Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica)

To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

Influence of jasmonic acid as potential activator of induced resistance against Karnal bunt in developing spikes of wheat

Induction of defense response against Karnal bunt (KB) by suppressing the pathogenesis was observed upon exogenous application of jasmonic acid (JA) as evident from decrease in the coefficient of infection and overall response value in both susceptible and resistant varieties of wheat. The ultra-structural changes during disease progression showed the signs of programmed cell death (PCD). However, JA strengthened the defense barrier by enhancing the lignifications of cell walls as observed in spikes of both varieties by histochemical analysis. Compared to the plants inoculated with pathogen alone, plants of resistant line (RJP) first treated with JA followed by inoculation with pathogen showed more lignifications and extracellular deposition of other metabolites on cells, which is supposed to prevent mycelial invasions. Contrary to this, susceptible (SJP) lines also showed lignifications but the invasion was more compared to resistant line. Induction of protease activity was higher in resistant variety than its corresponding susceptible variety. The protease activity induced during the colonization of the pathogen and its proliferation inside the host system gets inhibited by JA treatment as demonstrated by the quantitative and in-gel protease assay. The results indicate the role of JA signalling in inhibiting the proteases due to expression of certain protease inhibitor genes. SDS-PAGE analysis shows differential gene expression through induction and/or suppression of different proteins in wheat spikes of resistant and susceptible varieties under the influence of JA. Thus, exogenously applied JA provides the conditioning effect prior to the challenge of infection and induces defense against KB probably by maintaining a critical balance between proteases and protease inhibitors and/or coordinating induction of different families of new proteins.     

Expression and in silico characterization of Phenylalanine ammonium lyase against karnal bunt (Tilletia indica) in wheat (Triticum aestivum)

To investigate the lignifications process and its physiological significance under Karnal Bunt (KB), the changes in enzymes responsible for lignifications likes, phenylalanine ammonia lyase (PAL), were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The PAL gene was cloned and sequenced. The expression of PAL gene was measured by means of semi-quantitative RT-PCR. The enzyme was expressed constitutively in both the susceptible and resistant genotype. However, the activity was higher in all the developmental stages of resistant genotype, indicating that this genotype has a significant higher basal level of these enzymes as compared to the susceptible line and could be used as marker(s) to define KB resistance. The activity of PAL was significantly higher in WSv stage (Z=16). Structural comparisons based on alignments of all the protein sequences using the clustal W program and searches for conserved motifs using the MEME program have revealed broad conservation of main motifs characteristic of the plant PAL. MSA and phylogenetic analyses of different plants PAL demonstrate that all PAL cluster divided in to two main cluster. The PAL also possesses a specific consensus sequences [GS]- [STG]-[LIVM]-[STG]-[SAC]-S-G-[DH]-L-x-[PN]-L-[SA]-x(2,3)-[SAGVTL]. The pathway might be associated with the enhancement of structural defense barrier due to lignifications of cell wall as evident from the enhanced synthesis of lignin in all the stages of resistant genotype. Our results clearly indicate the possible role of enzymes of Phenyl propanoid pathway metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.     

Phenol metabolism in downy mildew resistant and susceptible genotypes of pearl millet

Biochemical characterisation of pearl millet genotypes was carried at pre- (45 DAS) and post-infection (57 DAS i.e. 7 days after infection) stages. Total phenol content at pre-infection stage did not show inherent resistance or susceptibility. While the total phenol content was found to be higher in susceptible genotypes at post-infection stage, qualitative analysis of phenol through high-performance thin layer chromatography showed absence of ferulic acid in resistant genotypes at pre-infection stage. Peroxidase (POX) activity was higher in susceptible genotypes at both the stages of analysis. Constitutive activity of phenylalanine ammonia lyase was higher in resistant genotype whereas induced activity was recorded higher in susceptible genotypes. Native poly-acrylamide gel electrophoresis isozyme banding pattern of POX showed some inducible band(s) due to disease infection in resistant and susceptible genotypes.     

Water Stress Induced Proline Accumulation in Contrasting Wheat Genotypes as Affected by Calcium and Abscisic Acid

Proline accumulation and mobilization in roots of 7-d-old seedlings of wheat genotypes varying in sensitivity towards water stress were compared. Water stress was induced by polyethylene glycol (PEG-6000; osmotic potential −1.5 MPa) in the presence of 0.1 mM abscisic acid (ABA), 1 mM calcium chloride, 0.5 mM verapamil (Ca²⁺ channel blocker), 0.5 mM fluridone (inhibitor of ABA biosynthesis). While both the genotypes did not differ in total proline accumulation, rate of proline accumulation and utilization was higher in tolerant genotype C 306 as compared to susceptible genotype HD 2380. The treatment with ABA and CaCl₂ caused further increase in proline accumulation during stress and reduced its mobilization during recovery. The membrane stability and elongation rate of roots was observed to be higher at ABA and calcium treatment in both the genotypes under stress. As was evident from inhibitor studies, the tolerant genotype was more responsive to ABA and the susceptible one to calcium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water stress effects on maize: Carbohydrate metabolism of resistant and susceptible cultivars ofZea mays L.

Carbohydrate changea in drought resistant and susceptible cultivars ofZea mays L. were studied at two different stages under imposed water stress in water culture experiments. The gradual decline in total carbohydrates with increasing stress levels was observed. At the 1st leaf stage starch content of the resistant cv. Agati-76 was lower than that of susceptible cv. Vijay but the reducing and non-reducing sugar content was much higher. At the 3rd leaf stage starch content was similar in two cultivars but sugar content was higher in the resistant cv. Agati-76. Differential changes in root and shoot carbohydrates as affected by increasing levels of stress were noticed. Carbohydrate changes have been discussed in relation to drought resistance.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

ABA信号转运调节的基因表达与源库动力学分析

通过对拟南芥NCED3、AA03及SDR1蛋白亚细胞定位分析及根系和叶片ABA池的动态库变化研究,结果表明气孔运动的有效ABA信号来自于保卫细胞之外,SDR与ABA前体加工和运输有关。胁迫处理后根系合成酶基因转录水平显著高于叶片,但叶片ABA水平是根系的10倍以上,离体叶片和附体叶片ABA含量测定表明,叶片ABA池的形成主要决定于根源ABA的输入。氟啶酮药剂阻断和遮荫实验说明根系ABA池受叶源类胡萝素前体供应影响。叶片ABA水平受根源ABA和叶源类胡萝素前体库双向转运调节,维管束组织系统可能协同和整合了这一复杂调节机制。该结论为逆境ABA信号转递机制研究和操纵内源ABA含量增强植物抗逆性的应用提供相关资料。     

Effects of Ozone Exposure on Resistance of Wheat Genotypes to Pyrenophora tritici-repentis

Three spring wheat genotypes, susceptible, moderately resistant or resistant to Pyrenophora tritici-repentis (tan spot fungus) were exposed to charcoal-filtered air and to approx. 80, 160, 240 (g m^?3 ozone for five consecutive days (7 h per day). Visible leaf injury on seedling plants (three-leaf stage) was only observed after fumigation with 160 or 240 (g m^?3 O₃. Amount of injury was four-fold and 10-fold on the susceptible genotype when compared to resistant or moderately resistant genotype at the two highest concentration of ozone, respectively. Genotypic differences to O₃ tolerance were detected at the seedling growth stage (three-leaf stage) and flowering stage but not at the stem elongation stage. A significant increase in tan spot lesion area was observed only on O₃ predisposed second top most leaves of the susceptible genotype at all the three levels of ozone. Predisposition did not enhance tan spot development in resistant and moderately resistant genotypes. In a test with 12 wheat genotypes, a highly significant positive correlation (r = 0· 986, p < 0· 0001) was observed between ozone sensitivity (percent leaf area damaged due to 240 (g m^?3 ozone exposure) and tan spot development (mm² lesion area) following inoculation with P. tritici-repentis. It indicates that wheat genotypes resistant to the tan spot fungus might be tolerant to ozone damage.     

Induction of alpha-amylase inhibitor synthesis in barley embryos and young seedlings by abscisic Acid and dehydration stress

An endogenous α-amylase inhibitor was found to be synthesized in embryos of developing barley grain (Hordeum vulgare cv Bonanza). Accumulation of this protein occurred late in development (stage IV), at the same time that endogenous abscisic acid (ABA) showed a large increase. The inhibitor could be induced up to 23-fold in isolated immature embryos (stage III) by culture in ABA. Precocious germination was also blocked in stage III embryos by ABA. Dehydration stress on the isolated immature embryos also induced higher levels of the inhibitor and ABA. An even greater response to dehydration stress was observed in young seedlings, where inhibitor content increased 20-fold and ABA increased 80-fold during water stress. The high degree of correlation between ABA and inhibitor contents in in situ embryos, dehydrated embryos and young seedlings, as well as the increase in inhibitor caused by exogenously applied ABA to isolated embryos, suggests that increased α-amylase inhibitor synthesis in response to dehydration stress is mediated by ABA.     

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.     

水稻细胞质雄性不育系及其保持系幼穗发育过程中内源激素的变化

利用酶联免疫测定技术研究了水稻细胞质雄性不育系珍汕97A及其保持系珍汕97B在幼穗发育过程中叶片、幼穗和花药中内源IAA、GA1＋4、ABA和iPAs含量的动态变化。结果表明,1）从幼穗发育的雌雄蕊形成期到三核花粉期,保持系叶片中IAA水平高于不育系,并在二核花粉期最高;在幼穗和花药中也以保持系为高。2）保持系与不育系叶片中GA1＋4含量变化趋势相似,均为先升后降,但从单核到三核花粉期以保持系为高;在幼穗和花药中也都以保持系为高。3）不育系叶片中ABA水平在幼穗发育早期明显高于保持系,中期与保持系相近,后期又高于保持系;在幼穗和花药中也都以不育系为高。4）保持系叶片、幼穗和花药中iPAs含量始终显著高于不育系。5）保持系叶片中IAA＋GA1＋4＋iPAs与ABA之比值也始终高于不育系。提示不育系叶片、幼穗和花药中IAA、GA1＋4、iPAs和ABA含量出现了异常,且IAA、GA1＋4和iPAs亏缺以及ABA盈积可能与水稻细胞质雄性不育发生有关。