. pyramidalis Beauv. species were grown in a constant-temperature room at 28 °C and 16 h day/8 h night and harvested as previously described ( Blomstedt et al. 1998a ). In addition, S. stapfianus leaf tissue detached from a fully hydrated plant was allowed to dry to the desired RWC in a constant-temperature room ( Blomstedt et al. 1998a ). Drying of leaf tissue detached from S. stapfianus does not confer desiccation tolerance and hence this material was used as an additional desiccation-sensitive sample. Treatment with ABA consisted of spraying leaves and watering fully hydrated plants with 2·5 m M ABA in water containing 0·2% Triton X-100 as a surfactant. Control plants were treated with water/Triton X-100 only.

cDNA library construction and screening

A cDNA library, constructed from mRNA of S. stapfianus at four stages of desiccation, 83%, 68%, 56% and 10% RWCs, using a ZAP-cDNA synthesis kit from Stratagene (La Jolla, California, USA) was screened as previously described ( Blomstedt et al. 1998b ). A large number of plaques that did not hybridize with either the fully hydrated or the 83% RWC probe were detected. These ‘cold plaques’ were selected for analysis. Secondary screening was conducted on amplified inserts following T3 and T7 primed PCR. The PCR products were run on 0·8% agarose gels, transferred to Hybond N+ (Amersham) and probed with cDNA prepared from S. stapfianus leaf material at different levels of RWC to determine the expression profile of the corresponding gene during desiccation. Clones deemed to be of interest were excised according to the instructions supplied by Stratagene, and the resulting plasmids designated pSDG (Sporobolus drought gene) clones.

RNA isolation and Northern analysis

Total RNA was isolated using a phenol extraction/LiCl precipitation method as detailed in Blomstedt et al. (1998a) . Poly(A+) RNA was isolated from the total RNA using Dynabeads Oligo (dT)25 magnetic beads according to the manufacturer’s instructions (Dynal, Oslo, Norway). Transcript levels in S. stapfianus were determined in both desiccation-tolerant tissue as well as desiccation-sensitive tissue. The latter was obtained by harvesting tissue from fully hydrated plants and then allowing the tissue to dehydrate. Tissue was also harvested from a desiccation-sensitive related species S. pyramidalis and included in the Northern analysis. Northern analysis was performed using total RNA as detailed in Blomstedt et al. (1998a) .

Sequence analysis

Plasmid DNA was isolated from selected clones using an alkaline lysis method ( Sambrook, Fritsch & Maniatis 1989) and sequenced using an ABI automated sequencer and the ABI dye terminator sequencing kit according to the manufacturer’s instructions (Perkin-Elmer). Analyses of sequence data and database searches were conducted utilizing programs accessed through the Australian National Genomic Information Service (ANGIS). Sequences isolated in this study have been entered into the EMBL nucleotide sequence database, accession numbers AJ242804, AJ242803, AJ242802, AJ242801, AJ242805, AJ242806 for clones SDG43c, SDG37c, SDG137c, SDG134c, SDG50c and SDG69c, respectively.

RESULTS

Screening and selection of low-abundance clones or clones which are expressed at severe stages of desiccation

The primary screening resulted in the selection of 155 ‘cold’ plaques which gave no signal with probe prepared from fully hydrated or desiccated (83% RWC) plants. No insert was detected in 41 of the 155 plaques following PCR amplification and these phage were discarded. PCR-amplified inserts from the remaining 114 phage stocks were hybridized with cDNA probes prepared from S. stapfianus leaf material from (i) fully hydrated plants and (ii) pooled RNA from plants harvested at 82%, 63%, 54%, 10% and < 5% RWC. The increased sensitivity of this reverse Northern technique resulted in the grouping of the selected clones into five categories ( Table 1). While clones contained in the first two categories in Table 1 may be considered of most interest as they detect transcripts that appear to accumulate during drying, several clones showed decreases in transcript levels during dehydration. A number of clones were selected from the first three categories in Table 1 for tertiary screening.

Table 1.  Summary of the results from secondary screening of selected clones
Category of cloneNumber of clones
  1. Total number of clones

  2. 114

  3. Inserts were amplified by PCR and probed with labelled cDNA prepared from fully hydrated plants and drought-stressed plants to determine which clones represent genes induced by desiccation.

