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.     

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH₂-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5 signal sequence, an open reading frame of 718 nucleotides, a 3 AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.     

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.     

Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.     

Structure of the dehydrin tas 14 gene of tomato and its developmental and environmental regulation in transgenic tobacco

We have isolated a genomic clone encoding tomato TAS14, a dehydrin that accumulates in response to mannitol, NaCl or abscisic acid (ABA) treatment. A fragment of tas14 gene containing the region from –2591 to +162 fused to -glucuronidase gene drives ABA- and osmotic stress-induced GUS expression in transgenic tobacco. Histochemical analysis of salt-, mannitol-and ABA-treated plants showed GUS activity mainly localized to vascular tissues, outer cortex and adventitious root meristems, coinciding with the previously observed distribution of TAS14 protein in salt-stressed tomato plants. In addition, GUS activity was also observed in guard cells, trichomes and leaf axils. Developmentally regulated gus expression was studied in unstressed plants and found to occur not only in embryos, but also in flowers and pollen. Tas14 expression in floral organs was confirmed by northern blots of tomato flowers.     

Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.).

We identified a novel salt-inducible soybean gene encoding an acidic-isoform of pathogenesis-related protein group 5 (PR-5 protein). The soybean PR-5-homologous gene, designated as Glycine max osmotin-like protein, acidic isoform (GmOLPa)), encodes a putative polypeptide having an N-terminal signal peptide. The mature GmOLPa protein without the signal peptide has a calculated molecular mass of 21.5 kDa and a pI value of 4.4, and was distinguishable from a known PR-5-homologous gene of soybean (namely P21 protein) through examination of the structural features. A comparison with two intracellular salt-inducible PR-5 proteins, tobacco osmotin and tomato NP24, revealed that GmOLPa did not have a C-terminal extension sequence functioning as a vacuole-targeting motif. The GmOLPa gene was transcribed constitutively in the soybean root and was induced almost exclusively in the root during 24 h of high-salt stress (300 mM NaCl). Interestingly, GmOLPa gene expression in the stem and leaf, not observed until 24 h, was markedly induced at 48 and 72 h after commencement of the high-salt stress. Abscisic acid (ABA) and dehydration also induced expression of the GmOLPa gene in the root; additionally, dehydration slightly induced expression in the stem and leaf. In fact, the 5'-upstream sequence of the GmOLPa gene contained several putative cis-elements known to be involved in responsiveness to ABA and dehydration, e.g. ABA-responsive element (ABRE), MYB/MYC, and low temperature-responsive element (LTRE). These results suggested that GmOLPa may function as a protective PR-5 protein in the extracellular space of the soybean root in response to high-salt stress and dehydration.     

Structure and expression of a cDNA encoding a putative neoxanthin cleavage enzyme (NCE), isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Oxidative cleavage of a 9-cis xanthophyll probably representsthe key regulatory step in abscisic acid (ABA) biosynthesis.A transposon tagged maize (Zea mays) mutant vp14, provided theoriginal DNA sequence data needed to design a VP14 fusion proteincapable of catalysing this reaction in vitro. A cDNA encodinga similar protein has now been isolated from a wilt-relatedtomato (Lycopersicon esculentum Mill.) library. The tomato cDNAand derived amino acid sequence have been compared to thoseof maize and of other enzymes catalysing broadly similar oxidativecleavage reactions. The results of Northern analysis in tomatoindicated that mRNA levels of this vp14 homologue increaseddramatically in response to water stress. Key words: ABA biosynthesis, oxidative cleavage step, tomato     

Er5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61% sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression.     

ABA- and ethylene-mediated responses in osmotically stressed tomato are regulated by the TSS2 and TOS1 loci

The study of mutants impaired in the sensitivity or synthesis of abscisic acid (ABA) has become a powerful tool to analyse the interactions occurring between the ABA and ethylene signalling pathways, with potential to change the traditional view of the role of ABA as just being involved in growth inhibition. The tss2 tomato mutant, which is hypersensitive to NaCl and osmotic stress, shows enhanced growth inhibition in the presence of exogenous ABA. The tos1 tomato mutant is also hypersensitive to osmotic stress, but in contrast to tss2, shows decreased sensitivity to ABA. Surprisingly, blocking ethylene signalling suppresses the growth defect of tss2 seedlings on ABA, NaCl, and osmotic stress, but not the osmotic hypersensitivity of tos1. The ethylene production of tss2 seedlings is increased compared with that of control seedlings under osmotic stress. In addition, the tss2 plants are hypersensitive to root growth inhibition by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This suggests that, in addition to ABA regulation, TSS2 acts as a negative regulator of endogenous ethylene accumulation. As previously shown in Arabidopsis, it is shown here that extensive cross-talk occurs between the ABA and ethylene signalling pathways in tomato and that the TSS2 and TOS1 loci appear as regulators of this cross-talk.     

PLC/PLD抑制剂对盐胁迫下玉米幼苗根系新的69.5kD热稳定蛋白表达调控的影响

逆境蛋白在植物抵抗逆境胁迫中具有重要作用。广泛存在于植物中的磷脂酶 (PhospholipaseC/D, PLC/PLD) 是一种逆境信号传递物质, 在逆境蛋白的诱导表达中具有重要意义。该文探讨了不同胁迫及PLC/PLD抑制剂处理后一种新发现的 6 9.5kD热稳定蛋白的表达情况, 为进一步研究PLC/PLD在逆境蛋白表达中的作用机理提供依据。以玉米 (Zeamays) ‘鲁单 90 0 2’幼苗为材料, 用 2 70mmol·L-1NaCl胁迫处理, 然后进行SDS _PAGE (Sodiumdo decylsulfate_Polyacrylamidegelelectrophoresis) 电泳, 从受胁迫的玉米幼苗根系中分离出一种新的 6 9.5kD热稳定蛋白, 其表达量随胁迫时间的延长逐渐增加。该蛋白在恢复正常条件后仍然表达, 表明其可能是一种胁迫适应性蛋白 ;2 0 μmol·L-1ABA处理也能引起该蛋白的表达, 但表达量远低于同期的 2 70mmol·L-1NaCl胁迫处理。NaCl+ABA处理后该蛋白表达量低于NaCl处理, 但高于ABA处理。PLC/PLD抑制剂硫酸新霉素 (Neomycinsulfate, NS) 和正丁醇 (n _Butylalcohol, BA) 对NaCl、ABA和NaCl+ABA诱导的 6 9.5kD热稳定蛋白表达均有明显的抑制作用, 且对ABA和NaCl+ABA两个处理蛋白表达的抑制作用均随抑制剂浓度升高或处理时间延长而逐渐增强, 但对NaCl处理蛋白表达的抑制作用则表现出复杂性。结果表明, 不同处理诱导 6 9.5kD热稳定蛋白表达的敏感程度不同, 而磷脂酶在不同处理诱导的信号传递途径中的作用机理有一定的差别。     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

Abscisic acid root and leaf concentration in relation to biomass partitioning in salinized tomato plants

Salinization is one of the most important causes of crop productivity reduction in many areas of the world. Mechanisms that control leaf growth and shoot development under the osmotic phase of salinity are still obscure, and opinions differ regarding the Abscisic acid (ABA) role in regulation of biomass allocation under salt stress. ABA concentration in roots and leaves was analyzed in a genotype of processing tomato under two increasing levels of salinity stress for five weeks: 100 mM NaCl (S10) and 150 mM NaCl (S15), to study the effect of ABA changes on leaf gas exchange and dry matter partitioning of this crop under salinity conditions. In S15, salinization decreased dry matter by 78% and induced significant increases of Na⁺ and Cl⁻ in both leaves and roots. Dry matter allocated in different parts of plant was significantly different in salt-stressed treatments, as salinization increased root/shoot ratio 2-fold in S15 and 3-fold in S15 compared to the control. Total leaf water potential (Ψ_w) decreased from an average value of approximately −1.0 MPa, measured on control plants and S10, to −1.17 MPa in S15. In S15, photosynthesis was reduced by 23% and stomatal conductance decreased by 61%. Moreover, salinity induced ABA accumulation both in tomato leaves and roots of the more stressed treatment (S15), where ABA level was higher in roots than in leaves (550 and 312 ng g⁻¹ fresh weight, respectively). Our results suggest that the dynamics of ABA and ion accumulation in tomato leaves significantly affected both growth and gas exchange-related parameters in tomato. In particular, ABA appeared to be involved in the tomato salinity response and could play an important role in dry matter partitioning between roots and shoots of tomato plants subjected to salt stress.