Ramesh Katam

iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry

Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening.

Analysis of peanut leaf proteome.

Peanut (Arachis hypogaea) is one of the most important sources of plant protein. Current selection of genotypes requires molecular characterization of available populations. Peanut genome database has several EST cDNAs which can be used to analyze gene expression. Analysis of proteins is a direct approach to define function of their associated genes. Proteome analysis linked to genome sequence information is critical for functional genomics. However, the available protein expression data is extremely inadequate. Proteome analysis of peanut leaf was conducted using two-dimensional gel electrophoresis in combination with sequence identification using MALDI/TOF to determine their identity and function related to growth, development and responses to stresses. Peanut leaf proteins were resolved into 300 polypeptides with pI values between 3.5 and 8.0 and relative molecular masses from 12 to 100 kDa. A master leaf polypeptide profile was generated based on the consistently expressed protein pattern. Proteins present in 205 spots were identified using GPS software and Viridiplantae database (NCBI). Identity of some of these proteins included RuBisCO, glutamine synthetase, glyoxisomal malate dehydrogenase, oxygen evolving enhancer protein and tubulin. Bioinformatical analyses showed that there are 133 unique protein identities. They were categorized into 10 and 8 groups according to their cellular compartmentalization and biological functionality, respectively. Enzymes necessary for carbohydrate metabolism and photosynthesis dominated in the set of identified proteins. The reference map derived from a drought-tolerant cv.Vemana should serve as the basis for further investigations of peanut physiology such as detection of expressed changes due to biotic and abiotic stresses, plant development. Furthermore, the leaf proteome map will lead to development of protein markers for cultivar identification at seedling stage of the plant. Overall, this study will contribute to improve our understanding of plant genetics and metabolism, and overall assist in the selection and breeding programs geared toward crop improvement.

Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.11 No.3, Issue of July 15, 2008

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress

UNLABELLED Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. SIGNIFICANCE Peanuts (Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.

Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using simple sequence repeat markers

Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent with the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies.

Characterization of Unique and Differentially Expressed Proteins in Anthracnose-Tolerant Florida Hybrid Bunch Grapes

Anthracnose is a major disease in Florida hybrid bunch grapes, caused by a fungus viz. Elsinoe ampelina. Florida hybrid bunch grapes are grown in southeastern USA for their superior wine characteristics. However, the effect of anthracnose on grape productivity and wine quality is a major concern to grape growers. Our research is aimed at determining biochemical basis of anthracnose tolerance in Florida hybrid bunch grape. Leaf samples were collected from the plants infected with E. ampelina at different periods and analyzed for differential protein expression using high throughput two-dimensional gel electrophoresis. Among the 32 differentially expressed leaf proteins, two were uniquely expressed in tolerant genotypes in response to E. ampelina infection. These proteins were identified as mitochondrial adenosine triphosphate synthase and glutamine synthetase, which are known to play a major role in carbohydrate metabolism and defense. Several proteins including ribulose 1-5 bisphosphate-carboxylase involved in photosynthesis were found to be suppressed in susceptible genotypes compared to tolerant genotypes following E. ampelina infection. The results indicate that the anthracnose-tolerant genotypes have the ability to up-regulate and induce new proteins upon infection to defend the invasion of the pathogen as well as maintain the normal regulatory processes.

Proteome analysis of muscadine grape leaves

Muscadine grapes are native to the southeastern United States and are used for making wine and consumed as fresh fruit. Grape berries, as ‘sink organs,’ rely on the use of available carbohydrate resources produced by photosynthesis to support their development and composition. A high throughput two-dimensional gel electrophoresis (2-DE) was conducted on muscadine (Vitis rotundifolia) grape leaf proteins to document complexity in their composition and to determine protein identity and function for enhancing photosynthetic efficiency of muscadine grape. 2-DE resolved muscadine leaf proteins into >258 polypeptides with pIs between 3.5 and 8.0 and molecular weight between 12,000 to 15,0000 Daltons. The consistently expressed proteins were excised and subjected to sequencing. Homology search of protein sequences showed 84% identity with Viridi plantae database. Identity of some of these proteins included RuBisCO, glutamine synthetase, pathogenesis-related protein, glyoxisomal malate dehydrogenase, ribonucleoprotein, chloroplast precursor, oxygen evolving enhancer protein. Comparative analysis of 10 muscadine cultivars showed quantitative differences in expression of 39 polypeptides among these genotypes. The results suggested that the polypeptide composition of muscadine grape leaf is complex, and polypeptide number and amount vary widely among muscadine genotypes, and these variations may be responsible for differences in their physiology, berry and stress tolerance characteristics.

