Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis.

The transcription factor superfamily, APETALA2/ethylene response factor, is involved in plant growth and development, as well as in environmental stress responses. Here, an uncharacterized gene of this family, AtERF019, was studied in Arabidopsis thaliana under abiotic stress situations. Arabidopsis plants overexpressing AtERF019 showed a delay in flowering time of 7 days and a delay in senescence of 2 weeks when comparison with wild type plants. These plants also showed increased tolerance to water deficiency that could be explained by a lower transpiration rate, owing to their smaller stomata aperture and lower cuticle and cell wall permeability. Furthermore, using a bottom-up proteomic approach, proteins produced in response to stress, namely branched-chain-amino-acid aminotransferase 3 (BCAT3) and the zinc finger transcription factor oxidative stress 2, were only identified in plants overexpressing AtERF019. Additionally, a BCAT3 mutant was more sensitive to water-deficit stress than wild type plants. Predicted gene targets of AtERF019 were oxidative stress 2 and genes related to cell wall metabolism. These data suggest that AtERF019 could play a primary role in plant growth and development that causes an increased tolerance to water deprivation, so strengthening their chances of reproductive success.

Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints.

Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana.

AtWRKY30 belongs to a higher plant transcription factor superfamily, which responds to pathogen attack. In previous studies, the AtWRKY30 gene was found to be highly and rapidly induced in Arabidopsis thaliana leaves after oxidative stress treatment. In this study, electrophoretic mobility shift assays showed that AtWRKY30 binds with high specificity and affinity to the WRKY consensus sequence (W-box), and also to its own promoter. Analysis of the AtWRKY30 expression pattern by qPCR and using transgenic Arabidopsis lines carrying AtWRKY30 promoter-ß-glucuronidase fusions showed transcriptional activity in leaves subjected to biotic or abiotic stress. Transgenic Arabidopsis plants constitutively overexpressing AtWRKY30 (35S::W30 lines) were more tolerant than wild-type plants to oxidative and salinity stresses during seed germination. The results presented here show that AtWRKY30 is responsive to several stress conditions either from abiotic or biotic origin, suggesting that AtWRKY30 could have a role in the activation of defence responses at early stages of Arabidopsis growth by binding to W-boxes found in promoters of many stress/developmentally regulated genes.

Contribution of a harpin protein from Xanthomonas axonopodis pv. citri to pathogen virulence.

Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citrus canker, contains a gene in the hrp [for hypersensitive response (HR) and pathogenicity] cluster that encodes a harpin protein called Hpa1. Hpa1 produced HR in the nonhost plants tobacco, pepper and Arabidopsis, whereas, in the host plant citrus, it elicited a weak defence response with no visible phenotype. Co-infiltrations of Xac with or without the recombinant Hpa1 protein in citrus leaves produced a larger number of cankers in the presence of the protein. To characterize the effect of Hpa1 during the disease, an XacΔhpa1 mutant was constructed, and infiltration of this mutant caused a smaller number of cankers. In addition, the lack of Hpa1 hindered bacterial aggregation both in solution and in planta. Analysis of citrus leaves infiltrated with Hpa1 revealed alterations in mesophyll morphology caused by the presence of cavitations and crystal idioblasts, suggesting the binding of the harpin to plant membranes and the elicitation of signalling cascades. Overall, these results suggest that, even though Hpa1 elicits the defence response in nonhost plants and, to a lesser extent, in host plants, its main roles in citrus canker are to alter leaf mesophyll structure and to aggregate bacterial cells, and thus increase virulence and pathogen fitness. We expressed the N-terminal and C-terminal regions and found that, although both regions elicited HR in nonhost plants, only the N-terminal region showed increased virulence and bacterial aggregation, supporting the role of this region of the protein as the main active domain.

Generation of hydrogen peroxide in chloroplasts of Arabidopsis overexpressing glycolate oxidase as an inducible system to study oxidative stress.

Arabidopsis (Arabidopsis thaliana) overexpressing glycolate oxidase (GO) in chloroplasts accumulates both hydrogen peroxide (H(2)O(2)) and glyoxylate. GO-overexpressing lines (GO plants) grown at 75 micromol quanta m(-2) s(-1) show retarded development, yellowish rosettes, and impaired photosynthetic performance, while at 30 micromol quanta m(-2) s(-1), this phenotype virtually disappears. The GO plants develop oxidative stress lesions under photorespiratory conditions but grow like wild-type plants under nonphotorespiratory conditions. GO plants coexpressing enzymes that further metabolize glyoxylate but still accumulate H(2)O(2) show all features of the GO phenotype, indicating that H(2)O(2) is responsible for the GO phenotype. The GO plants can complete their life cycle, showing that they are able to adapt to the stress conditions imposed by the accumulation of H(2)O(2) during the light period. Moreover, the data demonstrate that a response to oxidative stress is installed, with increased expression and/or activity of known oxidative stress-responsive components. Hence, the GO plants are an ideal noninvasive model system in which to study the effects of H(2)O(2) directly in the chloroplasts, because H(2)O(2) accumulation is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions.

Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes.

The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxide-responsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAAAAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H(2)O(2). Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves.

Investigating the role of plant heat shock proteins during oxidative stress.

Oxidative stress, arising from an imbalance in the generation and removal of reactive oxygen species (ROS), is a challenge faced by all aerobic organisms. In plants, different pathways sense ROS from extracellular sources or organelles such as mitochondria, chloroplast or peroxisome. In our recent paper on Plant Molecular Biology1 we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. Transcript profile analysis revealed that the expression level of various genes encoding heat shock proteins (Hsps), increased after a short term of oxidative stress treatment. Furthermore, there was an induction of heat shock transcription factors HsfA2 and HsfA4A that were reported to be regulators of genes involved in stress response of Arabidopsis.1,2In this addendum, we complement the expression analysis of two Hsp genes encoding Hsp70 and a 17.6 kDa class I small heat-shock protein (sHsp), and discuss their plausible role during oxidative stress, considering our data and other recently published papers.

Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom).

Tomato (Solanum lycopersicum) fruit ripening implies that chloroplastic proteins are degraded and new proteins are synthesized. Supplementary nutrition is frequently required when tomato plants begin to fruit and continues until the end of the plant's life cycle. Ammonium assimilation is crucial in these fruit maturation and ripening processes. Glutamine synthetase (GS; EC 6.3.1.2), the main ammonium-fixing enzyme in plants, could not be detected in red fruits of several tomato varieties when growing under standard nutrition. In this paper, we analyze the influence of the nutritional status on the ammonium assimilation capacity of ripe tomato (cv. Micro-Tom) fruit. For this purpose, GS expression and protein profiles were followed in mature green and red fruits harvested from plants grown under standard or supplemented nutrition. Under standard nutrient regime (weekly supplied with 0.5 x Hoagland solution) GS activity was found in chloroplasts (GS2) of mature green fruits, but it was not detected either in the chromoplasts or in the cytosol of red fruits. When plants were shifted to a supplemented nutritional regime (daily supplied with 0.5 x Hoagland solution), GS was found in red fruits. Also, cytosolic transcripts (gs1) preferentially accumulated in red fruits under high nutrition. These results indicate that mature green Micro-Tom fruits assimilate ammonia through GS2 under standard nutrition, while ripe red fruits accumulate GS1 under high nutrition, probably in order to assimilate the extra N-compounds made available through supplemented nutrition.