Characterization of mitogen activated protein kinases (MAPKs) in the Curcuma longa expressed sequence tag database

Mitogen activated protein kinase (MAPK) cascades are universal signal transduction modules that play crucial role in plant growth and development as well as biotic and abiotic stress responses. 20 and 17 MAPKs have been characterized in Arabidopsis and rice respectively, which are used for identification of the putative MAPKs in other higher plants. However, no MAPK gene sequences have yet been characterized for asexually reproducing plants. We describe the analysis of MAPK EST sequences from Curcuma longa (an asexually reproducible plant of great medicinal and economic significance). The four Curcuma MAPKs contains all 11 MAPK conserved domains and phosphorylation-activation motif, TEY. Phylogenetic analysis grouped them in the subgroup A and C as identified earlier for Arabidopsis. The Curcuma MAPKs identified showed high sequence homology to rice OsMPK3, OsMPK4 and OsMPK5 suggesting the presence of similar key element in signaling biotic and abiotic stress responses. Although further in vivo and in vitro analysis are required to establish the physiological role of Curcuma MAPKs, this study provides the base for future research on diverse signaling pathways mediated by MAPKs in Curcuma longa as well as other asexually reproducing plants.     

Survey and characterization of NBS-LRR (R) genes in Curcuma longa transcriptome

Resistance genes are among the most important gene classes for plant breeding purposes being responsible for activation of plant defense mechanisms. Among them, the nucleotide binding site-leucine rich repeat (NBS-LRR) class R-genes are the most abundant and actively found in all types of plants. Insilico characterization of EST database resulted in the detection of 28 NBS types R-gene sequences in Curcuma longa. All the 28 sequences represented the NB-ARC domain, 21 of which were found to have highly conserved motif characteristics and categorized as regular NBS genes. The Open Reading Frames varied from 361 (CL.CON.3566) to 112 (CL.CON.1267) with an average of 279 amino acids. Most alignment occurred with monocots (67.8%) with emphasis on Oryza sativa and Zingiber sequences. All best alignments with dicots occurred with Arabidopsis thaliana, Populus trichocarpa and Medicago sativa. These detected NBS type Rgenes from Curcuma longa can be used as a valuable resource for molecular marker development, molecular mapping of R-genes, and identification of resistance gene analogs and functional and evolutionary characterization of NBS-LRR-encoding resistance genes in asexually reproducing plants.     

Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a xyloglucan endotransglucosylase/hydrolase homolog, improves drought and salt tolerance in transgenic Arabidopsis plants

Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants.     

Diverse roles of glycine-rich RNA-binding protein 7 in the response of camelina (<Emphasis Type="Italic">Camelina sativa</Emphasis>) to abiotic stress

Camelina sativa L. is an oilseed crop used as a potential low-cost biofuel resource. Despite the economic and agricultural benefits of this crop, studies demonstrating the physiological and genetic response of camelina to changing environmental conditions are limited. In this study, three stress-responsive glycine-rich RNA-binding proteins (GRPs) in camelina—named CsGRP7a, CsGRP7b, and CsGRP7c—were isolated, and their functional roles in stress responses were characterized. The three CsGRP7 genes had similar nucleotide and deduced amino acid sequences, and contained an N-terminal RNA-recognition motif and a C-terminal glycine-rich region. The CsGRP7 genes were ubiquitously expressed in all plant tissues, and CsGRP7 proteins were localized to both the cytoplasm and the nucleus. The expression of CsGRP7 genes was markedly upregulated by cold stress, whereas their expression was only slightly affected by salt or dehydration stress. Analysis of CsGRP7a-expressing transgenic Arabidopsis thaliana and camelina plants revealed that CsGRP7a plays a positive role in cold stress tolerance, but a negative role in salt or drought stress tolerance. All three CsGRP7s harbored RNA chaperone activity. Collectively, these data indicate that the stress-responsive CsGRP7s harbor RNA chaperone activity and play different roles in the plant response to abiotic stresses.     

Activation of a mitochondrial ATPase gene induces abnormal seed development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activities. Other members constitute the 26S proteasome complexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fusion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)-dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.)

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.     

