Sheikh M. Basha

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.

Identification of drought-responsive transcripts in peanut (Arachis hypogaea L.)

We have used a reverse transcriptase polymerase chain reaction procedure (differential display) to identify cDNAs corresponding to transcripts affected by water stress in peanuts ( Arachis hypogaea L.). Using this method, we have identified several mRNA transcripts that are up- or down-regulated following water stress. With 21 primer combinations, a total of 1235 differential display products were observed in irrigated samples, compared to 950 differential display products in stressed samples. These products demonstrated qualitative and quantitative differences in the gene expression. The differentially expressed transcripts were collectively named PTRD (Peanut Transcripts Responsive to Drought). We have identified a total of 43 PTRD, which were significantly altered due to water stress. Slot blot analysis of 16 PTRD indicated that 12 were completely suppressed due to prolonged drought, two were down-regulated, and two were up-regulated under drought stress conditions. The 12 completely suppressed transcripts were studied further by RNA dot-blot analysis to compare their expression in drought tolerant and susceptible lines, which underwent three weeks of water stress. PTRD-1, -10, and -16 expressed for longer period in tolerant line compared to the susceptible line and can be used as molecular markers for screening peanut lines for drought tolerance.

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.

Compositional differences in the phenolics compounds of muscadine and bunch grape wines

Wines contain a large array of phenolic compounds, belonging to non-flavonoids, flavonoids and phenolic-protein-polysaccharide complexes. Phenolics in wine are responsible for wine color, astringency, and bitterness. This study evaluates phenolic composition of commercial and experimental wines derived from bunch (Vitis vinifera) and muscadine (Vitis rotundifolia) grapes to determine compositional differences in phenolics. HPLC analysis of wines showed that majority of phenolic compounds eluted during the first 30 min. Of the red wines tested, Château Cabrieres Chateauneuf de Pape (Rhone) showed the simplest phenolics profile while Cabernet Sauvignon (Vitis vinifera-California) showed the most complex profile. The phenolics composition of red and white wines varied greatly. Some white wines from bunch grape were devoid of any phenolics. Among muscadine white wines, some contained large number of phenolic compounds while the others showed smaller number of phenolic compounds. These data suggested that both the red and white wines contained a complex mixture of phenolic compounds whose content and composition varied by brand suggesting that the wine processing technique greatly influences phenolics composition of wines than color of the wine. Muscadine red wines were quite distinct than that of the bunch grapes, indicating that grape chemistry has a greater influence on wine phenolic composition.

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.

Resistance to Elsinoë Ampelina and Expression of Related Resistant Genes in Vitis Rotundifolia Michx. Grapes

Muscadine grapes (Vitis rotundifolia Michx) are considered as excellent genetic resources for grape breeding programs as they are known for their hardiness and resistance to pests and diseases. However, contrary to popular belief, our study indicated that not all muscadine cultivars are resistant to anthracnose disease. In order to identify a source of genetic tolerance towards anthracnose among muscadine cultivars, a series of in-situ and ex-situ experiments were conducted through strict and sensitive screening processes. Two consecutive years of field evaluation of 54 grape cultivars showed various levels of anthracnose incidence among the cultivars between a scale of 0 (tolerant) to 5 (highly-susceptible). Resistance bioassay by inoculation of different spore densities of Elsinoë ampelina on 40 cultivars presented similar results and was consistent with those obtained from the field test. A real-time PCR analysis was conducted to investigate differences of gene expression between susceptible and tolerant cultivars and to confirm results by phenotypic identification. Expression of genes encoding chalcone synthase, stilbene synthase, polygalacturonase-inhibiting protein, chitinase and lipid transfer-protein was only detected in tolerant cultivars. Resistant muscadine cultivars identified in this study could be excellent candidates for grape disease resistance breeding programs.

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety

Devaiah M. Kambiranda1, Hemanth KN. Vasanthaiah1, Ramesh Katam1, Athony Ananga2, Sheikh M. Basha1 and Karamthotsivasankar Naik3 1Plant Biotechnology Laboratory, College of Agriculture Florida A & M University, 6505 Mahan Drive, Tallahassee, Florida 32317 2Center for Viticulture, College of Agriculture Florida A & M University, 6505 Mahan Drive Tallahassee, Florida 32317 3Agriculture Research Station, ANGR Agricultural University, Kadiri, Andhra Pradesh 515591, 1,2USA 3India

Factors Affecting Phytoalexin Production in Peanut Seed

Summary Phytoalexins were elicited in the peanut ( Arachis hypogaea L.) seed by wounding (by slicing) and incubating the seed slices at 25 °C in the dark. Chromatographic analysis of the purified extract from 4-day incubated seed slices showed the presence of seven phytoalexin compounds. The relative content and ratios of these metabolites changed with increasing periods of incubation. During the incubation, a-amino nitrogen content of the seed increased while Peak V protein and soluble sugars decreased. Phytoalexin accumulation was highest at 100 % RH and decreased with decreasing humidity levels. Cycloheximide at a concentration of 100 µg/mL caused a 90 % decrease in the phytoalexin production, while actinomycin D (100 µg/mL) caused only a 10 % inhibition of the phytoalexin production.

Effect of Drought and Temperature Stress on Peanut (Arachis hypogaea L.) Seed Composition

Summary Peanut ( Arachis hypogaea L.) plants were subjected to drought and temperature stress for various periods, and the seeds from these plants were separated into Jumbo, Medium and No. 1 market size categories and analyzed for soluble and total carbohydrates, α-amino nitrogen, total protein, and oil. The results showed that soluble and total carbohydrate content of the seed increased due to drought and temperature stress, with Jumbo and Medium market categories showing the highest increase. The α-amino nitrogen content of the Jumbo market category decreased while that of the Number 1 market category increased following a 30-day stress exposure. Protein and oil content of all the market categories were not affected after exposure to drought and temperature stress. The protein and polypeptide profiles showed that a polypeptide with a molecular weight of 70,000 and a pi between 6.2 and 7.0 increased with increasing periods of drought and temperature stress. It is suggested that drought and temperature stresses cause increased accumulation and/or synthesis of carbohydrates and certain polypeptides thus may enhance Aspergillus invasion and aflatoxin production.

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.