Xerophyta viscosa (Baker) is a monocotyledonous resurrection plant that is capable of ..... Han B., Hughes D.W., Galau G.A., Bewely J.D. and Chermidae.
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Plant Growth Regulation 35: 137–145,©2001. ©©2001©Kluwer Academic Publishers. Printed in the Netherlands.
Molecular characterization of XVT8, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker Tozama Ndima 1, Jill Farrant 1, Jennifer Thomson 1 and Sagadevan Mundree 1,2,* 1
Microbiology Department, University of Cape Town, Private Bag, 7701, Rondebosch, South Africa; 2Botany Department, University of Cape Town, Private Bag, 7701, Rondebosch, South Africa; *Author for correspondence Received 10 March 2001
Abstract Xerophyta viscosa (Baker) is a monocotyledonous resurrection plant that is capable of tolerating extremes of desiccation. Upon rewatering, it rehydrates completely, assuming its full physiological activities. Studies on changes in gene expression associated with dehydration stress tolerance were conducted. A cDNA library was constructed from mRNA isolated from dehydrated X. viscosa leaves [85%, 37% and 5% relative water content (RWC)]. XVT8 represents one of 30 randomly selected clones that were differentially expressed when X. viscosa was dehydrated. Sequence analysis of XVT8 revealed that XVT8 exhibited 45% and 43% identity to dehydrin proteins from Arabidopsis thaliana and Pisum sativum respectively, at the amino acid level. XVT8 encodes a glycine -rich protein (27 kDa) which is largely hydrophilic and contains a hydrophobic segment at the C-terminus. Southern blot analysis confirmed the presence of XVT8 in the X. viscosa genome. XVT8 transcripts accumulated in X. viscosa plants that were exposed to heat, low temperature and dehydration stresses, and to exogenous abscisic acid and ethylene. These results provide direct evidence for the heat, low temperature, dehydration, abscisic acid and ethylene -dependent regulation of the XVT8 gene in X. viscosa. Introduction The desiccation tolerant plant Xerophyta viscosa Baker (Family Velloziaceae) belongs to a small group of angiosperms, referred to as “resurrection plants”, that are capable of tolerating extreme water loss (Bewely and Oliver 1992; Galau et al. 1986; Ingram and Bartels 1996; Vertucci and Farrant 1995). X. viscosa can be dehydrated to 5% relative water content (RWC) and upon rewatering, the desiccated plant rehydrates completely within 80 hours, resuming full physiological activities (Sherwin and Farrant 1996). This unique ability of tolerating severe water loss is shared with certain algae and bryophytes (Oliver and Bewely 1997), a few ferns (Reynolds and Bewely 1993a) and with specialized structures of higher plants such as seeds and pollen. It is not completely clear how resurrection plants tolerate desiccation. However, studies on the mechanisms involved have been derived mostly from the observations of cellular processes occurring during desiccation (Dace et al. 1998; Ingram and Bartels 1996; Oliver and Bewely
1997). It has been suggested that accumulation of sugars, proteins and other compatible solutes protects membranes against desiccation and play a role in the osmotic adjustment (Ingram and Bartels 1996; Oliver and Bewely 1997; Vertucci and Farrant 1995). The synthesis of new proteins such as late embryogenesis abundant (LEA) proteins, which protect the macromolecular functioning, have been shown to accumulate during maturational drying in seeds (Galau et al. 1986). LEA proteins are a subset of abscisic acid (ABA) responsive proteins. In angiosperms, these proteins are expressed mainly during embryo maturation (Galau et al. 1986). LEA proteins are extremely hydrophilic and have therefore been predicted to play various roles during dehydration, including the sequestration of ions, binding of water and functioning as molecular chaperones (Bray 1997; Dure et al. 1989; Xu et al. 1996). Six groups of LEA proteins have been identified based on common amino acid sequence domains (Bray 1993; Dure et al. 1989). At least five groups have been proposed to contribute towards desiccation tolerance in the embryo (Cohen et
