NaCI Reduces Indole-3-Acetic Acid Levels in the Roots of Tomato Plants Independent of Stress-Induced Abscisic Acid

Indole-3-acetic acid (IAA) was measured in leaves and roots of tomato (Lycopersicon esculentum) genotypes subjected to salt stress. An abscisic acid (ABA)-deficient mutant of tomato (sitiens), the genetic parent (Rheinlands Ruhm, RR), and a commercial variety (Large Cherry Red, LCR) of tomato were treated with 50 to 300 mM NaCl in nutrient culture. Both LCR and RR had significantly higher levels of IAA in the roots compared with that in sitiens prior to treatment. The initial levels of IAA in the roots of LCR and RR declined by nearly 75% after exposure to NaCl, whereas those in roots from the sitiens mutant remained unchanged. IAA levels in the leaves of all genotypes remained unchanged or increased slightly in response to NaCl. ABA was highest in leaves from the normal genotypes after exposure to NaCl. ABA levels in the roots of sitiens were similar to the levels in the normal genotypes, whereas levels in the leaves were only 10% of the levels found in normal genotypes regardless of the salt treatment. Treatment of LCR and sitiens with exogenous ABA increased the ABA levels in leaves and roots, but there were no measurable changes in endogenous IAA. Therefore, the reduction in IAA appears to result from an ABA-independent effect of NaCl on IAA metabolism in the roots of stressed plants.     

The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA

Abstract. Deuterium-labelled ABA-aldehyde was fed to various tomato genotypes. Normal and notabilis mutant plants incorporated substantial amounts of the label into ABA. In contrast, two ABA-deficient mutants, flacca and sitiens , reduced ABA-aldehyde to a mixture of cis- and trans -ABA alcohol rather than oxidizing it to ABA. It was concluded that ABA-aldehyde is the immediate precursor of ABA in higher plants. It appears that the flacca and sitiens lesions both act to block the last step of the ABA biosynthetic pathway. The mutant gene loci are likely to be involved in coding for different sub-units of the same dehydrogenase enzyme.     

Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying

The hypothesis that ABA produced by roots in drying soil is responsible for stomatal closure was tested with grafted plants constructed from the ABA-deficient tomato mutants, sitiens and flacca and their near-isogenic wild-type parent. Three types of experiments were conducted. In the first type, reciprocal grafts were made between the wild type and sitiens or flacca. Stomatal conductance accorded with the genotype of the shoot, not the root. Stomates closed in all of the grafted plants in response to soil drying, regardless of the root genotype, i.e. regardless of the ability of the roots to produce ABA. In the second type of experiment, wild-type shoots were grafted onto a split-root system consisting of one wild-type root grafted to one mutant (flacca or sitiens) root. Water was withheld from one root system, while the other was watered well so that the shoots did not experience any decline in water potential or loss of turgor. Stomates closed to a similar extent when water was withheld from the mutant roots or the wild-type roots. In the third type of experiment, grafted plants with wild-type shoots and either wild-type or sitiens roots were established in pots that could be placed inside a pressure chamber, and the pressure increased as the soil dried so that the shoots remained fully turgid throughout. Stomates closed as the soil dried, regardless of whether the roots were wild type or sitiens. These experiments demonstrate that stomatal closure in response to soil drying can occur in the absence of leaf water deficit, and does not require ABA production by roots. A chemical signal from roots leading to a change in apoplastic ABA levels in leaves may be responsible for the stomatal closure.     

Abscisic acid determines basal susceptibility of tomato to Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms

Abscisic acid (ABA) is one of the plant hormones involved in the interaction between plants and pathogens. In this work, we show that tomato (Lycopersicon esculentum Mill. cv Moneymaker) mutants with reduced ABA levels (sitiens plants) are much more resistant to the necrotrophic fungus Botrytis cinerea than wild-type (WT) plants. Exogenous application of ABA restored susceptibility to B. cinerea in sitiens plants and increased susceptibility in WT plants. These results indicate that ABA plays a major role in the susceptibility of tomato to B. cinerea. ABA appeared to interact with a functional plant defense response against B. cinerea. Experiments with transgenic NahG tomato plants and benzo(1,2,3)thiadiazole-7-carbothioic acid demonstrated the importance of salicylic acid in the tomato-B. cinerea interaction. In addition, upon infection with B. cinerea, sitiens plants showed a clear increase in phenylalanine ammonia lyase activity, which was not observed in infected WT plants, indicating that the ABA levels in healthy WT tomato plants partly repress phenylalanine ammonia lyase activity. In addition, sitiens plants became more sensitive to benzo(1,2,3)thiadiazole-7-carbothioic acid root treatment. The threshold values for PR1a gene expression declined with a factor 10 to 100 in sitiens compared with WT plants. Thus, ABA appears to negatively modulate the salicylic acid-dependent defense pathway in tomato, which may be one of the mechanisms by which ABA levels determine susceptibility to B. cinerea.     

