Sung Han Ok

Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco

The lipid signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. To investigate the biological functions of pepper lipid transfer protein (LTP), we carried out virus-induced gene silencing (VIGS) in pepper, constitutive expression of CALTPs and grafting experiments in the tobacco plant. Suppression of endogenous CALTPI and CALTPII by VIGS, respectively, resulted in enhanced susceptibility to Xanthomonas campestris pv. vescatoria and pepper mosaic mottle virus in pepper. On the other hand, the constitutive expression of CALTPI and CALTPII genes in tobacco plants showed enhanced resistance to oomycete pathogen, Phytophthora nicotianae and bacterial pathogen, Pseudomonas syringae pv. tabaci. Enhanced resistance is found to be associated with the enhanced CALTP transcript levels in the independent transgenic CALTPI or II tobacco lines. Induced resistance responses in grafted scion leaves revealed that LTP plays a role in long-distance systemic signaling in plants.

An Arabidopsis R2R3-MYB transcription factor, AtMYB20, negatively regulates type 2C serine/threonine protein phosphatases to enhance salt tolerance

We have characterized the function of a plant R2R3‐MYB transcription factor, Arabidopsis thaliana MYB20 (AtMYB20). Transgenic plants overexpressing AtMYB20 (AtMYB20‐OX) enhanced salt stress tolerance while repression lines (AtMYB20‐SRDX) were more vulnerable to NaCl than wild‐type plants. Following NaCl treatment, the expressions of ABI1, ABI2 and AtPP2CA, which encode type 2C serine/threonine protein phosphatases (PP2Cs) that act as negative regulators in abscisic acid (ABA) signaling, were suppressed in AtMYB20‐OX but induced in AtMYB20‐SRDX. The electrophoretic mobility shift assay results revealed that AtMYB20 binds to the promoter regions containing the MYB recognition sequence (TAACTG) and an ACGT core element of ABI1 and AtPP2CA. These findings suggest that AtMYB20 down‐regulates the expression of PP2Cs, the negative regulator of ABA signaling, and enhances salt tolerance.

OVEREXPRESSION OF HUMAN ERYTHROPOIETIN (EPO) AFFECTS PLANT MORPHOLOGIES: RETARDED VEGETATIVE GROWTH IN TOBACCO AND MALE STERILITY IN TOBACCO AND ARABIDOPSIS

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsisvia Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis

Characterization of the full-length sequences of phospholipase A2 induced during flower development

The suppression subtractive hybridization (SSH) method was used to isolate developmentally regulated genes during carnation flower maturation. Carnation flower maturation-related clones obtained by the SSH were serially assigned as CFMI (carnation flower maturation-induced) clones. Northern blot analysis showed that several CFMI clones were differentially expressed during flower development. One of the clones, CFMI-3, showed similarity to various animal secretory phospholipases A2 (PLA2). Since little is known about PLA2 gene sequence in plant species, the CFMI-3 clone was selected for further characterization by sequence analysis. Full sequence analysis reveals that the CFMI-3 contains a Ca2+ binding domain, a PLA2 active site, and 12 conserved Cys residues, which is a distinct characteristic of PLA2. Amino acid sequence alignment of CFMI-3 to various putative plant PLA2 confirmed that the CFMI-3 cDNA is the full-length putative PLA2 cDNA identified in plant species.

Endoplasmic Reticulum– and Golgi-Localized Phospholipase A2 Plays Critical Roles in Arabidopsis Pollen Development and Germination

This study shows that Arabidopsis PLA2-γ and -δ, which are specifically expressed in pollen, localize to the endoplasmic reticulum and/or Golgi and that the suppression of PLA2s disrupts the endomembrane and induces pollen collapse. The PLA2 product, 18-1:LPE, was found to be required for pollen tube germination. The phospholipase A2 (PLA2) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA2s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA2s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA2 paralogs (PLA2-β, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA2 using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA2 inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA2s that are conserved in other organisms.