138 al. 1991; Mundy and Chua 1988; Russouw et al. 1995; Swire-Clark and Meacock 1990). Dehydrins belong to a distinct family of proteins known as LEA D11, a subgroup of LEA proteins. Their expression has been shown to occur in most angiosperms and gymnosperms (Close et al. 1993; Close 1996; Dure 1993). Characteristically, dehydrins remain soluble even at high temperatures and are hydrophilic in nature. They are composed of different amounts of the K segment (lysine-rich repeat), the S segment (tract of Ser residues), and the Y segment (conserved N-terminal sequence) (Close 1996). The induction of expression of this protein has been shown to occur during environmental stresses such as drought, cold and salinity, and also in response to ABA (Blackman et al. 1991; Close 1996; Galau et al. 1987; Gee et al. 1994; Chermidae 1997). Although the physiological role of dehydrins has not been fully elucidated, it has been proposed that they play an important role in desiccation tolerance by interacting closely with other protectants such as oligosaccharides (Close 1996). The synthesis of many proteins during dehydration is regulated by the plant hormone abscisic acid (ABA) (Bray 1997; Ingram and Bartels 1996). ABA is produced under conditions of cold, drought and high salinity and is important in mediating physiological, cellular and molecular responses of plants to these stressed conditions (Nelson et al. 1994). Genes that are regulated positively or negatively by ABA and/or dehydration have been studied (Conley et al. 1997) and it has been reported that most genes require ABA under stressed conditions. In this paper, we describe the isolation and molecular characterization of a differentially-expressed cDNA clone (XVT8) that encodes a protein which has significant identity to dehydrins (Close et al. 1993).
Materials and methods Plant material and growth conditions Xerophyta viscosa Baker plants were collected from the Buffelskloof Nature Reserve (Mpumalanga Province, South Africa) and were grown under glasshouse conditions (Sherwin and Farrant 1996). The average midday light intensity during experimentation was 1 200 mol·m −2·s −1. Plant drying and determination of water content (on a dry mass basis) and
relative water content (RWC) was as described (Sherwin and Farrant 1996). Isolation of RNA Total RNA was extracted from X. viscosa leaves according to the protocol by (Chomczynski 1987). Three to four leaves were frozen in liquid nitrogen and ground with a mortar and pestle. 1 g of fine powder was transferred to a microcentrifuge tube and the tissue sample was homogenized in 1 ml of Trizol Reagent (Gibco BRL, U.S.A.). The mixture was vortexed for 1 min, and stored at room temperature for 5 min to permit complete dissociation of the nucleoprotein complex. 200 ml of chloroform was added and the contents mixed gently by inverting the tube for 15 sec. The resulting mixture was stored at room temperature for 5 min, following which it was centrifuged at 12000 × g for 15 min at 4 °C. The aqueous phase was then transferred to a new microcentrifuge tube and 500 ml of isopropanol was added to precipitate the RNA. Samples were incubated at room temperature for 15 min followed by centrifugation at 12000 × g for 15 min at 4 °C. The supernatant was discarded and the pellet was washed with 75% ethanol, and subsequently air dried. The pellet was resuspended in 50 l of diethyl pyrocarbonate (DEPC) treated water and stored at −70 °C. The samples were quantitated spectrophotometrically (Sambrook et al. 1989). Construction and screening of cDNA library Two g of poly(A) + RNA was extracted from X. viscosa leaves that were at 85%, 37% and 5% RWC. The RNA was pooled and used as a template to construct a cDNA library. The cDNA library was constructed in ZAPII (Stratagene, La Jolla, CA, USA) as described by the manufacturer. The amplified library was converted into an infective, packaged ss-pBluescript phagemid library (Short et al. 1988). 