Functional diversity of the plant glycine-rich proteins superfamily

The first plant glycine-rich proteins (GRPs) have been isolated more than 20 years ago based on their specific expression pattern and/or modulation by several biotic and abiotic factors. This superfamily is characterized by the presence of a glycine-rich domain arranged in (Gly)_n-X repeats. The presence of additional motifs, as well as the nature of the glycine repeats, groups them in different classes. The diversity in structure as well as in expression pattern, modulation and sub cellular localization have always indicated that these proteins, although classified as members of the same superfamily, would perform different functions in planta. Only now, two decades later, with the first functional characterizations of plant GRPs their involvement in diverse biological and biochemical processes are being uncovered. Here, we review the so far ascribed functions of plant GRPs.Key words: glycine-rich protein, cold-shock protein, RNA-binding protein, plant defense, flowering, cell elongation, RNA chaperone, signal transduction, oleosin, pollen competition     

Characterization of a stress-responsive ankyrin repeat-containing zinc finger protein of Capsicum annuum (CaKR1)

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain C(3)H(1) zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.     

姜黄根茎内生真菌区系分析

目的了解姜黄植物根茎中内生真菌群落组成和生态分布规律。方法采用表面消毒法分别于春、冬季从姜黄植物根茎中分离内生真菌,通过形态特征和基于ITS序列的系统发育分析初步确定其分类地位。结果分离获得51株内生真菌(春季33株、冬季18株),初步鉴定其分别归于5个纲、7个目、8个科、8个属(Fusarium,Gibberel-la,Alternaria,Phomopsis,Diaporthe,Nectria,Botryosphaeria,Mucor),其中镰刀菌属(Fusarium,51.0%)和赤霉属(Gibberel-la,17.6%)为优势菌群;春季分离的内生真菌分属于7个属(分离率为82.5%),而冬季的内生真菌仅归入4个属(分离率为45.0%)。结论姜黄植物中内生真菌具有较丰富的物种和系统发育多样性,在类群组成和分布上存在季节性差异,某些内生真菌(Fusariumsp.)具有宿主偏好性。     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Antiplatelet property of Curcuma longa L. rhizome-derived ar-turmerone

The antiplatelet activities of Curcuma longa L. rhizome-derived materials were measured using a platelet aggregometer and compared with those of aspirin as antiplatelet agent. The active constituent from the rhizome of Curcuma longa L. was isolated and characterized as ar-turmerone by various spectral analyses. At 50% inhibitory concentration (IC50) value, ar-turmerone was effective in inhibiting platelet aggregation induced by collagen (IC50, 14.4 microM) and arachidonic acid (IC50, 43.6 microM). However, ar-turmerone had no effect on platelet activating factor or thrombin induced platelet aggregation. In comparison, ar-turmerone was significantly more potent platelet inhibitor than aspirin against platelet aggregation induced by collagen. These results suggested that ar-turmerone could be useful as a lead compound for inhibiting platelet aggregation induced by collagen and arachidonic acid.     

The plant multigenic family of thiol peroxidases

Thiol peroxidases are ubiquitous recently characterized heme-free peroxidases, which catalyze the reduction of peroxynitrites and of various peroxides by catalytic cysteine residues and thiol-containing proteins as reductants. In plants, five different classes can be distinguished, according to the number and the position of conserved catalytic cysteines. Four classes are defined as peroxiredoxins and were already identified by phylogenetic sequence analysis, 1-Cys, 2-Cys, type II, and type Q peroxiredoxins, and the fifth is represented by glutathione peroxidases, which were recently shown to possess a thioredoxin-dependent activity in plants. Since the discovery of peroxiredoxins in plants in 1996, a lot of work has been devoted to the biochemical and functional characterization of the different peroxiredoxin isoforms, but in contrast, few structural data are available. The analysis of the Arabidopsis thaliana genome indicates that at least 17 isoforms of thioredoxin-dependent peroxidases are expressed in various plant compartments. The role of these proteins is discussed in terms of electron donor and substrate specificities and in light of their expression and localization. These enzymes are expressed in many plant tissues and are involved notably in the protection of the photosynthetic apparatus, in the response to various biotic or abiotic stresses by fighting reactive oxygen or nitrogen species and lipid peroxidation.     

Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production

Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from Curcuma amada, identified as Talaromyces pinophilus, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. Talaromyces pinophilus initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.