Single Cystathionine β-Synthase Domain–Containing Proteins Modulate Development by Regulating the Thioredoxin System in Arabidopsis

CBSX1 affects lignin deposition by regulating the H2O2 level in anthers and by modulating plant growth via regulation of photosynthesis. CBSX1 activates thioredoxins (Trxs) and further enhances the enzymatic activity of Trxs in the presence of AMP. CBSX1 is a redox regulator modulating Trxs for development and maintaining homeostasis under conditions that are threatening to the cell. Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain–containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H2O2 levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating Calvin cycle enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.

The Arabidopsis Calcium Sensor Calcineurin B-Like 3 Inhibits the 5′-Methylthioadenosine Nucleosidase in a Calcium-Dependent Manner

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. Sensing the calcium signals elicited by a variety of abiotic stresses, CBLs transmit the information to a group of serine/threonine protein kinases (CBL-interacting protein kinases [CIPKs]), which are currently known as the sole targets of the CBL family. Here, we report that the CBL3 member of this family has a novel interaction partner in addition to the CIPK proteins. Extensive yeast two-hybrid screenings with CBL3 as bait identified an interesting Arabidopsis (Arabidopsis thaliana) cDNA clone (named AtMTAN, for 5′-methylthioadenosine nucleosidase), which encodes a polypeptide similar to EcMTAN from Escherichia coli. Deletion analyses showed that CBL3 utilizes the different structural modules to interact with its distinct target proteins, CIPKs and AtMTAN. In vitro and in vivo analyses verified that CBL3 and AtMTAN physically associate only in the presence of Ca2+. In addition, we empirically demonstrated that the AtMTAN protein indeed possesses the MTAN activity, which can be inhibited specifically by Ca2+-bound CBL3. Overall, these findings suggest that the CBL family members can relay the calcium signals in more diverse ways than previously thought. We also discuss a possible mechanism by which the CBL3-mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are involved in plant growth and development as well as various stress responses.

Novel CIPK1-associated proteins in Arabidopsis contain an evolutionarily conserved C-terminal region that mediates nuclear localization.

Environmental stimuli, including light, pathogens, hormones, and abiotic stresses, elicit changes in the cytosolic Ca2+ signatures of plant cells. However, little is known about the molecular mechanisms by which plants sense and transmit the specific cytoplasmic Ca2+ signal into the nucleus, where gene regulation occurs to respond appropriately to the stress. In this study, we have identified two novel Arabidopsis (Arabidopsis thaliana) proteins specifically associated with Calcineurin B-Like-Interacting Protein Kinase1 (CIPK1), a member of Ser/Thr protein kinases that interact with the calcineurin B-like Ca2+-binding proteins. These two proteins contain a very similar C-terminal region (180 amino acids in length, 81% similarity), which is required and sufficient for both interaction with CIPK1 and translocation to the nucleus. Interestingly, the conserved C-terminal region was also found in many proteins from various eukaryotic organisms, including humans. However, none of them have been characterized so far. Taken together, these findings suggest that the two proteins containing the evolutionarily conserved C-terminal region (ECT1 and ECT2) may play a critical role in relaying the cytosolic Ca2+ signals to the nucleus, thereby regulating gene expression.

Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens

Physcomitrella patens is a model plant for studying gene function using a knockout strategy. To establish a proteome database for P. patens, we resolved over 1,500 soluble proteins from gametophore and protonema tissues by two-dimensional electrophoresis (2-DE) and obtained peptide mass fingerprints (PMFs) by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Using expressed sequence tags (ESTs), we were able to predict the identities of 90 protein spots. Most of these were related to energy or primary metabolism. Comparative proteome analysis was used to identify proteins specific for each of the tissue types. One of these was a metallothionein type-2 (PpMT2) protein that was highly upregulated in gametophore tissue. PpMT2 was induced in both the gametophore and protonema following culture on solid media and in response to various abiotic stresses such as copper, cadmium, cold, indole-3-acetic acid, and ethylene. We suggest that PpMT2 is not only involved in metal binding and detoxification, but also in many biological aspects as a metal messenger or a protein with additional functions.

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.