200 l of the ZAP cDNA library (average titer = 1.5 × 10 8 pfu/ml) was added to 200 l of XL-1-Blue cells (containing 10 11 cells; Stratagene) and 1l of Exassist helper phage (3 × 10 10 pfu/ml). After an incubation period of 15 min at 37 °C, 5 ml of 2 × YT medium (Sambrook et al. 1989) was added and the tube was incubated at 37 °C for 3 hours with shaking. The tube was subsequently heated at 70 °C for 20 min and centrifuged at 4,000 × g for 10 min to remove phage. The supernatant containing the infective ss-pBlue-
139 script phagemids was used to infect the DH5␣ strain of Escherichia coli. The infected DH5␣ bacterial cells were selected on LB/agar (Sambrook et al. 1989) plates, supplemented with 50 g/ml ampicillin. 192 colonies were isolated and their respective plasmids purified. A restriction digestion (EcoRI and XhoI) was carried out on each plasmid to verify the presence of a cDNA insert. 1 g of plasmid DNA was blotted in duplicate onto nylon membranes (MSI, 0.45 ) in a slot blot apparatus (Hoefer Scientific, San Francisco). 5 g of total RNA isolated from hydrated (100% RWC) and dehydrated (37% RWC) X. viscosa leaves were reversely transcribed separately, incorporating [ 32P] dCTP (Amersham, England) and used to probe the membranes separately. Filters were prehybridized at 68 °C for 4 hours and hybridized for 18 hours at 68 °C in 6 × SSC (1 × SSC is 150 mM NaCl, 17 mM Sodium Citrate), 5 × Denhardt’s solution, 0.1% SDS, and 100 g/ml herring sperm DNA. Filters were washed at room temperature in 2 × SSC, 0.1% SDS for 3 × 5 min, and finally at 68 °C in 0.1 × SSC, 0.1% SDS for 2 × 1 hour. The membranes were exposed to x-ray film at −70 °C for appropriate times. Differentially expressed genes were identified following autoradiography.
frozen powder were transferred into SS34 tubes. 15 ml of extraction buffer (100 mM Tris pH 8, 50 mM EDTA pH 8, 500 mM NaCl, 10 mM 2−mercaptoethanol) was added and the mixture was vortexed vigorously for 1 min. 1 ml of 20% SDS was added and the tubes were shaken vigorously. The mixture was then incubated at 65 °C for 10 min. 5 ml of 5 M potassium acetate was added to the mixture and the tube was shaken gently by inverting for 15 sec and thereafter incubated on ice for 20 minutes. The tubes were spun at 12000 rpm for 20 min and the supernatant was carefully transferred into a new set of SS34 tubes containing 10 ml of isopropanol. The tubes were incubated at −20 °C for 2 hours and then centrifuged at 10000 rpm for 15 minutes. The supernatant was discarded and pellet air-dried. The pellet was resuspended in 1 ml of TE buffer (Sambrook et al. 1989) and transferred into Eppendorf tubes. 10 l RNase (10 mg/ml) was added and the eppendorf tubes were incubated at 37 °C for 30 min. A phenol, phenol-chloroform and chloroform extraction was carried out and the DNA precipitated with isopropanol (Sambrook et al. 1989). 30 g of DNA was restricted with the endonucleases EcoRI and EcoRV. Northern and Southern blot analyses
DNA sequencing and analysis The nucleotide sequence of the cDNA clones were determined on both strands using the ALFexpress TM automated DNA Sequencer AMV3.0 (Pharmacia Biotech AB, Uppsala, Sweden). The sequencing reactions were carried out using the Thermo Sequenase Fluorescent Labeled Primer cycle sequencing kit (Amersham International, Buckinghamshire, England). The inferred amino acid sequences were used to search for homologies in protein sequence databases using the BLAST network service (Altschul et al. 1990). Amino acid comparisons were done with the CLUSTAL program of DNAMAN (Version 3.0, 1997). A hydropathic analysis of the deduced amino acid sequence of XVT8 using the algorithm of Kyte and Doolittle (1982) was conducted. Isolation of genomic DNA Chromosomal DNA was isolated from the leaves of X. viscosa using a plant minipreparation protocol (Dellaporta et al. 1983). Four to five young leaves were frozen in liquid nitrogen and ground into a fine powder using a mortar and a pestle. Aliquots of fine
