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.     

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.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

Basal expression of abscisic acid inducing immunity against Karnal bunt of wheat

In order to understand the intricate mechanism of differential immunity against Karnal bunt (KB), basal levels of carotenoids, abscisic acid (ABA), total protease and protease inhibitor were determined in resistant and susceptible genotypes during transition from vegetative to developing stages of wheat spikes. The lower levels of carotenoid precursor of ABA in resistant genotype than in susceptible genotype could be explained by more inter-conversion of carotenoids into ABA pool which was evident from the results of determining ABA by enzyme-linked sorbent assay. The ABA was significantly higher in resistant genotype at all stages than in susceptible genotype, while a sharp increase was observed at S2 stage. The activity of total protease was higher at initial stages of resistant genotype and gradually declined in later stages after anthesis. In contrast to the protease activity, a reverse trend was observed for the levels of cysteine protease inhibitor, suggesting a negative correlation with each other. Level of cysteine protease inhibitor was observed to be three-folds higher at S2 stage in the resistant genotype than in the susceptible genotype. The results provided the clue for the involvement of ABA-dependent pathways in upregulation of cystatin that leads to induction of differential immunity against the KB pathogen.     

小麦对赤霉病抗性不同品种的SOD活性

本研究对9个赤霉病抗性不同小麦品种采用赤霉病菌分生孢子悬浮液以单花针注法进行了田间和温室抗病性鉴定;测定了各品种的胚性愈伤组织和盛花期麦穗分别经赤霉病菌毒素和分生孢子接种前后SOD活性的变化。结果表明,各品种SOD活性与其对赤霉病抗性呈极显著的正相关。接种后寄主的SOD活性均有提高,抗病品种比感病品种提高幅度大,且有新的同工酶带出现。抗病品种望水白比感病品种Alondra“S”多出两条SOD同工酶谱带。SOD在小麦抗赤霉病上可能起积极作用,其活性有可能作为鉴定小麦抗赤霉病的一种生理生化指标。     

Cloning, in silico characterization and interaction of cysteine protease and cystatin for establishing their role in early blight disease in tomato

Cysteine protease (CP) and Cysteine protease inhibitor (CPI) or cystatin constitute a critical point in programmed cell death (PCD), a basic biological phenomenon which takes place in the plants, when they are exposed to varying biotic and abiotic stresses. In the present study we isolated and cloned cDNAs encoding cysteine protease and cystatin from early blight infected tomato plants. Using computational biology tools the sequence-structure-function relationships for the tomato cystatin and cysteine protease were elucidated. Interaction between the cystatin and cysteine protease of host and pathogen is higher as compared to interaction shown by cystatin and cysteine protease within the host. The interaction energy of (a)tomato cystatin—tomato cysteine protease, (b)tomato cystatin—fungal cysteine protease and (c)tomato cysteine protease—fungal cystatin are ?319.33 Kcal/mol, ?504.71 Kcal/mol and ?373.731 Kcal/mol respectively. Comparative protein sequence analysis with different plant cystatins and cysteine protease were also done with the sequences of cystatin and cysteine protease isolated from tomato. Structures for all the cystatin and cysteine protease were modeled along with their interactions with fungal cystatin and cysteine protease in order to explore the structural variability and its manifestation at the functional level. This helped to relate the already known functions of these proteins with their sequences as well as the predicted structures. This also served to better understand the CP-CPI interaction operational in developing this protein family and its implication in plant defense during fungal pathogenesis in tomato plants.     

中华鲟半胱氨酸蛋白酶抑制剂在毕赤酵母中的表达和活性分析

为研究鱼类半胱氨酸蛋白酶抑制剂（Cystatin）的功能并探索其在水产加工和病害防治中的应用潜力,将PCR改造后的编码成熟肽中华鲟（Acipenser sinensis）cystatin 基因亚克隆到毕赤酵母整合型表达载体pPICZαA,氯化锂法转化毕赤酵母菌株GS115,构建表达cystatin的酵母基因工程菌。经甲醇诱导、SDSPAGE检测培养基上清液,表明中华鲟cystatin在毕赤酵母中实现了高效表达,重组cystatin表达量约为215mg·L^-1。纯化后重组蛋白纯度达94.2%。生物活性检测结果表明,1μg重组中华鲟cystatin约能抑制15μg木瓜蛋白酶的水解活性。     

Interaction of L-amino Acids with the Fusion Structures of a Cysteine Proteinase/Cystatin Pair

In a basic investigation, the molecular interactions between 2 different fused products of Arabidopsis cysteine proteinase (CP) and cysteine proteinase inhibitor (CPI) pair “namely R1: H₂N-maltose-binding protein- CPI-CP-COOH and R2: H₂N-maltose binding protein-CP-CPI-COOH” and 4 different L-amino acids including L-Ala, L-Ser, L-Asp, and L-Phe were analyzed using experimental methods and computational tools. The activity of CP relatively increased in purified R2 product in which test L-amino acids tend to interact with CP/CPI pair. On the other hand, the functionality of R1 product (having no tendency to interact with CP/CPI pair) was not influenced by L-amino acids. Detection of the effect of L-enantiomers of amino acids on the fusion forms of 2 functionally related proteins such as CP and CPI is the first ever time work on this research area. As a research recommendation, manufacturing the switchable biological systems expressing the fused forms of CP/CPI pair was proposed to control the relative activities of proteolytic compounds in different environments in the future.     

Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.     

Molecular cloning of a new wheat calreticulin gene TaCRT1 and expression analysis in plant defense responses and abiotic stress resistance

Calreticulin proteins play essential roles in regulating various metabolic processes and in molecular signal transduction in animals and plants. Using homologous PCR, we screened a cDNA library of the wheat resistance gene Yr5 from a near-isogenic line in the susceptible common wheat variety Taichung 29, which was inoculated with an incompatible race CYR32 of Puccinia striiformis. We isolated a novel full-length cDNA encoding calreticulin protein, which we named TaCRT1. Sequence analyses indicated that TaCRT1 contains an open reading frame of 1287 bp in length; it was deduced to encode 428 amino acids. Clustering analysis showed that TaCRT1 belongs to group III of the calreticulin protein family. Semi-quantitative RT-PCR was used to analyze expression profiles of the isolated gene under biotic and abiotic stresses. Expression of TaCRT1 was suppressed by exogenous application of phytohormones, such as abscisic acid and methyl jasmonate, and by dehydration; but it was induced by CYR32 infection and cold treatment. Based on the expression patterns, we propose that TaCRT1 participates in regulatory processes involved in defense responses and stress resistance in wheat.     

Molecular Cloning and Expression of cDNA Encoding the Cysteine Proteinase Inhibitor from Upland Cotton

A cDNA encoding a novel cysteine proteinase inhibitor (CPI) was isolated from a gland mutant Xiangmian-18 of upland cotton during the pigments gland forming stage. The cDNA comprises 378 bp and encodes 125 amino acid residues with molecular mass of 13.8 kDa. It contains the conserved motif of cysteine protease inhibitors and belongs to the cystatin superfamily (Gln-Val-Val-Ala-Gly). The deduced amino acid sequences of the domains are highly similar to the normal upland cotton (96.8%). SDS-PAGE and western hybridization analysis showed that the expressed recombinant protein was recombinant CPI. The inhibitory activity of recombinant CPI was 46 u/μg which was measured by inhibiting the protease activity of papain. RT-PCR results indicated that the expression level of developing gland stage was higher than that of undeveloped gland stage.     

Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association

Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease‐mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions.     

Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato

The phytohormone jasmonic acid (JA) regulates the synthesis of secondary metabolites in a wide range of plant species. Here, we show that exogenous methyl-JA (MeJA) elicits massive accumulation of caffeoylputrescine (CP) in tomato leaves. A mutant (jai1) that is defective in jasmonate perception failed to accumulate CP in flowers and MeJA-treated leaves. Conversely, a transgenic tomato line (called 35S::PS) that exhibits constitutive JA signaling accumulated high levels of leaf CP in the absence of jasmonate treatment. RNA blot analysis showed that genes encoding enzymes in the phenylpropanoid and polyamine pathways for CP biosynthesis are upregulated in MeJA-treated wild-type plants and in untreated 35S::PS plants. These results indicate that CP accumulation in tomato is tightly controlled by the jasmonate signaling pathway, and provide proof-of-concept that the production of some plant secondary metabolites can be enhanced by transgenic manipulation of endogenous JA levels.     

Penetration and Development of Meloidogyne incognita in Roots of Resistant and Susceptible Corn Genotypes

Rates of penetration and development ofMeloidogyne incognita race 4 in roots of resistant (inbred Mp307, and S4 lines derived from the open-pollinated varieties Tebeau and Old Raccoon) and susceptible (Pioneer 3110) corn genotypes were determined. Seedlings grown in styrofoam containers were inoculated with 5,000 eggs of M. incognita. Roots were harvested at 3-day intervals starting at 3 days after inoculation (DAI) to 27 DAI and stained with acid fuchsin. Penetration of roots by second-stage juveniles (J2) at 3 DAI was similar for the four corn genotypes. Meloidogyne incognita numbers in Tebeau, Old Raccoon, Mp307, and Pioneer 3110 peaked at 12, 12, 15, and 27 DAI, respectively. Nematode development in the resistant genotypes was greatly suppressed compared to Pioneer 3110. Resistance to M. incognita in these genotypes appears to be expressed primarily as slower nematode development rather than differences in J2 penetration.     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.