Detected by ‘drought-stressed’ probe only19
Increased signal with ‘drought-stressed’6
probe
Detected or stronger signal with ‘fully6
hydrated’ probe
Similar signal with ‘fully hydrated’ and14
‘drought-stressed’ probes
Detected by neither probe69

The tertiary screen was conducted on selected clones following plasmid excision from the phage. Individual filters of EcoRI- and XhoI-digested plasmids were probed separately with cDNA probes made from fully hydrated plant material and plant material at mild drought stress (80% RWC), moderate drought stress (60% RWC) and extreme drought stress (< 10% RWC). The output from the tertiary screen of a subset of clones is depicted in Fig. 1. The results demonstrate that several of these inserts represent genes whose transcripts accumulate during severe dehydration. The expression patterns of selected clones were then analysed in desiccation-tolerant and desiccation-sensitive tissues. Two of the clones selected were found to represent novel members of the dehydrin gene family (unpublished results) and the characterization of these genes is not detailed here. The results from six of the selected clones are presented in this report.

Figure 1.

An example of a tertiary ‘reverse Northern’ screen of cold plaque inserts. Plasmid DNA from each clone was digested, separated by gel electrophoresis, blotted onto nylon membrane (Hybond N+) and probed with either fully hydrated probe (panel b) or probes from different stages of desiccation (80% RWC, panel c; 60% RWC, panel d; < 10% RWC, panel e).The plasmids in lanes 4 and 27 are controls which contain two previously characterized insert sequences from Sporobolus stapfianus that are constitutively expressed in fully hydrated tissue and in dehydrated tissue: lane 4, pSC40 encoding a sulphate transporter ( Ng et al. 1996 ); lane 27, pSDG3-18 encoding a thiol protease ( Blomstedt et al. 1998b ). The first lane (M) contains marker DNA with sizes (kb) as denoted.

Further characterization of six cold plaque clones

SDG137c: drought-inducible small glycine-rich protein

Two transcripts of 0·8 kb and 0·6 kb are detected in desiccation-tolerant tissue ( Fig. 2a) by the 562 bp insert of pSDG137c. Primary sequence data suggest that SDG137c encodes a putative protein of 95 amino acids ( Fig. 3) with a predicted isoelectric point of 10·2. Database searches did not reveal any strong similarity to previously identified genes. However, the S. stapfianus gene product does contain repeat regions rich in glycines and histidines which are similar to those present in the proteins encoded by drought-inducible genes from saltbush ( No et al. 1997 ) and the CORA gene and ABA- and environmental stress-inducible genes from Medicago sativa which are induced by drought and cold ( Luo et al. 1992 ; Laberge, Castonguay & Vezina 1993).

Figure 2.

Northern blot analysis of selected cold plaque clones using total RNA (20 μg lane−1) isolated from desiccation-tolerant and desiccation-sensitive Sporobolus leaf tissue at various stages of desiccation. ‘S. stap. intact’ and ‘S. pyram. intact’ indicate that RNA was isolated from leaf tissue harvested from whole plants of S. stapfianus (desiccation-tolerant) and S. pyramidalis (desiccation-sensitive), respectively. ‘S. stap. detached’ indicates that RNA was isolated from leaf tissue dried following excision (desiccation-sensitive). The letters above each lane indicate that RNA was isolated from tissue at the following RWCs: a, 100–90% (fully hydrated); b, 89–80% (mild drought stress); c, 79–60% (moderate drought stress); d, 59–40% (strong drought stress); e, 39–20% (severe drought stress); f, 19–11% (extreme drought stress); g, < 10% (air-dry). ABA+ indicates that RNA was isolated from fully hydrated S. stapfianus leaf tissue treated with abscisic acid. ABA indicates that RNA was isolated from fully hydrated S. stapfianus leaf tissue treated in the same manner without abscisic acid. Panel f is an ethidium bromide-stained representative sample of the gels, which were run concurrently, showing the relative amount of RNA loaded in each lane. Exposure times ranged from 2 to 8 d for filters(a)–(d) and overnight for filter (e).

Figure 3.

The nucleotide sequence of the S. stapfianus clone pSDG137c. The predicted protein sequence of 95 amino acids is shown below the nucleotide sequence. The regions rich in glycine and histidine which show some similarity to those found in an ABA- and environmentally stress-inducible gene (CORA) from Medicago sativa ( Luo et al. 1992 ) are underlined.

Plant proteins classified as glycine-rich (GRPs) exhibit an assortment of glycine-rich motifs diverging from GRPs containing contiguous glycine residues interspersed occasionally with alanine or serine residues (G-X, where X = G, S or A) to GRPs which contain no recognizable repeat motifs (reviewed in Showalter 1993). Many GRPs contain an N-terminal signal sequence and verification of localization in the cell wall has been obtained for bean and petunia GRPs ( Keller, Sauer & Lamb 1988; Condit, McLean & Meagher 1990). A subset of GRPs may also be located in the cytoplasm as they do not appear to have a signal peptide sequence and some GRP genes encode an RNA-binding sequence at the N-terminus ( Mortenson & Dreyfuss 1989; Sturm 1992). No signal sequence or RNA-binding sequence is evident in the SDG137c GRP product.