RNA isolated from hydrated (92% RWC) and dehydrated (42%, 32% and 4% RWC) X. viscosa leaves (five leaves from two separate pots, respectively for each RWC) were electrophoresed on 1.2% formaldehyde gels (Chomczynski 1987) in 20 mM Mops (3[N-morpholino]propanesulfonic acid) pH 7.0, and transferred to nylon membranes (MSI, 0.45. For the northern slot blot analysis of cold, heat, NaCl, ABA and ethylene treated X. viscosa plants, 4 g of total RNA was slot blotted onto nylon membranes (MSI, 0.45 ) in a slot blot apparatus (Hoefer Scientific, San Francisco). Endonuclease digested genomic DNA was electrophoresed on a 0.8% agarose gel and transferred to a nylon membrane as described above. The filters were prehybridized at 68 °C for 4 hours and hybridized for 18 hours at 68 °C in 6 × SSC, 5 × Denhardt’s solution, 0.1% SDS, and 100 g/mL herring sperm DNA. The XVT8 insert was labeled with [ 32P] dCTP by random primer labeling (Boehringer Mannheim, GmbH, Germany) and used as a probe. Filters were washed at room temperature in 2 × SSC, 0.1% SDS for 3 × 5 min, and finally at 68 °C in 0.1 × SSC, 0.1% SDS for 2 × 1 hour. The membranes were ex-
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Figure 1. Nucleotide and deduced amino acid sequence of XVT8. The highly conserved serine- and lysine- motifs are underlined. B. Hydropathy plot of XVT8 showing the largely hydrophilic protein.
posed to x-ray film at −70 °C for appropriate durations.
X. viscosa treatments X. viscosa was exposed to dehydration, cold, NaCl and heat stresses, and ABA and ethylene treatments. X. viscosa plants were dehydrated to 37% RWC under glasshouse conditions (described above). Plants
141 were subjected to low temperature (4 °C) and heat (42 °C) conditions, respectively for a duration of 7 days. For the low temperature treatment, plants were placed in a cold room (4 °C) with a photoperiod of 14 hours light. For the heat treatment, plants were placed in an incubator (42 °C) with a photoperiod of 14 hours light. Control samples were removed at time zero. Leaves were removed from the plants every 24 hours for RNA isolation. X. viscosa plants were sprayed with ABA (100 M) once daily and exposed to ethylene (100 ppm) constantly for a duration of 66 hours. Leaves were removed from the plants every 6 hours for RNA isolation and subsequent northern analysis.
Results Sequence analysis of XVT8 The XVT8 cDNA insert is 770 bp long, with an open reading frame of 732 bp (Figure 1A). The predicted protein has 244 amino acids, a calculated Mr of 27,000 and a pI of 9.5. The protein contains two highly conserved motifs, SSSSSSESDGEGGRRKK and KIKEKIPG, which are characteristic signature motifs of dehydrins (Figure 1A). Hydropathy analysis (Kyte and Doolittle 1982) predicted a largely hydrophilic protein, while a segment at the C-terminus was hydrophobic (Figure 1B). A computer search for homologies using the BLAST network service revealed that XVT8 has significant identity with several dehydrins. The highest identities were with ATDEHYDRIN and PSDEHYDRIN, dehydrins from Arabidopsis thaliana and Pisum sativum, in the order of 45% and 43% respectively (Figure 2). Northern and Southern analysis Northern blot analysis of total RNA isolated from hydrated (92% RWC) and dehydrated (42%, 32% and 4% RWC) X. viscosa leaves exhibited a single transcript of ⬇ 1.0 kb in dehydrated (42%, 32% RWC and 4%) leaves only (Figure 3). The lower band observed (Figure 3, lane 4) is probably due to degradation of RNA. Southern blot analysis of genomic DNA isolated from the leaves of X. viscosa and probed with the XVT8 insert produced two bands under high stringency conditions (Figure 4).
