Devaiah Kambiranda

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.

A Novel Pathogenic Mammalian Orthoreovirus from Diarrheic Pigs and Swine Blood Meal in the United States

ABSTRACT Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea. Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.

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

Genetic variation in sugar composition among muscadine, Florida hybrid bunch and bunch grape genotypes

Sugar content and composition of the bunch grape (Vitis vinifera) berry and its hybrids has been investigated extensively while limited information exists on the sugar content and composition of muscadine (Vitis rotundifolia) grapes. In this study, selected commercial grape cultivars belonging to muscadine, bunch, and Florida hybrid bunch market types were studied for content and composition of sugars in ripe berries for 2 consecutive years. Berry sugars were analyzed by high-performance liquid chromatography (HPLC), and the amount of sucrose, glucose, and fructose was quantified based on their peak area. Glucose and fructose were the predominant sugars in berries of bunch, and Florida hybrid bunch grape genotypes and were present in almost equal amounts. In contrast, the muscadine berry contained sucrose, glucose, and fructose. Sucrose was not detected in bunch and Florida hybrid bunch grape genotypes. Glucose constituted 46.0%–53.0% of the total berry sugars while fructose ranged between 47.0% and 53.0% among the bunch cultivars studied. In Florida hybrid bunch cultivars, the glucose percentage varied from 39.0% to 56.0% while fructose varied from 44.0% to 53.0%. In muscadine genotypes, sucrose constituted 17.0%–47.0%, glucose 27.0%–39.0%, and fructose 26.0%–47.0%. The proportion of glucose and fructose was almost equal in all three categories while the level of sucrose varied widely among the muscadine genotypes indicating that variable sucrose accumulation and/or hydrolysis may influence the berry sucrose content among the grape genotypes.

In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv. Carlos Reveals Proteins Associated with Flavor and Aroma Compounds

Ripening in nonclimacteric fruits such as grape involves complex chemical changes that have a profound influence on the accumulation of flavor and aroma compounds distinct to a particular grape genotype. In this study, proteome characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos), primarily grown in the Southeastern United States was performed during berry ripening. Stage-specific protein expression was obtained among different stages of berries. Differential analysis showed the expression of 522 proteins that regulate diverse biological processes and metabolic pathways. Of these, 30 proteins are associated with the production of key phenolic compounds, whereas 25 are associated with the production of muscadine aroma compounds. These proteins are involved in the phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin, and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of muscadine grape to improve its flavor and aroma properties.

Prospects for Transgenic and Molecular Breeding for Cold Tolerance in Canola (Brassica napus L.)

Canola is cultivated both during winter and spring seasons in the United States and this exposes the crop to winter kill, frost, and high temperatures, during the reproductive period. The temperatures during winter and spring are known to influence all the crucial steps of the reproductive cycle including gametogenesis, pollination, fertilization and embryogenesis (Angadi, 2000). Winter rapeseed has been successfully grown in the Pacific Northwest, southern Great Plains, Midwest, and southeast regions of the USA. The hardiest cultivars will routinely survive winters in the north east of USA but survival is inconsistent further south (Rife et al., 2001). Winter-grown canola (Brassica napus L.) production is limited mostly by frost and winter-kill in the southern canola-growing regions of the United States (Singh et al., 2008). For instance, the late freeze in 2007 resulted in significant damage to most of the winter canola cultivars at the National Winter Canola Variety Trials in Alabama, U.S. (Cebert and Rufina, 2007). Winter hardiness and freezing tolerance are a major concern for improving production consistency in many regions of the canola growing countries.

Proteome Profile of American Hybrid Grape cv. Blanc du Bois during Ripening Reveals Proteins Associated with Flavor Volatiles and Ethylene Production

The study of key control points in ripening is essential to improve grape wine quality. Molecular basis of ripening is still far from being understood from the Pierces disease (PD)‐tolerant grapes predominantly grown in the southeastern United States. To identify proteins expressed during Blanc du Bois grape berry green and ripening stages, proteome analysis from five different stages revealed 1091, 1131, 1078, 1042, and 1066 proteins. Differential expression analysis revealed 551 common proteins across different stages of maturity that are involved in various biochemical and metabolic pathways. The proteins identified were associated with phenylpropanoids, isoquinoline alkaloids, fatty acids, unsaturated fatty acids, and furanones. Our data provide the first step to understand the complex biochemical changes during ripening of PD‐tolerant American hybrid grapes that are popular for their aroma and flavor profile in the southeastern United States. Proteomics data are deposited to the ProteomeXchange PXD004157.