Both the 0·8 and 0·6 kb transcripts accumulate during dehydration in desiccation-tolerant tissue samples from intact S. stapfianus plants, reaching a maximum in tissue at severe drought stress. At further extremes of drought stress, the transcript levels decrease but remain higher than those observed in tissues with RWCs above 60–79% ( Fig. 2a). Neither transcript is detected in desiccation-sensitive S. stapfianus tissues which were detached before drying. The 0·6 kb transcript appears to accumulate transiently in desiccation-sensitive S. pyramidalis tissue during the mid stages of drying; however, at lower RWCs, the transcript levels decrease and are not detectable in air-dry tissue (< 10% RWC). The level of the 0·8 kb transcript appears to be unaffected by the application of ABA to fully hydrated S. stapfianus plants, whilst the level of the 0·6 kb transcript appears to be decreased by ABA treatment ( Fig. 2a).

SDG43c: glycine- and proline-rich protein

pSDG43c contains a cDNA insert of 855 bp which hybridizes to a transcript of similar size ( Fig. 2b) and encodes a putative protein of 197 amino acids ( Fig. 4) comprised mainly of glycine (29%), proline (15%) and histidine (13%), with alanine, lysine and tyrosine also being abundant at 8% each. Together, these six amino acids constitute 81% of the deduced protein. The putative gene product contains two repeating motifs. The first motif, located in the N-terminal region, consists of repeating units made up largely of proline, glycine, tyrosine, glutamine and alanine ( Fig. 4). Another S. stapfianus gene (SDG7), previously isolated from drought-stressed plants ( Blomstedt et al. 1998a ), also encodes a putative glycine-rich protein with similar repeating units. The second motif, which is not present in the SDG7 protein, is located in the C-terminal region of SDG43c and is comprised of glycine and histidine residues in an imperfect repeat with a core amino acid sequence of ‘GHG’ ( Fig. 4). The glycine–histidine repeats are similar to those found in the protein encoded by SDG137c. The putative SDG43c protein has a predicted molecular mass of 19–20 kDa and an isoelectric point of 11.

Figure 4.

The nucleotide sequence of pSDG43c. The predicted protein sequence is shown below the nucleotide sequence. The regions rich in proline and glycine are single-underlined, while the glycine- and histidine-rich motifs are double-underlined.

Northern blot analysis ( Fig. 2b) shows that the SDG43c probe detects two transcript both of which accumulate throughout desiccation. It is likely that the larger 1·0 kb transcript represents SDG43c and the smaller 0·7 kb transcript represents cross-hybridization of the SDG43c probe with the SDG7 transcript. The larger transcript is not detected in desiccation-sensitive tissue, nor do the levels of this transcript increase in fully hydrated S. stapfianus plants following the application of exogenous ABA.

SDG134c: protein initiation factor 1 (eIF1)

The clone pSDG134c contains an insert of 796 bp which hybridizes to a transcript of similar size ( Fig. 2c). Translation of this sequence reveals a putative protein of 115 amino acids showing 97% identity to the protein initiation factor 1 (eIF1/Sui1) from rice ( de Pater et al. 1992 ).

To our knowledge, the up-regulation of eIF1/Sui1 has not previously been linked to any form of stress in plants or other organisms. Northern blot analysis shows that the SDG134c transcript is present at very low levels in fully hydrated tissue of S. stapfianus and also in desiccation-sensitive tissue. As the plants experience escalating levels of drought stress, increased transcript levels are observed only in the desiccation-tolerant S. stapfianus leaf tissue. The highest levels of transcript are present in the air-dry desiccation-tolerant samples ( Fig. 2c). The application of the phytohormone ABA appears to have no effect on the levels of SDG134c transcript ( Fig. 2c).

SDG69c: early light-inducible protein (ELIP)

Translation of the small 264 bp insert of pSDG69c reveals a partial open reading frame which encodes a putative gene product with over 70% identity to the C-terminal end of early light-inducible proteins (ELIPs) from several species including barley ( Grimm, Kruse & Kloppstech 1989) and the desiccation-tolerant dicot C. plantagineum ( Bartels et al. 1992 ). We have subsequently isolated another gene encoding a similar but not identical ELIP (SDG17s) from S. stapfianus indicating that ELIPs are encoded by a multi-gene family in S. stapfianus. ELIPs have been shown to possess two conserved hydrophobic regions ( Ouvrard et al. 1996 ) and the second of these is present in both the S. stapfianus ELIPs.

