A probable lipid transfer protein gene is induced by NaCl in stems of tomato plants

A full-length tomato cDNA clone, TSW12, which is developmentally and environmentally regulated, has been isolated and characterized. TSW12 mRNA is accumulated during tomato seed germination and its level increases after NaCl treatment or heat shock. In mature plants, TSW12 mRNA is only detected upon treatment with NaCl, mannitol or ABA and its expression mainly occurs in stems. The nucleotide sequence of TSW12 includes an open reading frame coding for a basic protein of 114 amino acids; the first 23 amino acids exhibit the sequence characteristic of a signal peptide. The high similarity between the TSW12-deduced amino acid sequence and reported lipid transfer proteins suggests that TSW12 encodes a lipid transfer protein.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, bandc, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

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.     

A new DEAD-box helicase ATP-binding protein (OsABP) from rice is responsive to abiotic stress

The DEAD-box RNA helicase family comprise enzymes that participate in every aspect of RNA metabolism, associated with a diverse range of cellular functions including response to abiotic stress. In the present study, we report on the identification of a new DEAD-box helicase ATP-binding protein (OsABP) from rice which is upregulated in response e to multiple abiotic stress treatments  including NaCl, dehydration, ABA, blue and red light. It possesses an ORF of 2772 nt, encoding a protein of 923 aa, which contains the DEAD and helicase C-terminal domains, along with the nine conserved motifs specific to DEAD-box helicases. The in silico putative interaction with other proteins showed that OsABP interacts with proteins involved in RNA metabolism, signal transduction or stress response. These results imply that OsABP might perform important functions in the cellular response to specific abiotic stress.     

The expression of a rab-related gene,rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh

We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.     

Tissue-specific expression of Pa18, a putative lipid transfer protein gene,during embryo development in Norway spruce (Picea abies)

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.     

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: A model for a potential in vivo lipid carrier protein

The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 ų without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160–171, 1998. © 1998 Wiley-Liss, Inc.     

The expression of the salt-responsive gene salT from rice is regulated by hormonal and developmental cues

The expression pattern of the salT gene was analyzed in different cell types and organs of rice (Oryza sativa L.) in response to saline and hormonal treatments to obtain detailed information on the physiological cues controlling gene expression. Gel blot analysis of RNA and in-situ hybridization performed on seedlings grown for 10 ds in the presence of 1% NaCl revealed that salT was expressed mainly in the younger tissues of the plant. In contrast, 6-week-old plants exhibited maximal salT mRNA accumulation in sheaths of older leaves. In addition, salT was normally expressed in rapidly dividing suspension-cultured cells, but not in quiescent ones. Altogether, these results may indicate that salT expression in each region of the plant is dependent on the metabolic activity of the cells as well as on whether or not they are stressed. The effects of two growth regulators, abscisic acid (ABA) and gibberellic acid, were investigated in combination with the effects of NaCl. Gibberellic acid had a synergistic effect on the induction of the salT gene when combined with 0.5% NaCl, but did not induce salT on its own. At 10 μM, ABA induced salT both in the absence of NaCl and in its presence. Whereas 1 μM ABA acted additively with NaCl to induce gene expression, 5 μM ABA with NaCl was only as effective as NaCl alone. This may indicate that the two stimuli act independently and possibly through antagonistic signal transduction pathways. Received: 26 March 1998 / Accepted: 11 July 1998