Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audiences of the project are the grape growers, consumers, wineries and grape breeders, who are interested in the outcome of this research. The research findings were presented at the FAMU grape harvest festival, field days and Florida Grape Growers’ Association Meeting. The research data provided insight into the synthetic and accumulation pattern of metabolites in the developing and ripening berry that are responsible for disease tolerance and nutraceutical characteristics of the berry and would be of great interest to target audience. The project data will aid in selecting and grouping grape cultivars based on expression level of nutraceutically and enologically important berry components, and will be valuable for growers to select genotypes suitable to their target market. This research also helped train four minority undergraduate students in laboratory techniques and research methods, and currently two African American graduate students are conducting thesis research on this topic and thus is a great means for providing experiential learning to minorities for improving their career opportunities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided training and hands-on experiential learning to faculty and students across the College of Agriculture and Food Science as well as among the other FAMU Colleges. Several undergraduate and graduate students from across the campus were recruited into the project and provided training in various biochemical and cellular laboratory techniques, data analysis, preparing reports and platform presentations. Further, this research also increased collaboration and cooperative research among FAMU faculty across the Campus. The following is the list of presentations made by faculty and students across the Colleges. Oral Presentations Scientists Sheikh M Basha, 2013. Molecular and Cellular Approach to Understand Sugar Metabolism in Muscadine Grape. ARD Meeting, Jacksonville, FL, April 6-9. Sheikh M Basha Devaiah Kambiranda, Katam R, 2013. Global Protein Profiling for Insight into Secondary Metabolite Synthesis in Muscadine Grape. Plant and Animal Genome conference, San Diego, CA. Devaiah Kambiranda, Sheikh M Basha, 2013. Proteomic Analysis of Ripening Muscadine Berry to Identify and Characterize Proteins Associated with Metabolite Synthesis Responsible for Superior Enological Characteristics of Muscadine Grape Berry. ARD Meeting, Jacksonville, FL, April 6-9. Student Presentation Johnson K, Gordon K, Kambiranda D, Basha SM. 2012. Estimation of total Amino Acids, Sugars, Proteins and Carbohydrates in Florida Hybrid grapes/Muscadine grapes. Presented at RATLR symposium, FAMU-CAFS - Secured third Price. How have the results been disseminated to communities of interest? The results of this research were presented at the Annual Grape Growers Meeting, International Plant and Animal Genome Conference and 1890 Agricultural Researach Directors Meeting as well as to the college and high school students, consumers and faculty visiting the laboratory, hobbyist wine makers and shared with scientists interested in developing muscadine genotypes with high nutraceutical value and disease resistance. What do you plan to do during the next reporting period to accomplish the goals? Proteome and metabolome analysis will be expanded to a wider range of grape genotypes with diverse characteristics, and to the disease infected berries and leaf tissue from muscadine and Florida hybrid bunch grape genotypes. Metabolite analysis will be carried out using HPLC among the Anthracnose and Pierces disease-tolerant and -susceptible genotypes of Florida hybrid bunch grapes to determine if tolerant genotypes produce higher amount of stilbene compounds/pathogenesis-related proteins to exhibit their anti-fungal and antibacterial activity. Metabolite analysis will also reveal genetic variation among diverse grape genotypes. High throughput proteome analysis will be carried out using infected and control leaf and berry tissues. Proteome analysis using shotgun technique will provide greater coverage and reveal low abundance proteins that might play crucial role in disease tolerance. The results obtained through proteome analysis will be confirmed by Real time PCR. Evaluation of additional genotypes with diverse characteristics will validate the expression of disease resistance proteins and will be useful for developing superior genotypes using conventional and molecular breeding. Leaf samples from different muscadine genotypes will be used for proteome analysis to identify variation in photosynthesis related proteins and its effect on their photosynthetic efficiency. Unique and differentially expressed proteins will be sequenced to know their identity and function. Further real time PCR will be performed for selected photosynthesis related proteins to monitor changes in their mRNA level. Enzyme analysis will be carried out using muscadine genotypes that contain high and low berry sugar content to determine genetic variation in their Invertase, sucrose synthase and sucrose phosphate synthase enzyme levels during berry ripening. Invertase-inhibitor gene will be characterized to determine copy number and variation among Vitis species. Real time PCR will be performed using various berry developmental stages and genotypes to determine differences in the expression pattern of the Invertase-inhibitor among muscadine genotypes. RNA-Seq will be performed on developing and ripening muscadine berry to reveal global gene expression pattern of enologicaly important genes and its regulation. Small RNAs will be identified for genes involved in disease tolerance, sugar and secondary metabolism to modify/suppress gene expression. Studies will be also conducted to develop innovative protocols for rapidly increasing nutraceutical compound level to improve their market value using various abiotic and biotic stress agents and management practices. These activities are envisioned to help increase the level of phenolics as well as other metabolites associated with superior berry characteristics. Further, the synthetic and accumulation pattern of enologically-important components will be studied among muscadine berry of diverse characteristics to help improve wine characteristics and shelf life of muscadine grape products. Additional studies will be conducted to develop functional foods using berry tissue and juice from high phytochemical-containing grape genotypes to increase their market value, consumer acceptance and grower profit.