ELIPs are nuclear-encoded proteins which are associated with thylakoid membranes in the chloroplast and are believed to be involved in the protection of the photosystem during UV stress ( Ouvrard et al. 1996 ). A number of desiccation-related genes encoding ELIPs have been isolated, including the dsp-22 gene from C. plantagineum ( Bartels et al. 1992 ; Alamillo & Bartels 1996) and sdi-1 from a drought-tolerant line of sunflower ( Ouvrard et al. 1996 ). In sunflower, sdi-1 transcripts are detectable in unstressed plants and increase in both drought-sensitive and drought-tolerant lines during water deficit, with higher levels of transcript attained in the drought-tolerant line. Application of exogenous ABA does not elevate the level of sdi-1 transcripts ( Ouvrard et al. 1996 ). By contrast, the dsp-22 transcript is not detected in unstressed plants but forms one of the most abundant desiccation-induced transcripts ( Bartels et al. 1992 ). Application of ABA also raises the transcript level in C. plantagineum, and transcript accumulation during either drought stress or ABA treatment is markedly less in the absence of light ( Alamillo & Bartels 1996).

Northern blot analysis shows that the S. stapfianus ELIP transcript is detectable only in desiccation-tolerant tissue of S. stapfianus which has experienced severe drought stress ( Fig. 2d) and does not accumulate to very high levels. No signal is detected in fully hydrated plants or in desiccation-sensitive tissue experiencing water deficit. The hormone ABA appears to have no effect on transcript accumulation.

SDG50c: gamma tonoplast-intrinsic protein (γTIP)

Characterization of the insert contained in the pSDG50c cold plaque reveals that the S. stapfianus SDG50c gene encodes a protein with similarity to gamma tonoplast-intrinsic proteins (γTIP) from several species ( Fig. 5), with the strongest similarity (87%) being to a γTIP from rice ( Liu, Umeda & Uchimiya 1994). The expression of tonoplast-intrinsic proteins (TIPs) has also been associated with stress in a number of plant species. TIPs, along with plasma membrane-intrinsic proteins (PIPs), are subsets of channel proteins called the major intrinsic proteins (MIPs) which allow the translocation of small solutes across membranes. Those MIPs which act as water channels are called aquaporins and are believed to be associated with maintaining cell turgor ( Maurel 1997). Plant MIPs characterized so far segregate into three classes: TIPs, PIPs and nodulin 26 ( Weig, Deswarte & Chrispeels 1997). TIPs and PIPs can be differentiated by signature sequences which are present in both the N- and C-terminal ends of the protein as well as around the conserved asparagine–proline–alanine (NPA) motifs ( Schäffner 1998). The S. stapfianus gene product possesses two perfectly conserved NPA motifs and six putative membrane-spanning domains ( Fig. 5) which are distinctive characteristics of TIPs ( Higuchi et al. 1998 ). The presence of these characteristic features strongly suggest that SDG50c encodes a γTIP.

Figure 5.

Alignment of SDG50c with several γTIPs from different species, including rice ( Liu et al. 1994 ), barley ( Schunmann & Ougham 1996), Arabidopsis ( Höfte et al. 1992 ), iceplant (U43291, unpublished data) and tobacco ( Yamamoto et al. 1991 ). Identical residues in the six sequences are indicated by asterisks. The six putative membrane-spanning domains are single-overlined and the two NPA motifs are double-overlined.

An increase in the levels of transcript encoding the S. stapfianusγTIP is only evident in desiccation-tolerant S. stapfianus tissue suffering water deficit. Very high levels of transcript accumulate in the air-dry desiccation-tolerant tissue ( Fig. 2e). While low levels of the transcript are present in fully hydrated tissue of S. stapfianus harvested intact and in both fully hydrated and dehydrated desiccation-sensitive tissue, no increase in transcript levels is observed in the drought-sensitive tissue as the plant material dries. An increase in the level of the γTIP transcript is observed following application of exogenous ABA to fully hydrated S stapfianus plants ( Fig. 2e).

SDG37c: serine/threonine phosphatase type 2c

The insert in pSDG37c is 1·1 kb long and does not represent the entire transcript based on Northern blot analysis. Sequence data show that SDG37c has a high level of similarity to serine/threonine phosphatases type 2C ( Fig. 6). The highest level of similarity (76%) is to the protein phosphatase type 2C (MP2C) from alfalfa ( Meskiene et al. 1998a ).

Figure 6.