Osmotin gene expression is controlled by elicitor synergism

Arachidonic acid (AA), a fatty-acid fungal elicitor, and a cellulase preparation from Aspergillus niger , a protein-type fungal elicitor, induced osmotin gene expression. Both elicitors activated the osmotin promoter fused to a β-glucuronidase (GUS) reporter gene in a tissue-specific manner in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38). The cellulase preparation was more effective than AA at the concentrations tested and, unlike AA, also induced the accumulation of osmotin mRNA and protein. Combinations of AA and the cellulase preparation had a greater than additive effect on the activation of the osmotin promoter and the accumulation of osmotin mRNA and protein. Both AA and the cellulase preparation, when applied separately, were virtually ineffective in the induction of the osmotin promoter in cotyledon tissues. However, together they were able to induce synergistically GUS fused to the osmotin promoter. Increases in osmotin-promoter-driven GUS activity and accumulation of osmotin mRNA induced by AA, the cellulase preparation or their combination were reversed by norbornadiene, an ethylene action inhibitor, indicating that ethylene is involved in the induction of the osmotin gene by these elicitors.     

Growth of tomato and an ABA-deficient mutant (sitiens) under saline conditions

To determine whether ABA accumulation inhibits or promotes shoot growth under stress, an ABA-deficient mutant tomato, sitiens, and its wild-type, the cultivar Rheinlands Rhum, were exposed to moderate salinity stress. Plants were grown at 75 m M NaCl for 2 weeks under conditions of moderate or high relative humidity (70% and 95% RH, respectively). At 70% RH, shoot DW and relative growth rate were reduced more in sitiens than in the cultivar, but the major difference between genotypes was in the degree of injury suffered by older leaves. Most leaves of sitiens died after 2 weeks, but those of the cultivar remained alive. When plants were grown at 95% RH, to maximize the leaf water status of both genotypes, there was no significant effect of salt on shoot DW of either genotype. However, there was still considerable leaf death in sitiens whereas no visible injury appeared in the cultivar. Cl^– accumulated to higher levels in leaf tissues than Na⁺, but to similar concentrations in both genotypes, and so could not explain the injury in the sitiens leaves. The results indicate that ABA maintains rather than inhibits new growth under stress, and has a major effect on preservation of older leaves.     

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.     

Abscisic Acid and C10 Dicarboxylic Acids in Wilty Tomato Mutants

Linforth, R. S. T., Taylor, I. B. and Hedden, P. 1987. Abscisicacid and C10 dicarboxylic acids in wilty tomato mutants.—J.exp. Bot. 38: 1734–1740. The concentration of C₁₀ dicarboxylic acids in wilty tomatomutants was investigated. Three of the genotypes studied (flacca,sitiens and the double mutant homozygote flacca/sitiens) werefound to have higher concentrations of 2,7-dimethyl-2,4-octadienedioicacid (ODA) than the isogenic normal form. In contrast, the othergenotypes (notabilisand the double mutant homozygotes notabilis/flaccaand notabilis/sitiens) were found to have lower concentrationsof ODA than the isogenic normal form. The concentration of ODAin flacca plants was increased by water stress and reduced byexogenously applied abscisic acid (ABA). A second structurallyrelated compound, 2,7-dimethyl-4-octenedioic acid (OEA) wasalso quantified, but it showed no clear genotype-related pattern. The concentration of ABA in the wilty tomato mutants was alsoinvestigated. As expected in the light of previously publishedresults, it was reduced in the single mutants relative to theisogenic control plants. In the double mutant flacca/sitiensABA levels were similar to those of the single mutant sitiens.However, in the two double mutants notabilis/flacca and notabilis/sitiensABA was substantially lower than those in any other genotypeinvestigated. Key words: Abscisic acid, 2,7-dimethyl-2,4-octadienedioic acid, 2,7-dimethyl-4-octenedioc acid, tomato, wilty mutants     

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.     

Ectopic expression of a conifer <Emphasis Type="Italic">Abscisic Acid Insensitive3</Emphasis> transcription factor induces high-level synthesis of recombinant human α-<Emphasis Type="SmallCaps">l</Emphasis>-iduronidase in transgenic tobacco leaves

We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-L-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 microM S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs.