Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Nitrate or NaCl regulates floral induction in Arabidopsis thaliana

Flowering, the transition from the vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Exposure to an extended period of stress (such as low nitrate or NaCl) can also promote flowering in many species, but little is known about how these forms of stress regulate floral induction. In this study, we found that stress induced by low concentrations of nitrate or NaCl activated the biosynthesis of gibberellin (GA) as evidenced by increased expression of the GA biosynthetic enzyme GA1. Expression of CO and SOC1 were also enhanced, leading to an acceleration of flowering. The effects of nitrate and NaCl on the photoperiod pathway were distinct, however. Two genes related to the photoperiod pathway, CCA1 and LHY, were repressed only under low NaCl treatment, while expression was unaltered by nitrate. Therefore, we suggest that the biosynthesis of gibberellin (GA) may play an important role in integrating signals induced by exogenous stress to regulate flowering in Arabidopsis.     

CbCBF from Capsella bursa-pastoris enhances cold tolerance and restrains growth in Nicotiana tabacum by antagonizing with gibberellin and affecting cell cycle signaling

Plant cells respond to cold stress via a regulatory mechanism leading to enhanced cold acclimation accompanied by growth retardation. The C-repeat binding factor (CBF) signaling pathway is essential for cold response of flowering plants. Our previously study documented a novel CBF-like gene from the cold-tolerant Capsella bursa-pastoris named CbCBF, which was responsive to chilling temperatures. Here, we show that CbCBF expression is obviously responsive to chilling, freezing, abscisic acid, gibberellic acid (GA), indoleacetic acid or methyl jasmonate treatments and that the CbCBF:GFP fusion protein was localized to the nucleus. In addition, CbCBF overexpression conferred to the cold-sensitive tobacco plants enhanced tolerance to chilling and freezing, as well as dwarfism and delayed flowering. The leaf cells of CbCBF overexpression tobacco lines attained smaller sizes and underwent delayed cell division with reduced expression of cyclin D genes. The dwarfism of CbCBF transformants can be partially restored by GA application. Consistently, CbCBF overexpression reduced the bioactive gibberellin contents and disturbed the expression of gibberellin metabolic genes in tobacco. Meanwhile, cold induced CbCBF expression and cold tolerance in C. bursa-pastoris are reduced by GA. We conclude that CbCBF confers cold resistance and growth inhibition to tobacco cells by interacting with gibberellin and cell cycle pathways, likely through activation of downstream target genes.     

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice

Two benzenoid esters, methyl salicylate (MeSA) and methyl benzoate (MeBA), were detected from insect-damaged rice plants. By correlating metabolite production with gene expression analysis, five candidate genes encoding putative carboxyl methyltransferases were identified. Enzymatic assays with Escherichia coli-expressed recombinant proteins demonstrated that only one of the five candidates, OsBSMT1, has salicylic acid (SA) methyltransferase (SAMT) and benzoic acid (BA) methyltransferase (BAMT) activities for producing MeSA and MeBA, respectively. Whereas OsBSMT1 is phylogenetically relatively distant from dicot SAMTs, the three-dimensional structure of OsBSMT1, which was determined using homology-based structural modeling, is highly similar to those of characterized SAMTs. Analyses of OsBSMT1 expression in wild-type rice plants under various stress conditions indicate that the jasmonic acid (JA) signaling pathway plays a critical role in regulating the production and emission of MeSA in rice. Further analysis using transgenic rice plants overexpressing NH1, a key component of the SA signaling pathway in rice, suggests that the SA signaling pathway also plays an important role in governing OsBSMT1 expression and emission of its products, probably through a crosstalk with the JA signaling pathway. The role of the volatile products of OsBSMT1, MeSA and MeBA, in rice defense against insect herbivory is discussed.     

Isolation and expression profiles of gibberellin metabolism genes in developing male and female cones of Pinus tabuliformis

Gibberellins (GAs) are important in the floral regulatory networks of angiosperm plants. Several lines of evidence suggest that GAs also play a pivotal role in conifer male and female cone development. To gain new insights into the GA metabolism pathway in conifer trees and the role of GA metabolism in male and female cone development, we identified GA metabolism genes in Pinus tabuliformis. These included one PtCPS gene, one PtKS gene, one PtKO gene, TWO PtKAO genes, one PtGA20ox gene, two PtGA3ox genes and 12 PtGA2ox genes. According to phylogenetic analysis, the GA biosynthesis pathway evolved after the divergence of mosses from ferns, but the GA-deactivating gene family underwent divided expansion after divergence of the angiosperms from gymnosperms. However, the active sites of all GA metabolism enzymes were conserved during the evolution of land plants. During male and female cone development of P. tabuliformis, the expression of most of the PtGA2ox genes, especially PtGA2ox10, was higher than GA biosynthesis genes. However, the expression of PtKAO1 in cones peaked at a very early developmental stage. The expression pattern of GA metabolism genes indicated that GAs play different roles at the early and late stages of cone development.     

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Drought resistance is increased in plants by the absence of the hormone gibberellic acid (GA) or by a lack of GA sensitivity. We studied the effects of tissue-specific reduction in GA levels on drought tolerance, on recovery from drought stress, and on primary and secondary growth using transgenic tobacco plants expressing the GA-inactivating gene PtGA2ox ₁ (GA 2-oxidase) specifically in leaves, stems, or roots. Localized reduction of bioactive GA₁ levels was achieved by tissue-specific expression of the PtGA2ox ₁ gene in leaves using the rbcs promoter (LD plants), in roots using the TobRB7 promoter (RD plants), and in stems using the LMX5 promoter (SD plants). In response to drought stress, all transgenic tobacco plants exhibited reduced primary and secondary growth and increased drought tolerance with a corresponding reduction in malondialdehyde levels, higher relative water content, increased proline and sugar content, and elevated peroxidase, superoxide dismutase, and catalase activities relative to wild-type plants. The highest level of drought tolerance and the most rapid recovery from stress was achieved by localized reduction of GA₁ in the roots of the RD transgenic plants. In addition, although the total bioactive GA₁ content in RD and LD plants was essentially identical, the heights of LD plants were significantly greater and drought tolerance was significantly less than in RD plants. It is possible that the site of gibberellin-related gene expression plays an important role in the balance between growth and drought tolerance.     

Overexpression of GASA5 increases the sensitivity of Arabidopsis to heat stress

Basal thermotolerance is very important for plant growth and development when plants are subjected to heat stress. However, little is known about the functional mechanism of gibberellins (GAs) in the basal thermotolerance of plants. In the present work, we provide molecular evidence that a member of the gene family encoding the GA-stimulated Arabidopsis (GASA) peptides, namely GASA5, is involved in the regulation of seedling thermotolerance. The GASA5-overexpressing plants displayed a weak thermotolerance, with a faster cotyledon-yellowing rate, lower seedling-survival rate, and slower hypocotyl elongation, in comparison to the wild-type and GASA5 null-mutant (gasa5-1) plants, after heat-stress treatment. The short-hypocotyl phenotype of GASA5-overexpressing plants could be rescued by the exogenous application of salicylic acid (SA), the hormone found to protect plants from heat stress-induced damage. GASA5 expression was inhibited by heat stress but unaffected by the application of exogenous SA. However, expression of the gene encoding the noexpresser of PR genes 1 (NPR1), a key component of the SA-signaling pathway, was downregulated by GASA5 overexpression. Importantly, when different GASA5-genotype plants were treated with heat stress, several genes encoding heat-shock proteins, including HSP101, HSP70B, HSP90.1, HSP17.6-C1, and HSP60, were inhibited by GASA5 overexpression. Meanwhile, hydrogen peroxide was accumulated at high levels in heat stress-treated GASA5-overexpressing plants. These results suggest that the Arabidopsis GASA5 gene acts as a negative regulator in thermotolerance by regulating both SA signaling and heat shock-protein accumulation.