A comparison of the deduced partial amino acid sequence of the protein phosphatase type 2C from S. stapfianus with MP2C from alfalfa ( Meskiene et al. 1998a ) and ABI1 from Arabidopsis ( Meyer et al. 1994 ; Leung et al. 1997 ). The nucleotide sequence of pSDG37c from S. stapfianus is shown with the deduced amino acid sequence directly beneath. Amino acids that are identical in the MP2C sequence shown below the SDG37c amino acid sequence, and in the ABI1 sequence underneath, are in bold. The dots indicate where gaps have been introduced into the sequences to allow alignment. Deduction of the amino acid sequence of SDG37c as depicted required removal from the sequence of an 88 nt intron at the position marked with a triangle (see text).

The amino acid sequence of SDG37c was deduced following removal from the sequence of an 88 nt intron at the position marked with a triangle ( Fig. 6). Perfect matches were found for the fully conserved 5′ and 3′ intron splice sites and seven of the eight most highly conserved surrounding nucleotides ( Luehrsen, Taha & Walbot 1994). In addition, the sequence removed contains a high AU content (65%) shown to be important for intron recognition in plants ( Brown & Simpson 1998), and an intron prediction program ( Hebsgaard et al. 1996 ) indicates that the chosen splice sites have a confidence value > 95%. Environmental stresses including heat shock, heavy metal exposure and anaerobiosis have been reported as inhibiting but not blocking pre-mRNA splicing in plants (reviewed in Simpson & Filipowicz 1996). The presence of this intron suggests that this transcript may have been extracted before processing was complete, possibly as a result of the exposure of the plant material to drought stress.

The putative SDG37c PP2C protein has 38% and 37% identity with the Arabidopsis PP2Cs ABSCISIC ACID-INSENSITIVE1 and 2 (ABI1 and ABI2), respectively ( Leung, Merlot & Giraudat 1997). The MED and DGH sequences (positions 141–143 and 177–179, respectively, in the ABI1 protein) which are located at the PP2C active site ( Das et al. 1996 ) are conserved in SDG37c, as is the G residue which is converted to a D (position 180) in the Arabidopsis abi1 and abi 2 mutations ( Leung et al. 1997 ). Interestingly, the SDG37 product also has a G residue at the equivalent position to G174 in ABI1. This residue has been proposed as essential for the binding of target proteins ( Sheen 1998). These residues are also conserved in the alfalfa MP2C protein.

The transcript from the SDG37c gene decreases dramatically in desiccation-tolerant S. stapfianus tissue as the plants experience severe drought stress ( Fig. 7). A similar drop is observed in desiccation-sensitive tissue of S. pyramidalis (data not shown). The application of ABA or cold treatment does not appear to significantly affect the transcript level in S. stapfianus.

Figure 7.

Northern blot analysis using total RNA (50 μg lane−1) isolated from desiccation-tolerant S. stapfianus following cold and ABA treatment and from leaf tissue at various stages of desiccation. ‘S. stap. intact’ indicates that RNA was isolated from leaf tissue harvested from whole plants of S. stapfianus. The letters above each lane indicate that RNA was isolated from tissue at the following RWCs: a, 100–90% (fully hydrated); b, 89–80%; d, 59–40%; e, 39–20%; f, 19–11%; g, < 10% (air-dry). For lanes labelled ‘S. stap. ABA tmt’, ‘+’ indicates that RNA was isolated from fully hydrated S. stapfianus leaf tissue treated with abscisic acid, while ‘–’ indicates that RNA was isolated from fully hydrated S. stapfianus leaf tissue treated in the same manner without abscisic acid. For the lane labelled ‘S. stap. Cold tmt’, 4° indicates that RNA was isolated from leaf tissue following 4 °C treatment of fully hydrated S. stapfianus for 20 h. The lower panel is an ethidium bromide staining of the gel showing the relative amount of RNA loaded in each lane.

DISCUSSION

We have previously reported the isolation of several S. stapfianus genes whose transcripts increase in abundance in the early stages of water loss and remain at relatively high abundance throughout the dehydration process ( Blomstedt et al. 1998a , b). While many of the conventional differential screening techniques are suited to those genes whose transcripts are produced in abundance, they do not easily allow direct isolation of genes with low transcript levels. In the present study, we have utilized the cold plaque strategy to detect lowly transcribed genes represented in a cDNA library prepared from S. stapfianus undergoing dehydration, with the aim of identifying medium- to low-level transcripts which alter in abundance at severe stages of drought stress.

In the primary screen, 155 cold plaques were selected. Almost half of the PCR-amplified inserts from these cold plaques were still not detected after probing, and are likely to represent genes expressed at very low abundance at all stages of water stress. However, tertiary screening of a selected subset of clones indicated that several cold plaques represented genes expressed at medium to low levels whose transcripts alter in abundance at severe stages of drought stress. Further characterization indicated that increases observed for several of the transcripts occur only in desiccation-tolerant tissue during severe drought stress, and thus these genes may be of particular interest in the study of desiccation tolerance in S. stapfianus.

