Expression,tissue distribution and subcellular localization of dehydrin TAS14 in salt-stressed tomato plants

We previously isolated and characterized TAS14, an mRNA that is induced in tomato upon osmotic stress or abscisic acid (ABA) treatment and that shares expression and sequence characteristics with other dehydrin genes in different species. Affinity-purified antibodies against TAS14 protein were used to study the expression of TAS14 protein, both in seedlings and mature plants, its tissue distribution and its subcellular localization. TAS14 protein was not detected in 4-day-old seedlings but accumulated after ABA, NaCl or mannitol treatments. In NaCl-treated seedlings, some protein was detectable after 6 h of treatment and reached maximal levels between 24 and 48 h. Concentrations ranging from 5 to 12.5 g/l NaCl induced the protein to similar levels. In salt-stressed mature plants, TAS14 was expressed abundantly and continuously in aerial parts, but only slightly and transiently in roots. Immunocytochemical analysis of salt-treated plants showed TAS14 accumulated in adventitious root primordia and associated to the provascular and vascular tissues in stems and leaves. Immunogold electron microscopy localized TAS14 protein both in the cytosol and in the nucleus, associated to the nucleolus and euchromatin. Since TAS14 is a phosphoprotein in vivo, the classes of protein kinases potentially responsible for its in vivo phosphorylation were tested in in vitro phosphorylation assays. TAS14 protein was phosphorylated in vitro by both casein kinase II and cAMP-dependent protein kinase.The first two authors contributed equally to this paper.     

Gene note. Isolation of a cDNA corresponding to a low temperature- and ABA-responsive gene encoding a putative glycine-rich RNA-binding protein in Solanum commersonii

A cDNA encoding a putative RNA-binding glycine-rich protein, SCRGP-1, was isolated from the wild potato species Solanum commersonii. The amino acid sequence of the deduced protein revealed that the protein contains a consensus RNA-binding domain and has a glycine-rich carboxy-terminal domain. The corresponding gene is induced by low temperature, ABA, wounding, and drought in both Solanum commersonii and Solanum tuberosum. The response of this putative RNA-binding protein gene to low temperature and ABA treatments in Solanum sp. suggests that the SCRGP-1 protein might participate in the adaptation process leading to increased freezing tolerance.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEA D-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Expression and promoter activity of the desiccation-specific Solanum tuberosum gene, StDS2

Environmental stresses induce the expression of several plant genes via multiple and cross‐talking signalling pathways. Previously it was shown that ScDS2, a gene of the wild potato species, Solanum chacoense, is highly inducible by dehydration but not by abscisic acid (ABA), the mediator of many plant stress responses. Herein it is shown that ScDS2‐related genes are present in the cultivated potato, Solanum tuberosum (StDS2) and also in the non‐tuberizing Solanum species, Solanum brevidens (SbDS2). We show that expression of StDS2 is dehydration‐specific, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of ABA. Signalling of StDS2 induction, however, is dependent on the synthesis of novel proteins because cycloheximide can block StDS2 expression. To analyse the promoter region of StDS2 a genomic library of Solanum tuberosum was established and 1140 and 498 bp regions of the StDS2 promoter were isolated. The promoter fragments were fused to the β‐glucuronidase (GUS) reporter gene and tested in transgenic potato plants. Both promoter fragments were able to induce GUS activity in response to dehydration. This result suggests that drought‐specific cis‐elements are located within 498 bp upstream to the StDS2 coding sequence.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display a significant increase in freezing tolerance during periods of cold acclimation (CA). To gain a better understanding of the molecular mechanisms of CA, it is important to unravel the functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEAD-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed. The text was submitted by the authors in English.