Proteomic Approach to Screen Peanut Genotypes with Enhanced Nutritional Qualities

Peanut is an important food legume grown in semi- arid tropics of the world. Aspergillus fungus thrives in drought prevalent regions producing immunosuppressive carcinogenic aflatoxins in peanut seed. Identification and development of drought- tolerant genotype/s is the potential means to reduce aflatoxin contamination. We have determined differences in biochemical and molecular responses of peanut genotypes with varying degree of drought tolerance. Changes in protein and mRNA composition of seed in response to drought stress were measured using 2-dimensional electrophoresis and differential display RT PCR. Mass spectroscopy analysis revealed down-regulation of methionine rich proteins (MRPs) and arachin proteins in drought- susceptible (DS) genotypes, while these proteins continue to express in drought-tolerant (DT) genotypes. Up-regulation of mRNA transcripts in DT genotypes indicated their association with stress tolerance. Continued expression of these proteins seems to enhance drought tolerance, reduce aflatoxin level and enhance nutritional value of peanut.

Overexpression of a Plasma Membrane Bound Na+/H+ Antiporter-Like Protein (SbNHXLP) Confers Salt Tolerance and Improves Fruit Yield in Tomato by Maintaining Ion Homeostasis

A Na+/H+ antiporter-like protein (NHXLP) was isolated from Sorghum bicolor L. (SbNHXLP) and validated by overexpressing in tomato for salt tolerance. Homozygous T2 transgenic lines when evaluated for salt tolerance, accumulated low Na+ and displayed enhanced salt tolerance compared to wild-type plants (WT). This is consistent with the amiloride binding assay of the protein. Transgenics exhibited higher accumulation of proline, K+, Ca2+, improved cambial conductivity, higher PSII, and antioxidative enzyme activities than WT. Fluorescence imaging results revealed lower Na+ and higher Ca2+ levels in transgenic roots. Co-immunoprecipitation experiments demonstrate that SbNHXLP interacts with a Solanum lycopersicum cation proton antiporter protein2 (SlCHX2). qRT-PCR results showed upregulation of SbNHXLP and SlCHX2 upon treatment with 200 mM NaCl and 100 mM potassium nitrate. SlCHX2 is known to be involved in K+ acquisition, and the interaction between these two proteins might help to accumulate more K+ ions, and thus maintain ion homeostasis. These results strongly suggest that plasma membrane bound SbNHXLP involves in Na+ exclusion, maintains ion homeostasis in transgenics in comparison with WT and alleviates NaCl stress.

Peanut Bioinformatics: Tools and Applications for Developing More Effective Immunotherapies for Peanut Allergy and Improving Food Safety

Advanced tools of bioinformatics have been employed to assess the features critically required for allergenicity and cross-reactivity. A tremendous accumulation of data on plant proteins in recent years has made it possible to classify allergens in different protein families, with most food allergens grouped into four protein families. These families can be grouped together into superfamilies by comparing sequences and related structures. This information makes it possible to identify a wide range of related proteins that may result in the development of multiple food allergies that initiate the development of cross-reactive antibodies in susceptible individuals. Since peanut allergies are responsible for most episodes of food-induced anaphylaxis, a detailed immunological and molecular characterization of these allergenic components is essential to develop suitable immunotherapies. This would also allow us to screen transgenic plants for the possible development of allergens similar to those allergenic components in peanuts. Homology modeling in combination with residue-wise solvent accessibility of monomers and biological assemblies of allergens certainly gives valuable information about antigenic determinants on protein allergens. Through this review, we discuss the applications of bioinformatics tools toward the mitigation of peanut allergy.