Impacts
What was accomplished under these goals?
Our studies have revealed that during berry development and ripening, muscadine grapes express large amount of proteins which are responsible for the synthesis of nutraceutical components as well as those involved in disease resistance. To investigate biosynthetic and accumulation pattern of various phenolic compounds we have conducted a detailed proteome profiling study of the developing berry using isotope labeling followed by mass spectrometry analysis. Raw files obtained after protein sequencing were used to analyze quantitative variation in protein profiles among the disease tolerance and nutraceutical-related proteins. The protein identities thus obtained were fed to various annotation tools for revealing their function and role in various biological processes and metabolic pathways, and their regulation during berry development and ripening. The data revealed that over 60 proteins that are involved in the synthesis of secondary metabolites and disease tolerance are expressed during berry development and ripening. Up-regulation of proteins involved in the synthesis of secondary metabolites and those involved in disease tolerance was mainly observed during berry maturation and ripening suggesting that their expression is developmentally regulated. The majority of proteins identified were known to be involved in terpenoid, flavonoid and anthocyanin biosynthetic pathways. Further, the identified disease resistance-related proteins were found to belong to the PR10, PR-4, MLP and 14-3-3 like protein family and are constitutively expressed during berry development indicating their role in maintaining disease resistance in developing and ripening muscadine berry. These proteins appear to play an important role not only in disease tolerance but also affect berry quality and its enological characteristics. Further, to investigate genetic variation in accumulation of metabolites responsible for disease tolerance and nutraceutical properties, muscadine berries were collected from different genotypes and analyzed. Berry metabolites were extracted with methanol and the extracts were analyzed for their stilbenes content and composition by HPLC coupled to a UV detector. Identity of stilbenes was determined using known standards. The results revealed that muscadine genotypes contained five different stilbenes and their analogs viz. piceid, pterostilbene, trans-resveratrol, cis- and trans-stilbenes in variable amounts among the dark, bronze and pink skinned berries. Overall the proteome and metabolome data revealed interrelationship between accumulation of pathogenesis-related proteins and stilbenes in muscadine grapes. Proteomics study was also conducted to increase our knowledge of Vitis leaf proteome for increasing photosynthetic efficiency and sugar content of muscadine berry to improve its enological and disease tolerance characteristics. Two-dimensional gel electrophoresis (2-DE) was conducted on muscadine, bunch and hybrid bunch leaf proteins to reveal genetic variation in their protein content and composition. Unique and differentially expressed proteins were sequenced and their function was determined using gene ontology tools. Comparative analysis of leaf proteome showed that 54 polypeptides varied qualitatively and quantitatively among the three Vitis species studied. Of these, seven proteins were found to be unique to muscadine, two proteins were present in both muscadine and bunch, while 28 proteins were common to all the three species. Further, functional analysis of these proteins showed that they are involved in signal transduction pathway, transport of metabolites, energy metabolism, protein trafficking, photosynthesis and defense. The study also identified proteins unique to muscadine grape that are involved in defense and stress tolerance functions. In addition, the results also showed that photosynthesis-related proteins were found to be more abundant in bunch (Vitis vinifera) grape compared to muscadine grapes (Vitis rotundifolia).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Katam R, Sheikh MB, Parks C, Sakata K, Latinwo L. 2012 Identification and characterization of Differentially Expressed Leaf Proteins among Vitis Species. Annals of Applied Biology, 160: 273290.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Muscadine grape products are perceived to be inferior compared to bunch grapes and are not readily acceptable to consumer. Hence, our research goal is to expand utilization of muscadine products through better understanding of the role of berry components on disease and drought tolerance and other value-added characteristics of grape products. One of the components that influence muscadine product quality is its low sugar content and occurrence of high levels of sucrose. Our data has shown equal amounts of sucrose, glucose and fructose in muscadine genotypes while bunch grapes contain mainly glucose and fructose, and little to none sucrose. During veraison sucrose generated in leaves is transported to the berry where it is cleaved to its constituent's glucose and fructose. In muscadine grapes sucrose accumulates in the berry due to