Wound- and systemin-inducible calmodulin gene expression in tomato leaves

Using a calmodulin (CaM) cDNA as a probe in northern analyses, transgenic tomato plants that overexpress the prosystemin gene were found to express increased levels of CaM mRNA and protein in leaves compared to wild-type plants. These transgenic plants have been reported previously to express several wound-inducible defense-related genes in the absence of wounding. Calmodulin mRNA and protein levels were found to increase in leaves of young wild-type tomato plants after wounding, or treatment with systemin, methyl jasmonate, or linolenic acid. CaM mRNA appeared within 0.5 h after wounding or supplying young tomato plants with systemin, and peaked at 1 h. The timing of CaM gene expression is similar to the expression of the wound- or systemin-induced lipoxygenase and prosystemin genes, signal pathway genes whose expression have been reported to begin at 0.5–1 h after wounding and 1–2 h earlier than the genes coding for defensive proteinase inhibitor genes. The similarities in timing between the synthesis of CaM mRNA and the mRNAs for signal pathway components suggests that CaM gene expression may be associated with the signaling cascade that activates defensive genes in response to wounding.     

Evidence that abscisic acid does not regulate a centralized whole-plant response to low soil-resource availability

It has been suggested that abscisic acid (ABA) regulates a centralized response of plants to low soil resource availability that is characterized by decreased shoot growth relative to root growth, decreased photosynthesis and stomatal conductance, and decreased plant growth rate. The hypothesis was tested that an ABA-deficient mutant of tomato (flacca; flc) would not exhibit the same pattern of down-regulation of photosynthesis, conductance, leaf area and growth, as well as increased root/shoot partitioning, as its near isogenic wild-type in response to nitrogen or water deficiency, or at least not exhibit these responses to the same degree. Plants were grown from seed in acid-washed sand and exposed to control, nutrient stress, or water stress treatments. Additionally, exogenous ABA was sprayed onto the leaves of a separate group of flc individuals in each treatment. Growth analysis, based on data from frequent harvests of a few individuals, was used to assess the growth and partitioning responses of plants, and gas exchange characteristics were measured on plants throughout the experiment to examine the response of photosynthesis and stomatal conductance. Differences in growth, partitioning and gas exchange variables were found between flc and wild-type individuals, and both nutrient and water treatments caused significant reductions in relative growth rate (RGR) and changes in biomass partitioning. Only the nutrient treatment caused significant reductions in photosynthetic rates. However, flc and wild-type plants responded identically to nutrient and water stress for all but one of the variables measured. The exception was that flc showed a greater decrease in the relative change in leaf area per unit increase of plant biomass (an estimate of the dynamics of leaf area ratio) in response to nutrient stress—a result that is opposite to that predicted by the centralized stress response model. Furthermore, addition of exogenous ABA to flc did not significantly alter any of the responses to nutrient and water stress that we examined. Although it was clear that ABA regulated short-term stomatal responses, we found no evidence to support a pivotal role for ABA, at least absolute amounts of ABA, in regulating a centralized whole-plant response to low soil resource availability.     

The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.     

NFX1-LIKE2 (NFXL2) suppresses abscisic acid accumulation and stomatal closure in Arabidopsis thaliana

The NFX1-LIKE1 (NFXL1) and NFXL2 genes were identified as regulators of salt stress responses. The NFXL1 protein is a nuclear factor that positively affects adaptation to salt stress. The nfxl1-1 loss-of-function mutant displayed reduced survival rates under salt and high light stress. In contrast, the nfxl2-1 mutant, defective in the NFXL2 gene, and NFXL2-antisense plants exhibited enhanced survival under these conditions. We show here that the loss of NFXL2 function results in abscisic acid (ABA) overaccumulation, reduced stomatal conductance, and enhanced survival under drought stress. The nfxl2-1 mutant displayed reduced stomatal aperture under all conditions tested. Fusicoccin treatment, exposition to increasing light intensities, and supply of decreasing CO(2) concentrations demonstrated full opening capacity of nfxl2-1 stomata. Reduced stomatal opening presumably is a consequence of elevated ABA levels. Furthermore, seedling growth, root growth, and stomatal closure were hypersensitive to exogenous ABA. The enhanced ABA responses may contribute to the improved drought stress resistance of the mutant. Three NFXL2 splice variants were cloned and named NFXL2-78, NFXL2-97, and NFXL2-100 according to the molecular weight of the putative proteins. Translational fusions to the green fluorescent protein suggest nuclear localisation of the NFXL2 proteins. Stable expression of the NFXL2-78 splice variant in nfxl2-1 plants largely complemented the mutant phenotype. Our data show that NFXL2 controls ABA levels and suppresses ABA responses. NFXL2 may prevent unnecessary and costly stress adaptation under favourable conditions.     