Genes which exhibited transcript accumulation specifically in drought-stressed desiccation-tolerant tissue were found to encode an eIF1 translation initiation factor, two drought stress-inducible glycine-rich proteins, a tonoplast-intrinsic protein (TIP) and an early light-inducible protein (ELIP). A serine/threonine phosphatase type 2C gene was also isolated whose transcript was present during moderate drought stress but dropped below detectable levels during severe water deficit.

This is the first report suggesting that a gene encoding an eIF1 translation initiation factor may have a role in the drought stress response of plants. Although accumulation of protein initiation factor eIF1 transcripts during environmental stress has not previously been reported, accumulation of transcripts encoding translation elongation factor eEF1A during cold treatment have been reported in barley and maize ( Dunn et al. 1993 ; Berberich et al. 1995 ) and during wounding and exposure to low oxygen levels in potato ( Morelli, Shewmaker & Vayda 1994; Vayda, Shewmaker & Morelli 1995). It has been proposed that an increase in eEF1A may allow rapid synthesis of proteins required to respond to those stresses ( Kidou & Ejiri 1998). The appearance of higher levels of eIF1 transcripts during the mid stage of dehydration in S. stapfianus coincides with the phase of novel protein synthesis previously reported to occur at this level of drought stress in this resurrection plant ( Kuang et al. 1995 ). It is possible that SDG134c encodes a novel isoform of eIF1 that is capable of operating during severe drought stress; however, this seems unlikely as there are only two amino acid differences between the S. stapfianus sequence and that of maize and only one difference with the rice sequence. In the desiccation-tolerant tissue of S. stapfianus, the presence of this transcript persists, with the highest levels being observed in completely dehydrated plants. It is generally assumed that in desiccation-tolerant plants many of the molecular components essential for re-initiation of metabolic activity have survived the desiccation process and are present in the dried tissue. It is likely that rapid protein synthesis is also required during the rehydration process which leads to replacement or repair of damaged or lost components and full metabolic activity being achieved within several hours.

The protein encoded by SDG137c contains several glycine-rich GHGG repeat motifs, is highly hydrophilic and does not appear to contain an N-terminal signal sequence. A number of GRPs, some of which lack signal sequences, have been shown to be expressed in response to a variety of environmental stress conditions including drought and low temperature ( Luo et al. 1992 ; Showalter 1993) and may be associated with stabilizing membranes against damage during dehydration ( Thomashow 1998).

The glycine-rich motifs found in SDG137c are similar to those occurring in the C-terminal domain of the drought-responsive SDG43c gene product which has several glycine-rich GGHG repeat motifs interspersed with a KFK repeat motif. The N-terminal domain of SDG43c, however, contains 11 proline-rich repeats which are identical (GYPPQ) or similar (GAYPPPP) to those found in a previously isolated S. stapfianus drought-responsive gene, SDG7 ( Blomstedt et al. 1998a ). Plant proteins containing GYPPQ repeats had not previously been linked to drought stress. Similar GYP repeat regions are found in the cytosolic domain of several membrane-associated animal proteins ( Döring, Schleicher & Noegel 1991; Ovchinnikov et al. 1988 ). The N- and C-terminal domains of the SDG43c protein are separated by a run of alanines which form the basis of a central hydrophobic domain of 21 amino acids. This hydrophobic region is likely to form a helical structure according to the Chou and Fasman prediction ( Chou & Fasman 1974).

This SDG43c gene product has 66% identity at the amino acid level with a glycine- and proline-rich protein (GPRP) encoded by a gene isolated from Arabidopsis ( Marty et al. 1996 ). SDG43c also has a similar level of identity to an unpublished carrot sequence (EMBL X72383) which appears to encode a truncated version of a similar protein. Neither of these dicot sequences have yet been associated with drought stress. There appears to be no extracellular signal sequence encoded by SDG43c, nor is there one encoded by the Arabidopsis GPRP; however, Marty et al. (1996) have shown that the Arabidopsis GPRP interacts with membranes, presumably via the central hydrophobic domain. This has led to the suggestion that the GPRP may have a role in vesicle-associated intracellular transport or in linking cellular components through the plasma membrane with the cell wall ( Marty et al. 1996 ). Several studies have proposed that tyrosine residues in repeat regions similar to those occurring in SDG7 and the N-terminus of SDG43c may be involved in the formation of cross-links with GRPs and other cell wall components to reduce cell wall elasticity in response to various stresses, including water deficit ( Bradley, Kjellbom & Lamb 1992; Meier et al. 1992 ; No et al. 1997 ). This presents an attractive hypothesis for a proposed role for the S. stapfianus GPRP in drought tolerance, whereby interaction of the GPRP with the plasma membrane and cell wall may help to maintain the integrity of the cell during dehydration or rehydration.

In S. stapfianus, the SDG69c ELIP transcript is not detected in fully hydrated plants or in plants undergoing mild to moderate drought stress, and the transcript is only detected at low levels as the plant is exposed to very severe water deficit. ELIPs are homologous to the cbr gene isolated from Dunaliella bardawil ( Lers, Levy & Zamir 1991). This gene has been associated with zeaxanthin-related non-photochemical fluorescence quenching under stress conditions ( Braun et al. 1996 ). A number of studies suggest that ELIPs bind the carotenoid zeaxanthin during drought stress and provide a mechanism for non-photochemical quenching of fluorescence without the requirement for a pH gradient across the thylakoid membrane. The quenching of excess light energy during water-deficit conditions may help prevent oxidative damage to the photosynthetic apparatus during dehydration ( Casper, Eickmeier & Osmond 1993).

The expression patterns of ELIPs in response to drought stress and the application of ABA suggest that there are significant differences in ELIP gene regulation, not only between drought-sensitive and drought-tolerant plants but also between some drought-tolerant resurrection species. Drought stress-induced ELIP transcript accumulation appears to be significantly greater in C. plantagineum than S. stapfianus. Differences in regulation of ELIP transcript levels between species may reflect differences in the biological and morphological mechanisms which protect these plants from photoinhibitory damage during dehydration. Several studies have suggested that some resurrection plants, including S. stapfianus, initiate a partial systematic disassembly of the photosynthetic apparatus at an early stage in the dehydration process, thus avoiding potential photoinhibitory damage ( Casper et al. 1993 ; Di Blasi et al. 1998 ). In S. stapfianus, non-photochemical quenching was observed not to increase significantly until a RWC of 40% was reached ( Di Blasi et al. 1998 ). This finding correlates well with the water-deficit level at which the ELIP transcript is observed to increase ( Fig. 2d). It is noteworthy that field-dried S. stapfianus accumulates a high level of a water-soluble pigment that colours the outer leaf an intense purple–black which may protect the photosynthetic apparatus from excessive light intensities, whereas much less pigmentation is observed in drying C. plantagineum (light purple to grey).

Genes encoding water-specific or solute channel proteins have been shown to be inducible by osmotic stress in pea ( Guerrero, Jones & Mullet 1990), rice ( Liu et al. 1994 ), Arabidopsis ( Höfte et al. 1992 ; Yamaguchi-Shinozaki et al. 1992 ) and C. plantagineum ( Mariaux et al. 1998 ). A comparison of the expression patterns of γTIP transcripts during dehydration of S. stapfianus and the resurrection dicot C. plantagineum reveals significant differences. In C. plantagineum, the expression of the γTIP is transient, reaching a maximum after only 1 h of stress and then decreasing to less than control levels after 4 h ( Mariaux et al. 1998 ). By contrast, the S. stapfianus transcript accumulates progressively throughout the dehydration process and reaches a very high level in air-dry tissue. This suggests that these genes are regulated differently in the two desiccation-tolerant species, a situation which may reflect a difference in function. Alternatively, the differences in the expression patterns in the two resurrection species may merely have resulted from differences in the experimental treatment of sample tissues. High-level accumulation of the γTIP transcript in S. stapfianus is confined to desiccation-tolerant tissue and suggests that the S. stapfianus SDG50c γTIP may be required for the cell to accommodate the large deviations in osmotic potential that are experienced during dehydration and rehydration and to facilitate the rehydration process by raising the water permeability across the tonoplast membrane.

The S. stapfianus SDG37c gene encoding the type 2C protein Ser/Thr phosphatase (PP2C) has identity with a PP2C gene, MP2C, from alfalfa ( Meskiene et al. 1998a ) as well as the Arabidopsis PP2C genes ABI1 and ABI2 ( Leung et al. 1997 ; Meyer, Leube & Grill 1994). The Arabidopsis ABI PP2Cs play a role in signal transduction associated with stress-related ABA-dependent gene expression, whereas the alfalfa PP2C acts in an ABA-independent pathway. The PP2C activity of the Arabidopsis proteins can negatively regulate the ABA signal transduction pathway controlling both induction and repression of gene transcription in response to ABA ( Sheen 1998).

