Yang Do Choi

Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant “resurrection plants,” trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3–10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mg/g fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.

Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses

Methyl jasmonate is a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses. We have cloned the novel gene JMT encoding an S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Arabidopsis thaliana. Recombinant JMT protein expressed in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with Km value of 38.5 μM. JMT RNA was not detected in young seedlings but was detected in rosettes, cauline leaves, and developing flowers. In addition, expression of the gene was induced both locally and systemically by wounding or methyl jasmonate treatment. This result suggests that JMT can perceive and respond to local and systemic signals generated by external stimuli, and that the signals may include methyl jasmonate itself. Transgenic Arabidopsis overexpressing JMT had a 3-fold elevated level of endogenous methyl jasmonate without altering jasmonic acid content. The transgenic plants exhibited constitutive expression of jasmonate-responsive genes, including VSP and PDF1.2. Furthermore, the transgenic plants showed enhanced level of resistance against the virulent fungus Botrytis cinerea. Thus, our data suggest that the jasmonic acid carboxyl methyltransferase is a key enzyme for jasmonate-regulated plant responses. Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and interplant communications.

Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions.

Drought poses a serious threat to the sustainability of rice (Oryza sativa) yields in rain-fed agriculture. Here, we report the results of a functional genomics approach that identified a rice NAC (an acronym for NAM [No Apical Meristem], ATAF1-2, and CUC2 [Cup-Shaped Cotyledon]) domain gene, OsNAC10, which improved performance of transgenic rice plants under field drought conditions. Of the 140 OsNAC genes predicted in rice, 18 were identified to be induced by stress conditions. Phylogenic analysis of the 18 OsNAC genes revealed the presence of three subgroups with distinct signature motifs. A group of OsNAC genes were prescreened for enhanced stress tolerance when overexpressed in rice. OsNAC10, one of the effective members selected from prescreening, is expressed predominantly in roots and panicles and induced by drought, high salinity, and abscisic acid. Overexpression of OsNAC10 in rice under the control of the constitutive promoter GOS2 and the root-specific promoter RCc3 increased the plant tolerance to drought, high salinity, and low temperature at the vegetative stage. More importantly, the RCc3:OsNAC10 plants showed significantly enhanced drought tolerance at the reproductive stage, increasing grain yield by 25% to 42% and by 5% to 14% over controls in the field under drought and normal conditions, respectively. Grain yield of GOS2:OsNAC10 plants in the field, in contrast, remained similar to that of controls under both normal and drought conditions. These differences in performance under field drought conditions reflect the differences in expression of OsNAC10-dependent target genes in roots as well as in leaves of the two transgenic plants, as revealed by microarray analyses. Root diameter of the RCc3:OsNAC10 plants was thicker by 1.25-fold than that of the GOS2:OsNAC10 and nontransgenic plants due to the enlarged stele, cortex, and epidermis. Overall, our results demonstrated that root-specific overexpression of OsNAC10 enlarges roots, enhancing drought tolerance of transgenic plants, which increases grain yield significantly under field drought conditions.

Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis

AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 transcript accumulation within 30 min. The gene was also activated under various abiotic stresses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an AtMYB44 promoter-driven β-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more sensitive to ABA and has a more rapid ABA-induced stomatal closure response than wild-type and atmyb44 knockout plants. Transgenic plants exhibited a reduced rate of water loss, as measured by the fresh-weight loss of detached shoots, and remarkably enhanced tolerance to drought and salt stress compared to wild-type plants. Microarray analysis and northern blots revealed that salt-induced activation of the genes that encode a group of serine/threonine protein phosphatases 2C (PP2Cs), such as ABI1, ABI2, AtPP2CA, HAB1, and HAB2, was diminished in transgenic plants overexpressing AtMYB44. By contrast, the atmyb44 knockout mutant line exhibited enhanced salt-induced expression of PP2C-encoding genes and reduced drought/salt stress tolerance compared to wild-type plants. Therefore, enhanced abiotic stress tolerance of transgenic Arabidopsis overexpressing AtMYB44 was conferred by reduced expression of genes encoding PP2Cs, which have been described as negative regulators of ABA signaling.

Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Transgenic Rice Plants Increases Trehalose Accumulation and Abiotic Stress Tolerance without Stunting Growth

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) ofEscherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight−1. This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with eitherTPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPSand/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Methyl jasmonate as a vital substance in plants

The plant floral scent methyl jasmonate (MeJA) has been identified as a vital cellular regulator that mediates diverse developmental processes and defense responses against biotic and abiotic stresses. The pleiotropic effects of MeJA have raised numerous questions about its regulation for biogenesis and mode of action. Characterization of the gene encoding jasmonic acid carboxyl methyltransferase has provided basic information on the role(s) of this phytohormone in gene-activation control and systemic long-distance signaling. Recent approaches using functional genomics and bioinformatics have identified a whole set of MeJA-responsive genes, and provide insights into how plants use volatile signals to withstand diverse and variable environments.

OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice

Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice.

Methyl Jasmonate Reduces Grain Yield by Mediating Stress Signals to Alter Spikelet Development in Rice

Jasmonic acid (JA) is involved in plant development and the defense response. Transgenic overexpression of the Arabidopsis (Arabidopsis thaliana) jasmonic acid carboxyl methyltransferase gene (AtJMT) linked to the Ubi1 promoter increased levels of methyl jasmonate (MeJA) by 6-fold in young panicles. Grain yield was greatly reduced in Ubi1:AtJMT plants due to a lower numbers of spikelets and lower filling rates than were observed for nontransgenic (NT) controls. Ubi1:AtJMT plants had altered numbers of spikelet organs, including the lemma/palea, lodicule, anther, and pistil. The loss of grain yield and alteration in spikelet organ numbers were reproduced by treating NT plants with exogenous MeJA, indicating that increased levels of MeJA in Ubi1:AtJMT panicles inhibited spikelet development. Interestingly, MeJA levels were increased by 19-fold in young NT panicles upon exposure to drought conditions, resulting in a loss of grain yield that was similar to that observed in Ubi1:AtJMT plants. Levels of abscisic acid (ABA) were increased by 1.9- and 1.4-fold in Ubi1:AtJMT and drought-treated NT panicles, respectively. The ABA increase in Ubi1:AtJMT panicles grown in nondrought conditions suggests that MeJA, rather than drought stress, induces ABA biosynthesis under drought conditions. Using microarray and quantitative polymerase chain reaction analyses, we identified seven genes that were regulated in both Ubi1:AtJMT and drought-treated NT panicles. Two genes, OsJMT1 and OsSDR (for short-chain alcohol dehydrogenase), are involved in MeJA and ABA biosynthesis, respectively, in rice (Oryza sativa). Overall, our results suggest that plants produce MeJA during drought stress, which in turn stimulates the production of ABA, together leading to a loss of grain yield.

Expression of an Expansin Gene Is Correlated with Root Elongation in Soybean

Expansin is a family of proteins that catalyze long-term expansion of cell walls and has been considered a principal protein that affects cell expansion in plants. We have identified the first root-specific expansin gene in soybean (Glycine max),GmEXP1, which may be responsible for root elongation. Expression levels of GmEXP1 were very high in the roots of 1- to 5-d-old seedlings, in which rapid root elongation takes place. Furthermore, GmEXP1 mRNA was most abundant in the root tip region, where cell elongation occurs, but scarce in the region of maturation, where cell elongation ceases, implying that its expression is closely related to root development processes. In situ hybridization showed that GmEXP1 transcripts were preferentially present in the epidermal cells and underlying cell layers in the root tip of the primary and secondary roots. Ectopic expression ofGmEXP1 accelerated the root growth of transgenic tobacco (Nicotiana tabacum) seedlings, and the roots showed insensitivity to obstacle-touching stress. These results imply that theGmEXP1 gene plays an important role in root development in soybean, especially in the elongation and/or initiation of the primary and secondary roots.

Overexpression of salicylic acid carboxyl methyltransferase reduces salicylic acid-mediated pathogen resistance in Arabidopsis thaliana

We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomycesorontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants.