Jong-Joo Cheong

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.

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.

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.

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.

Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from Brevibacterium helvolum

ABSTRACT A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. RecombinantBvMTS, BvMTH, and fusion geneBvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of α-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.

Non-specific phytohormonal induction of AtMYB44 and suppression of jasmonate-responsive gene activation in Arabidopsis thaliana

The Arabidopsis thaliana transcription factor gene AtMYB44 was induced within 10 min by treatment with methyl jasmonate (MeJA). Wound-induced expression of the gene was observed in local leaves, but not in distal leaves, illustrating jasmonate-independent induction at wound sites. AtMYB44 expression was not abolished in Arabidopsis mutants insensitive to jasmonate (coi1), ethylene (etr1), or abscisic acid (abi3-1) when treated with the corresponding hormones. Moreover, various growth hormones and sugars also induced rapid AtMYB44 transcript accumulation. Thus, AtMYB44 gene activation appears to not be induced by any specific hormone. MeJA-induced activation of jasmonate-responsive genes such as JR2, VSP, LOXII, and AOS was attenuated in transgenic Arabidopsis plants overexpressing the gene (35S:AtMYB44), but significantly enhanced in atmyb44 knockout mutants. The 35S:MYB44 and atmyb44 plants did not show defectiveness in MeJA-induced primary root growth inhibition, indicating that the differences in jasmonate-responsive gene expression observed was not due to alterations in the jasmonate signaling pathway. 35S:AtMYB44 seedlings exhibited slightly elevated chlorophyll levels and less jasmonate- induced anthocyanin accumulation, demonstrating suppression of jasmonate-mediated responses and enhancement of ABA-mediated responses. These observations support the hypothesis of mutual antagonistic actions between jasmonate- and abscisic acid-mediated signaling pathways.

Expression of the Arabidopsis AtMYB44 gene confers drought/salt-stress tolerance in transgenic soybean

AtMYB44, a member of the subgroup 22 R2R3 MYB transcription factors, positively regulates abscisic acid signaling to induce stomatal closure, thus conferring drought/salt-stress tolerance in Arabidopsis thaliana. In this study, AtMYB44 was transformed into soybean [Glycine max (L.) Merrill] using the cotyledonary-node method. The resulting homozygous lines were shorter than the non-transgenic controls (Bert) throughout the growth period when grown in a greenhouse. The transgenic soybeans exhibited significantly enhanced drought/salt-stress tolerance, as observed in Arabidopsis. In field cultivation studies, the transgenic soybean plants showed reduced growth, but much higher yields upon seed harvest, demonstrating improved environmental stress tolerance. The amino acid and fatty acid compositions were not significantly altered in seeds harvested from the transgenic lines. These results suggest that the interaction of AtMYB44 with specific sequences in target gene promoters and/or specific proteins activates a tolerance mechanism that is conserved in Arabidopsis and soybean.

AtCPL5, a novel Ser‐2‐specific RNA polymerase II C‐terminal domain phosphatase, positively regulates ABA and drought responses in Arabidopsis

Arabidopsis RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatases regulate stress-responsive gene expression and plant development via the dephosphorylation of serine (Ser) residues of the CTD. Some of these phosphatases (CTD phosphatase-like 1 (CPL1) to CPL3) negatively regulate ABA and stress responses. Here, we isolated AtCPL5, a cDNA encoding a protein containing two CTD phosphatase domains (CPDs). To characterize AtCPL5, we analyzed the gene expression patterns and subcellular protein localization, investigated various phenotypes of AtCPL5-overexpressors and knockout mutants involved in ABA and drought responses, performed microarray and RNA hybridization analyses using AtCPL5-overexpressors, and assessed the CTD phosphatase activities of the purified AtCPL5 and each CPD of the protein. Transcripts of the nucleus-localized AtCPL5 were induced by ABA and drought. AtCPL5-overexpressors exhibited ABA-hypersensitive phenotypes (increased inhibition of seed germination, seedling growth, and stomatal aperture), lower transpiration rates upon dehydration, and enhanced drought tolerance, while the knockout mutants showed weak ABA hyposensitivity. AtCPL5 overexpression changed the expression of numerous genes, including those involved in ABA-mediated responses. In contrast to Ser-5-specific phosphatase activity of the negative stress response regulators, purified AtCPL5 and each CPD of the protein specifically dephosphorylated Ser-2 in RNAPII CTD. We conclude that AtCPL5 is a unique CPL family protein that positively regulates ABA-mediated development and drought responses in Arabidopsis.

An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography-chemical ionization-mass spectrometry.

We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.