Hak Soo Seo

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.

LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1

Far-red light regulates many aspects of seedling development, such as inhibition of hypocotyl elongation and the promotion of greening, acting in part through phytochrome A (phyA). The RING motif protein COP1 is also important because cop1 mutants exhibit constitutive photomorphogenesis in darkness. COP1 is present in the nucleus in darkness but is gradually relocated to the cytoplasm upon illumination. Here we show that COP1 functions as an E3 ligase ubiquitinating both itself and the myb transcription activator LAF1, which is required for complete phyA responses. In transgenic plants, inducible COP1 overexpression leads to a decrease in LAF1 concentrations, but is blocked by the proteasome inhibitor MG132. The coiled-coil domain of SPA1, a negative regulator of phyA signalling, has no effect on COP1 auto-ubiquitination but facilitates LAF1 ubiquitination at low COP1 concentrations. These results indicate that, in darkness, COP1 functions as a repressor of photomorphogenesis by promoting the ubiquitin-mediated proteolysis of a subset of positive regulators, including LAF1. After the activation of phyA, SPA1 stimulates the E3 activity of residual nuclear COP1 to ubiquitinate LAF1, thereby desensitizing phyA signals.

The Senescence-Induced Staygreen Protein Regulates Chlorophyll Degradation

Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase–impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

This work identifies COP1 as the ubiquitin E3 ligase for not only phytochrome B but also other members of the stable phytochrome family and shows that PIF transcription factors enhance phyB ubiquitination by COP1 in vitro. It provides a molecular mechanism for the termination of red light signal transduction. Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.

Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg2+ into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.

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.

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

ABSTRACT To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The Km values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, thekcat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (kcat/Km). The Km and kcat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-d-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1

Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis.

SPL28 encodes a clathrin‐associated adaptor protein complex 1, medium subunit μ1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa)

To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3′-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T1 plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.