Effect of cold and heat stresses on XVT8 expression Northern slot blot analysis of cold and heat treated X. viscosa plants revealed that the XVT8 transcripts were detected 24 hours after the above treatments, respectively (Figure 5A and B). The abundance of XVT8 transcripts increased significantly 48 and 72 hours after exposure to 4 °C (Figure 5A). At 96 hours, there appeared to be basal levels of XVT8 transcripts, and further exposure to the low temperature stress resulted in a gradual increase in transcript levels. The abundance of XVT8 transcripts following exposure to heat stress was not as dramatic as observed for cold stress (Figure 5B). A gradual increase in XVT8 transcripts was observed up until 72 hours of heat stress, followed by a slight decrease and subsequent increase in transcript levels. No XVT8 transcripts were detected under control conditions prior to the cold and heat treatments. The blots were probed with -actin to verify equal loading of RNA samples. Effect of ABA and ethylene on XVT8 expression Northern slot blot analysis of ethylene and ABA treated plants revealed that XVT8 expression was induced after 12 and 6 hours of ethylene and ABA treatments, respectively (Figure 6A and B). A gradual increase in XVT8 transcripts was observed following ABA treatment, until a saturation level was reached (36 hours) (Figure 6B). This level of XVT8 transcripts was observed for the following 30 hours. XVT8 transcripts observed 12 hours after ethylene treatment remained the same for the following 24 hours (Figure 6A). A significant increase in XVT8 transcript levels was observed 42 hours after ethylene treatment, followed by a gradual decrease. A similar pattern of XVT8 expression was observed at 60 and 66 hours where an increase and subsequent decrease in transcript levels was observed. The blots were probed with -actin to verify equal loading of RNA samples.
Discussion The strategy of differential screening was used for the isolation of cDNA clones from X. viscosa. The cDNA library represented genes that were expressed in X. viscosa leaves during dehydration (85%, 37% and 5% RWC). The ZAPII vector allowed for the directional cloning of cDNAs and the efficient rescue of phagemids from this vector (Short et al. 1988). This
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Figure 2. Amino acid comparision of XVT8 with related proteins. An asterisk (*) indicates identity with the XVT8 sequence while a dot(·) indicates similarity. Percentages following the sequences indicate the percentage identity to XVT8 as obtained from a computer search using the BLAST network service. The sequences are: ATHYDRIN, a dehydrin protein from Arabidopsis thaliana (Lang and Palva 1992); PSHYDRIN, a dehydrin protein from Pisum sativum (U91969). Gaps(-) were introduced to optimise sequence alignment.
strategy was used to screen 192 randomly selected cDNA clones from the X. viscosa cDNA library. Total RNA isolated from hydrated (100% RWC) and dehydrated (37% RWC) X. viscosa leaves were reverse transcribed and used to probe one replicate of the 192 cDNA clones, respectively. 30 cDNAs showed higher expression levels when the plant was dehydrated, while 20 cDNA clones exhibited higher levels of expression in the hydrated plant relative to the dehydrated condition. XVT8 represents one of them. The nucleotide sequence of the XVT8 cDNA has an open reading frame of 732 bp and shows high levels of identity with dehydrins. XVT8 is a glycine-rich protein (27 kDa) which is largely hydrophilic and contains the highly conserved serine- and lysine-rich
motifs of dehydrins. It shows significant identity to ATDEHYDRIN and PSDEHYDRIN, dehydrins from Arabidopsis thaliana and Pisum sativum (Lang and Palva 1992). In addition, it also has significant identity to dehydrins from Hordeum vulgare, Sorghum bicolor and Helianthus annus (data not shown). The properties described above together with the results from the BLAST search, supported our hypothesis that XVT8 is likely to be a dehydrin. The size of dehydrins varies from 14 to 85 kDa according to sequence data (Close et al. 1993) and their molecular organization is more akin to that of seed storage proteins than is typical of enzymes (Close 1996). The role of dehydrins is therefore likely to be structural rather than enzymatic. Southern blot analysis con-
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Figure 3. Northern blot analysis of hydrated (92%RWC; lane 1) and dehydrated (42%, 32% and 4% RWC; lanes 2, 3 and 4) X. viscosa leaves. 10 g of total RNA was fractionated on a 1.2% agarose gel and transferred to a nylon membrane. The blot was probed with 32P-labelled XVT8 insert. Equivalent amounts of RNA were loaded as determined from gel photograph.
