Cloning and characterization of pathogenesis-related protein 4 gene from Panax ginseng

The family of pathogenesis-related protein 4 (PR4) is a group of proteins with a Barwin domain in C-terminus and generally thought to be involved in plant defense responses. In the present study, PR4 (designated as PgPR4) cDNA was isolated from the leaf of Panax ginseng C.A. Meyer. and characterized. The ORF is 513 bp with a deduced amino acid sequence of 170 residues. A GenBank BlastX search revealed that the deduced amino acid of PgPR4 shares the highest sequence similarity to PR4 of Sambucus nigra (72% identity). Sequence and structural analysis indicated that PgPR4 belongs to class II of PR4 proteins. This is the first report on the isolation of PR4 gene from the P. ginseng genome. The high-level expression of PgPR4 was observed in the root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR4 expression could be up-regulated by pathogen infection, salt, wounding, and hormone stresses. These results suggest that PgPR4 could play a role in the molecular defense response of ginseng to abiotic stress and pathogen attack.     

Expression of the ginseng PgPR10-1 in Arabidopsis confers resistance against fungal and bacterial infection

Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Among three previously identified PR-10 homologs from ginseng (PgPR10-1, PgPR10-2, and PgPR10-3), we found that the exact same sequence of PgPR10-2 exist in all tested nine cultivars. But a deletion and SNP was found in American ginseng (Panax quinquefolius). PR-10 proteins are known to be small and cytosolic, and showed similar three-dimensional structure. Here we show that the heterologous overexpression of PgPR10-1 in Arabidopsis showed enhanced resistance against Pseudomonas syringe, Fusarium oxysporum, and Botrytis cinerea and in-frame tagging with fluorescent protein showed its cytoplasm and nucleus localization. Protein–protein interaction of PgPR10-2 with PgPR10-1, PgPR10-2 and PgPR10-3 suggests that the PgPR10 proteins might form multimeric complexes in different cellular compartments to function in development and in defense-related mechanism. Differential response of PgPR10-1 and PgPR10-2 against different sets of biotic stresses in ginseng plant supports this notion.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro _RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.     

Overexpression of VaCBF4, a Transcription Factor from Vitis amurensis, Improves Cold Tolerance Accompanying Increased Resistance to Drought and Salinity in Arabidopsis

Environmental stress has a great impact on fruit yield and quality in grapes. Understanding mechanisms underlying stress tolerance in plants is useful for grape breeding. Here, a CBF gene, designated VaCBF4, was identified in V. amurensis. The expression of VaCBF4 was induced by several abiotic stresses, including cold, exogenous abscisic acid (ABA), drought, salinity, and cold-drought conditions. A yeast one-hybrid assay demonstrated that VaCBF4 protein could bind to a conserved DRE cis-element, which contains the core sequence ACCGAC and regulates cold- and dehydration-responsive. Transgenic Arabidopsis seedlings overexpressing VaCBF4 showed enhanced tolerance to cold, drought, and salinity when compared with wild-type controls. LT₅₀, a chilling temperature required to cause 50 % electrolyte leakage in leaves, was 4 °C lower in transgenic Arabidopsis lines than that in non-cold-acclimated wild-type seedlings. Moreover, two stress-responsive genes, AtRD29A and AtCOR47, also showed higher levels of expression in the transgenic lines than in wild-type seedlings under normal growth condition. Taken together, all these results clearly indicate that VaCBF4 is involved in the response to abiotic stresses, and it may be a good candidate gene for genetic improvement to develop stress-tolerant varieties in grapes.     

GhMPK17, a Cotton Mitogen-Activated Protein Kinase,Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H₂O₂ level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Identification of ERF genes in peanuts and functional analysis of AhERF008 and AhERF019 in abiotic stress response

Ethylene-responsive factor (ERF) play an important role in regulating gene expression in plant development and response to stresses. In peanuts (Arachis hypogaea L.), which produce flowers aerially and pods underground, only a few ERF genes have been identified so far. This study identifies 63 ERF unigenes from 247,313 peanut EST sequences available in the NCBI database. The phylogeny, gene structures, and putative conserved motifs in the peanut ERF proteins were analysed. Comparative analysis revealed the absence of two subgroups (A1 and A3) of the ERF family in peanuts; only 10 subgroups were identified in peanuts compared to 12 subgroups in Arabidopsis and soybeans. AP2/ERF domains were found to be conserved among peanuts, Arabidopsis, and soybeans. Outside the AP2/ERF domain, many soybean-specific conserved motifs were also detected in peanuts. The expression analysis of ERF family genes representing each clade revealed differential expression patterns in response to biotic and abiotic stresses. Overexpression of AhERF008 influenced the root gravity of Arabidopsis, whereas overexpression of AhERF019 enhanced tolerance to drought, heat, and salt stresses in Arabidopsis. The information generated in this study will be helpful to further investigate the function of ERFs in plant development and stress response.