Salt stress activation of wound-related genes in tomato plants

Plants respond to various stresses by expressing distinct sets of genes. The effects of multiple stresses on plants and their interactions are not well understood. We have discovered that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato (Lycopersicon esculentum) plants. Salt stress was also found to enhance the plant's response to wounding locally and systemically. The tomato mutant (def-1), which has an impairment in the octadecanoid pathway, displayed a severe reduction in the accumulation of proteinase inhibitors under salt stress, indicating that salt stress-induced accumulation of proteinase inhibitors was jasmonic acid dependent. The analysis of salt stress in another tomato mutant, spr-1, which carries a mutation in a systemin-specific signaling component, and transgenic tomato plants that express an antisense-prosystemin cDNA, showed that prosystemin activity was not required for the salt-induced accumulation of proteinase inhibitors, but was necessary to achieve maximal levels. These results suggest that a prosystemin independent- but jasmonic acid-dependent pathway is utilized for proteinase inhibitor accumulation in response to salt stress.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid beta-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.     

Does root-sourced ABA have a role in mediating growth and stomatal responses to soil compaction in tomato (Lycopersicon esculentum)?

Isogenic wild-type (Ailsa Craig) and abscisic acid (ABA)-deficient mutant (flacca) genotypes of tomato were used to examine the role of root-sourced ABA in mediating growth and stomatal responses to compaction. Plants were grown in uniform soil columns providing low to moderate bulk densities (1.1–1.5 g cm^?3), or in a split-pot system, which allowed the roots to divide between soils of the same or differing bulk density (1.1/1.5 g cm^?3). Root and shoot growth and leaf expansion were reduced when plants were grown in compacted soil (1.5 g cm^?3) but leaf water status was not altered. However, stomatal conductance was affected, suggesting that non-hydraulic signal(s) transported in the transpiration stream were responsible for the observed effects. Xylem sap and foliar ABA concentrations increased with bulk density for 10 and 15 days after emergence (DAE), respectively, but were thereafter poorly correlated with the observed growth responses. Growth was reduced to a similar extent in both genotypes in compacted soil (1.5 g cm^?3), suggesting that ABA is not centrally involved in mediating growth in this severely limiting ‘critical’ compaction stress treatment. Growth performance in the 1.1/1.5 g cm^?3 split-pot treatment of Ailsa Craig was intermediate between the uniform 1.1 and 1.5 g cm^?3 treatments, whereas stomatal conductance was comparable to the compacted 1.5 g cm^?3 treatment. In contrast, shoot dry weight and leaf area in the split-pot treatment of flacca were similar to the 1.5 g cm^?3 treatment, but stomatal conductance was comparable to uncompacted control plants. These results suggest a role for root-sourced ABA in regulating growth and stomatal conductance during ‘sub-critical’ compaction stress, when genotypic differences in response are apparent. The observed genotypic differences are comparable to those previously reported for barley, but occurred at a much lower bulk density, reflecting the greater sensitivity of tomato to compaction. By alleviating the severe growth reductions induced when the entire root system encounters compacted soil, the split-pot approach has important applications for studies of the role of root-sourced signals in compaction-sensitive species such as tomato.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.     

Overexpression of <Emphasis Type="Italic">SlERF1</Emphasis> tomato gene encoding an ERF-type transcription activator enhances salt tolerance

Ethylene-responsive factors (ERFs) play an important role in plant responses to stresses. For this study, we obtained sense- and antisense-SlERF1 transgenic tomato plants to analyze the function of the SlERF1 gene in tomato plants. Overexpression of SlERF1 in tomato plants enhanced salt tolerance during tomato seedling root development. In addition, tomato seedlings overexpressing SlERF1 showed the higher relative water content and lower MDA content and electrolyte leakage; they accumulated more free proline and soluble sugars, as compared with wild-type and antisense-SlERF1 transgenic tomato plants under salt stress. Moreover, SlERF1 activated the expression of stress-related genes, including LEA, P5CS, DREB3-1, and ltpg2 in tomato plants under salt stress. Thus, SlERF1 played a positive role in the salt tolerance of tomato plants.     

Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid

The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric 'Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.     

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors () is Clarence A. Ryan.     

The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.