The alfalfa PP2C, MP2C, was found to deactivate a mitogen-activated protein kinase (MAPK) pathway ( Meskiene et al. 1998b ). This stress-activated MAP kinase (SAMK) pathway is activated in response to drought stress as well as wounding, cold and touch, and may act independently of ABA to allow rapid induction of cold- and drought-related gene expression ( Jonak et al. 1996 ). Jonak et al. (1996) have also suggested that the SAMK pathway may induce ABA synthesis by activating synthetic enzymes either directly or via induction of ABA synthesis genes. The expression of alfalfa MP2C follows a peak in SAMK activity, suggesting that it acts as a negative regulator of the SAMK pathway. Using transcription and translation inhibitors, Meskiene et al. (1998b) have shown that inactivation of the SAMK pathway in alfalfa requires de novo expression of the MP2C gene which is thought to result from the activity of the SAMK pathway itself and thus provide a mechanism to attenuate the stress signal by a negative feedback mechanism.

SDG37c has significantly closer identity to alfalfa MP2C than to any of the Arabidopsis PP2Cs. The identity between SDG37c and MP2C is 58%, which is 8% higher than between the Arabidopsis PP2Cs, ABI1 and AtPP2C ( Kuromori & Yamamoto 1994), which have been shown to have partially redundant functions ( Sheen 1998). Notably, transcription of the ABI1 and ABI2 genes is induced by ABA ( Leung et al. 1997 ), whereas the SDG37c gene is not. While the shared identity between the S. stapfianus PP2C and the alfalfa PP2C suggests that the SDG37c gene product may act as a negative regulator in an ABA-independent MAPK pathway in S. stapfianus, the pattern of SDG37c transcript accumulation in S. stapfianus indicates that these genes may not have completely orthologous functions. The alfalfa MP2C gene operates in a drought/cold-activated pathway and is induced following stress to attenuate the stress response, whereas the S. stapfianus SDG37c transcript is present at detectable levels in fully hydrated plants and does not appear to accumulate during cold-treatment. It is likely however, that more detailed experiments to detect transient increases in SDG37c transcript levels are required. SDG37c transcripts are not detectable below 44% RWC, which suggests that this gene product does not operate during severe water deficit. This may indicate that the signalling pathway with which SDG37c may be associated is permanently activated during continuing severe water loss, or alternatively that the pathway has ceased operation at this level of severe water deficit. The latter hypothesis may be more likely given that the homologous transcript levels in the desiccation-sensitive species S. pyramidalis drop below detectable levels at a much earlier stage of water loss (around 80% RWC, unpublished observation). Experiments are underway to examine transcript levels during rehydration and to obtain direct evidence of water-deficit related MAPK-dependent gene expression in S. stapfianus.

In the majority of higher plants, only the seed and pollen have the ability to withstand complete desiccation, and a number of the genes activated in the early stages of dehydration in resurrection plants are similar to those expressed in the desiccating seed of most plants ( Blomstedt et al. 1998a , b). This has led to the hypothesis that resurrection plants may possess regulatory genes which allow the desiccation processes of the seed and pollen to occur in the vegetative tissue. Previous gene expression studies in this laboratory have also shown that many of the early responses to mild dehydration are similar in desiccation-sensitive and desiccation-tolerant plants, with several genes showing a fairly rapid increase in abundance in response to moderate water stress ( Blomstedt et al. 1998a , b). However, several of these genes have elevated transcript levels which persist throughout the entire drying process in the desiccation-tolerant S. stapfianus, but not in comparative desiccation-sensitive tissues ( Blomstedt et al. 1998a ). This suggests that these gene products may be required to protect the plant from severe dehydration or to allow successful rehydration and the restoration of metabolic activity.

The desiccation-tolerant tissue of S. staphianus appears to retain the ability to increase transcript levels of specific genes even at extreme levels of dehydration, with several of the drought-responsive genes identified in this study showing significant expression only at severe stages of desiccation. The comparative studies suggest that this ability is largely restricted to desiccation-tolerant tissue and that these gene products may be associated with the ability of S. stapfianus to withstand levels of extreme water loss. The putative functions of these genes also provide some understanding of the molecular mechanisms which may be associated with conferring desiccation tolerance in this resurrection species. The alteration in transcript levels of these genes, which the present study indicates can occur almost throughout the entire dehydration process, suggests that there may be several unique key regulatory genes capable of operating at successive levels of water loss to confer desiccation tolerance in the vegetative tissue of this resurrection plant. Current studies using those SDG genes expressed specifically in desiccation-tolerant tissue at severe stages of water loss are aimed at identifying regulatory genes controlling their expression and addressing the question of whether similar mechanisms operate during the desiccation stage of seed development.

ACKNOWLEDGMENTS

We are grateful for the financial support provided by the Meat Research Corporation, Australia (UMON:004) and the Australian Research Council (A19230441) which allowed this work to be carried out.

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