Hyeong Cheol Park

Calmodulin interacts with MLO protein to regulate defence against mildew in barley.

In plants, defence against specific isolates of a pathogen can be triggered by the presence of a corresponding race-specific resistance gene, whereas resistance of a more broad-spectrum nature can result from recessive, presumably loss-of-regulatory-function, mutations. An example of the latter are mlo mutations in barley, which have been successful in agriculture for the control of powdery mildew fungus (Blumeria graminis f. sp. hordei; Bgh). MLO protein resides in the plasma membrane, has seven transmembrane domains, and is the prototype of a sequence-diversified family unique to plants, reminiscent of the seven-transmembrane receptors in fungi and animals. In animals, these are known as G-protein-coupled receptors and exist in three main families, lacking sequence similarity, that are thought to be an example of molecular convergence. MLO seems to function independently of heterotrimeric G proteins. We have identified a domain in MLO that mediates a Ca2+-dependent interaction with calmodulin in vitro. Loss of calmodulin binding halves the ability of MLO to negatively regulate defence against powdery mildew in vivo. We propose a sensor role for MLO in the modulation of defence reactions.

Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor

The Ca2+-binding protein calmodulin mediates cellular Ca2+ signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions −858 and −728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.

Variation in Molybdenum Content Across Broadly Distributed Populations of Arabidopsis thaliana Is Controlled by a Mitochondrial Molybdenum Transporter (MOT1)

Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping.

SIZ1 Small Ubiquitin-Like Modifier E3 Ligase Facilitates Basal Thermotolerance in Arabidopsis Independent of Salicylic Acid

Small ubiquitin-like modifier (SUMO) conjugation/deconjugation to heat shock transcription factors regulates DNA binding of the peptides and activation of heat shock protein gene expression that modulates thermal adaptation in metazoans. SIZ1 is a SUMO E3 ligase that facilitates SUMO conjugation to substrate target proteins (sumoylation) in Arabidopsis (Arabidopsis thaliana). siz1 T-DNA insertional mutations (siz1-2 and siz1-3; Miura et al., 2005) cause basal, but not acquired, thermosensitivity that occurs in conjunction with hyperaccumulation of salicylic acid (SA). NahG encodes a salicylate hydroxylase, and expression in siz1-2 seedlings reduces endogenous SA accumulation to that of wild-type levels and further increases thermosensitivity. High temperature induces SUMO1/2 conjugation to peptides in wild type but to a substantially lesser degree in siz1 mutants. However, heat shock-induced expression of genes, including heat shock proteins, ascorbate peroxidase 1 and 2, is similar in siz1 and wild-type seedlings. Together, these results indicate that SIZ1 and, by inference, sumoylation facilitate basal thermotolerance through processes that are SA independent.

Identification of rice blast fungal elicitor-responsive genes by differential display analysis.

In order to study molecular interactions that occur between rice and rice blast fungus upon infection, we isolated fungal elicitor-responsive genes from rice (Oryza sativa cv. Milyang 117) suspension-cultured cells treated with fungal elicitor prepared from the rice blast fungus (Magnaporthe grisea) employing a method that combined mRNA differential display and cDNA library screening. Data base searches with the isolated cDNA clones revealed that the OsERG1 and OsERG2 cDNAs share significant similarities with the mammalian Ca2+-dependent lipid binding (C2) domains. The OsCPX1 cDNA is highly homologous to peroxidases. The OsHin1 cDNA exhibits homology to the tobacco hin1 gene, whose expression is induced by avirulent pathogens. The OsLPL1 and OsMEK1 cDNAs share homologies with lysophospholipases and serine/threonine mitogen-activated protein (MAP) kinase kinases, respectively. The OsWRKY1 and OsEREBP1 cDNAs are homologous to transcription factors, such as the WRKY protein family and the AP2/EREBP family, respectively. Transcripts of the OsERG1, OsHin1, and OsMEK1 genes were specifically elevated only in response to the avirulent race KJ301 of the rice blast fungus. Our study yielded a number of elicitor-responsive genes that will not only provide molecular probes, but also contribute to our understanding of host defense mechanisms against the rice blast fungus.

Comparative proteomic analysis of the short-term responses of rice roots and leaves to cadmium.

Cadmium (Cd) is a non-essential heavy metal that is recognized as a major environmental pollutant. While Cd responses and toxicities in some plant species have been well established, there are few reports about the effects of short-term exposure to Cd on rice, a model monocotyledonous plant, at the proteome level. To investigate the effect of Cd in rice, we monitored the influence of Cd exposure on root and leaf proteomes. After Cd treatment, root and leaf tissues were separately collected and leaf proteins were fractionated with polyethylene glycol. Differentially regulated proteins were selected after image analysis and identified using MALDI-TOF MS. A total of 36 proteins were up- or down-regulated following Cd treatment. As expected, total glutathione levels were significantly decreased in Cd-treated roots, and approximately half of the up-regulated proteins in roots were involved in responses to oxidative stress. These results suggested that prompt antioxidative responses might be necessary for the reduction of Cd-induced oxidative stress in roots but not in leaves. In addition, RNA gel blot analysis showed that the proteins identified in the proteomic analysis were also differentially regulated at the transcriptional level. Collectively, our study provides insights into the integrated molecular mechanisms of early responses to Cd in rice.

Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species

Cadmium (Cd) is a non-essential toxic heavy metal that influences normal growth and development of plants. However, the molecular mechanisms by which plants recognize and respond to Cd remain poorly understood. We show that, in Arabidopsis, Cd activates the mitogen-activated protein kinases, MPK3 and MPK6, in a dose-dependent manner. Following treatment with Cd, these two MAPKs exhibited much higher activity in the roots than in the leaves, and pre-treatment with the reactive oxygen species (ROS) scavenger, glutathione, effectively inhibited their activation. These results suggest that the Cd sensing signaling pathway uses a build-up of ROS to trigger activation of Arabidopsis MPK3 and MPK6.

Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis

Environmental challenges to plants typically entail retardation of vegetative growth and delay or cessation of flowering. Here we report a link between the flowering time regulator, GIGANTEA (GI), and adaptation to salt stress that is mechanistically based on GI degradation under saline conditions, thus retarding flowering. GI, a switch in photoperiodicity and circadian clock control, and the SNF1-related protein kinase SOS2 functionally interact. In the absence of stress, the GI:SOS2 complex prevents SOS2-based activation of SOS1, the major plant Na(+)/H(+)-antiporter mediating adaptation to salinity. GI overexpressing, rapidly flowering, plants show enhanced salt sensitivity, whereas gi mutants exhibit enhanced salt tolerance and delayed flowering. Salt-induced degradation of GI confers salt tolerance by the release of the SOS2 kinase. The GI-SOS2 interaction introduces a higher order regulatory circuit that can explain in molecular terms, the long observed connection between floral transition and adaptive environmental stress tolerance in Arabidopsis.

Identification of a Calmodulin-Regulated Soybean Ca2+-ATPase (SCA1) That Is Located in the Plasma Membrane

Ca2+-ATPases are key regulators of Ca2+ ion efflux in all eukaryotes. Animal cells have two distinct families of Ca2+ pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non–plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca2+-ATPase 1), that encodes Ca2+-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca2+-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K1/2 ∼10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca2+ pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane–localized Ca2+ pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.

Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage

We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.