Water-deficit induction of a tomato H1 histone requires abscisic acid

Many genes are induced by periods of water deficit, and a subset of these are dependent on elevated ABA content for expression. A number of drought-induced genes are not induced in leaves of the ABA-deficient mutant flacca from tomato (Lycopersicon esculentum) but are induced in detached, wilted wild-type leaves and ABA-treated leaves of both genotypes. The nucleotide sequence of the cDNA and corresponding genomic DNA fragment of one of these genes, his1-s (formerly called le20), encodes an amino acid sequence that is rich in Lys, Ala, and Ser. The predicted protein contains the tripartite structure of H1 histone and is similar to other H1 histones, especially in the globular domain. Since, his1-s is more closely related to a stress-induced gene from Lycopersicon pennellii than to another H1 histone in the tomato genome it is considered a stress-induced variant of H1 histone. his1-s mRNA accumulated in vegetative plants in response to other abiotic stress treatments, including application of polyethylene glycol, and salt. The mRNA preferentially accumulated in leaves as compared to roots. his1-s mRNA accumulation was controlled during development; the level was higher in developing seeds of mature green fruit than in detached wilted leaves. H1 histones have been implicated in the general repression of gene expression and in the regulation of specific genes. The rapid accumulation of his1-s mRNA during stress may indicate that this unique, stress-induced H1 histone is involved in controlling gene expression during plant stress.     

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

The effect of salicylic acid on barley response to water deficit

The effect of a moderate (PEG −0.75 MPa) and severe (PEG −1.5 MPa) water deficit on SA content in leaves and roots as well as the effect of pre-treatment with SA on reaction to water stress were evaluated in two barley genotypes — the modern cv. Maresi and a wild form of Hordeum spontaneum. Water deficit increased SA content in roots, whereas SA content in leaves did not change. The level of SA in the roots of control plants was about twofold higher in ‘Maresi’ than in H. spontaneum. After 6 hours of a moderate stress the level of SA increased about twofold in H. spontaneum and about two and a half-fold in ‘Maresi’. Under severe stress conditions the level of SA increased about twofold in the both genotypes, but not before 24 hrs of the stress. Plant treatment with SA before stress reduced a damaging action of water deficit on cell membrane in leaves. A protective effect was more noticeable in H. spontaneum than in ‘Maresi’. SA treatment increased ABA content in the leaves of the studied genotypes. An increase of proline level was observed only in H. spontaneum. The obtained results suggest that ABA and proline can contribute to the development of antistress reactions induced by SA.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen

Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% ¹⁸O₂ and 80% N₂ indicates that one atom of ¹⁸O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing ¹⁸O₂ indicates that one atom of ¹⁸O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

     

Spider toxin (Hvt) gene cloned under phloem specific RSs1 and RolC promoters provides resistance against American bollworm (Heliothis armigera)

Spider venoms are neurotoxin proteins that can kill insects. Spider toxin Hvt gene was cloned under two phloem specific RSs1 and RolC promoters, transformed into tobacco plants through Agrobacterium-mediated transformation and tested against Heliothis armigera larvae. Transgenic plants were confirmed through PCR. First instar larvae of H. armigera were released on detached leaves of transformed and non-transformed plants. Insect bioassays showed 93–100% mortality of H. armigera larvae within 72 h on the leaves of transgenic plants while all larvae survived and continued feeding on detached leaves from non-transformed control plants. The Hvt gene expressing under phloem specific RSs1 and RolC promoters could therefore be used for developing H. armigera-resistant, genetically-modified crops.     

Dithiothreitol and PEG Induced Combined Stress May Affect the Expressions of ABA Aldehyde Oxidase,Sucrose Synthase and Proline Metabolic Genes in Maize Seedlings

The endoplasmic reticulum (ER) is an organelle in the cell where proteins are created and folded. Folding is a very elaborate process that is often interrupted by various biotic and abiotic stresses, leading to the formation of unfolded and misfolded proteins called ER stress. Dithiothreitol (DTT)-induced unfolded protein response (UPR) in endoplasmic reticulum (ER) has been recently reported in plants. Also, previous studies demonstrated that treatment with polyethylene glycol (PEG₆₀₀₀) could stimulate water deficit in crops. However, further researches should be conducted to elucidate the molecular mechanism of ER stress response and the relationship between water deficiency and ER. In this study, we examined the expressions of sucrose synthase (SuS) gene, proline metabolic genes and abscisic aldehyde oxidase (AAO3) gene in maize seedlings that were subjected to DTT and PEG induced combined stresses by using quantitative real-time RT-PCR. Three weeks old detached maize seedlings were treated with or without DTT and PEG₆₀₀₀ for 12 h. The treatment with DTT increased about 2-fold the expression of gene encoding proline synthesis enzyme, pyrroline-5-carboxylate synthetase (P5CS) but no statistically affected the proline catabolism enzyme, proline dehydrogenase (ProDH) in comparison with un-treated seedlings. PEG treatment was also up-regulated P5CS while it was down-regulated ProDH. The relative expression levels of SuS and AAO3 genes statistically enhanced about 2.5 fold under the DTT-induced ER stress. Likewise, the expression levels of SuS and AAO3 genes were up-regulated in the detached seedlings exposed to PEG-induced water deficit. Conversely, the induced gene expressions were down-regulated under the combined stress, the DTT-induced ER stress and PEG-induced water deficit in comparison with the singular stress responses (DTT or PEG). The results indicated that the expressions of genes, related to the synthesis of some signal osmolyte compounds such as proline and sucrose can be suppressed when ER stress occurred under water deficiency in maize seedlings. The changes in the expressions of genes involved in osmolyte and ABA metabolism can be related to ER stress response as well as variations in water status.     

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.