Figure 4. Southern blot analysis of genomic DNA from X. viscosa leaves (lanes 1 and 2). 15g of DNA was restricted with EcoRI (lane 1) and EcoRV (lane 2), electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane and probed with XVT8 insert.
firmed the presence of XVT8 in the X. viscosa genome. It has been well documented that dehydrins accumulate in response to dehydration (Close 1996; Dure 1993; Han et al. 1997; Chermidae 1997) and also cold and salt stress (Close 1996). We therefore tested the effects of dehydration, low temperature and heat
Figure 5. Northern slot blot analysis of low temperature (A) and heat (B) stressed X. viscosa plants. 1 = control; 2–8 = RNA taken from plants at 24h intervals following the start of treatment. 4 g of total RNA was slot blotted onto nylon membranes in a slot blot apparatus (Hoefer Scientific, San Francisco). The blot was probed with 32P-labelled XVT8 insert. Equivalent amounts of RNA were loaded as determined by using a -actin probe (A and B).
Figure 6. Northern slot blot analysis of ethylene (A) and ABA (B) treated X. viscosa plants. 1 = control. 2–12 = RNA taken from plants at 6 h intervals following the start of treatment. 4 g of total RNA was slot blotted onto nylon membranes in a slot blot apparatus (Hoefer Scientific, San Francisco). The blot was probed with 32Plabelled XVT8 insert. Equivalent amounts of RNA were loaded as determined by using a -actin probe (A and B).
stress, and the effect of exogenous ABA and ethylene on XVT8 expression in the resurrection plant X. viscosa. The XVT8 transcripts accumulated in response to each of the above treatments. There was particularly abundant accumulation of the transcripts when plant was dehydrated to 37% RWC, suggesting that XVT8 does indeed play a role in tolerance of water loss in this plant as has been reported for other desiccation tolerant organisms (Close 1996; Dure 1993;
144 Han et al. 1997; Chermidae 1997). The precise nature of that role is still unclear (Black et al. 1999). The accumulation of XVT8 transcripts in response to low temperature stress is consistent with results from studies on other plants (Close et al. 1993; Lang and Palva 1992). The two-phase induction of XVT8 expression could imply that there is a turnover of the dehydrin proteins. The accumulation of XVT8 transcripts in response to heat stress is significant, as to date, there has been no previous report of the response of dehydrins to heat stress. Nevertheless, since dehydrins have been found to accumulate in response to any environmental stress that has a dehydration component (Close 1996), this is not a surprising result. X. viscosa plants grow in shallow soils on rock (predominantly granitic) surfaces, in environments in which it would be exposed to high summer temperatures (Gaff 1971). Thus the presence of a dehydrin that accumulates during heat stress would facilitate its survival of elevated temperatures. This raises the possibility of another signal that is involved in XVT8 expression. The transcription of dehydrin genes is widely known to be inducible by ABA-dependent and ABAindependent signals (Robertson and Chandler 1994). We have observed that XVT8 expression was induced 6 hours after the exposure to exogenous ABA. This indicates that the XVT8 promoter responds to ABA at the whole plant level. The induction of XVT8 expression in X. viscosa leaves by dehydration probably was associated with dehydration-induced increases in endogenous ABA. A number of studies have revealed an increase in endogenous ethylene in plants exposed to hypoxia (Atwell et al. 1988; Drew 1997; He et al. 1996). We observed that the exposure of X. viscosa plants to exogenous ethylene resulted in the accumulation of XVT8 transcripts, with the maximum accumulation occurring after 42 hours of treatment (Figure 6A). This suggests that ethylene could be a signal for XVT8 expression in X. viscosa. In summary, the high sequence identity observed with known dehydrins, the presence of the conserved serine- and lysine-rich motifs and the hydropathy analysis data clearly support the view that XVT8 is a dehydrin. Our results provide direct evidence for the dehydration, low temperature, heat, ABA and ethylene-dependent regulation of the XVT8 gene in X. viscosa.
Acknowledgements TBN is grateful to the NRF for an MSc fellowship. JMF acknowledges John and Sandie Burrows for the supply of plant material.
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