reduced invertase activity resulting in sucrose buildup. The high level of sucrose appears to cause feedback inhibition/substrate inhibition which prevents further sugar transport from leaf to berry. In addition, the observed low invertase activity in muscadine berry appears to be because of the presence of invertase-inhibitor which causes sucrose buildup due to poor sucrose hydrolysis. These metabolic events appear to be primarily responsible for low sugar accumulation in muscadine berry. Muscadine grapes contain certain unique phenolics compounds such as ellagic acid, ellagitannins, terpenes, etc., which act synergistically with resveratrol to provide disease tolerance in muscadine grapes. These components also contribute to the superior nutraceutical value of muscadine grapes. Hence, attempts were made to develop various value-added products from muscadine berry and juice. For this purpose grape berries of different maturities and ripening were collected and various products were prepared employing heat treatment and addition of sugar, salt and other ingredients to soften the skin and seed tissue for increasing their palatability. Using this approach we have developed and tested various berry products such as beer nuts, preserves, resins, bakery ingredients, etc. These products were found to be highly acceptable to the taste panel and appear to have potential commercial value. The disease research was aimed at characterizing the uniquely expressed xylem proteins by PD-tolerant genotypes. A high throughput two-dimensional gel electrophoresis was conducted on muscadine, hybrid bunch and bunch grape xylem proteins. The differentially expressed proteins were excised, and analyzed in MALDI/TOF mass spectrometer. Comparative analysis of xylem proteome showed that highest amount of protein is present in muscadine, moderate amount in Florida hybrid bunch and lowest in bunch grape, and are associated with disease resistance, energy metabolism, protein processing and degradation, biosynthesis, stress related functions, cell wall biogenesis, signal transduction, and ROS detoxification. Further, β-1, 3-glucanase, 10-deacetyl baccatin III-10-O-acetyl transferase-like, COP9, aspartyl protease nepenthesin precursor were found to be uniquely expressed in muscadine grape while they are absent in PD-susceptible bunch grape. PARTICIPANTS: Devaiah Kambiranda, Ramesh Katam, Kundai Chibanguza, Sheikh M Basha, Lekan M Latinwo, Virginia Gottschalk, Jarvis Jackson TARGET AUDIENCES: Grape growers, grape industry, scientists, researchers, students PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Muscadine grape products are perceived to be inferior compared to bunch grapes and are not readily acceptable to consumer. Hence, our goal is to expand utilization of muscadine products through better understanding of the role of berry components on disease and drought tolerance and other value-added characteristics of grape products. One of the components that influence muscadine product quality is its low sugar content and occurrence of high levels of sucrose. Our data has shown equal amounts of sucrose, glucose and fructose in muscadine genotypes while bunch grapes contain mainly glucose and fructose, and little to none sucrose. During veraison sucrose generated in leaves is transported to the berry where it is cleaved to its constituent's glucose and fructose. In muscadine grapes sucrose accumulates in the berry due to reduced invertase activity resulting in sucrose buildup. The high level of sucrose appears to cause feedback inhibition/substrate inhibition which prevents further sugar transport from leaf to berry. In addition, the observed low invertase activity in muscadine berry appears to be because of the presence of invertase-inhibitor which causes sucrose buildup due to poor sucrose hydrolysis. These metabolic events appear to be primarily responsible for low sugar accumulation in muscadine berry. Muscadine grapes contain certain unique phenolics compounds such as ellagic acid, ellagitannins, terpenes, etc., which act synergistically with resveratrol to provide disease tolerance in muscadine grapes. These components also contribute to the superior nutraceutical value of muscadine grapes. Hence, attempts were made to develop various value-added products from muscadine berry and juice. For this purpose grape berries of different maturities and ripening were collected and various products were prepared employing heat treatment and addition of sugar, salt and other ingredients to soften the skin and seed tissue for increasing their palatability. Using this approach we have developed and tested various berry products such as beer nuts, preserves, resins, bakery ingredients, etc. These products were found to be highly acceptable to the taste panel and appear to have potential commercial value. The disease research was aimed at characterizing the uniquely expressed xylem proteins by PD-tolerant genotypes. A high throughput two-dimensional gel electrophoresis was conducted on muscadine, hybrid bunch and bunch grape xylem proteins. The differentially expressed proteins were excised, and analyzed in MALDI/TOF mass spectrometer. Comparative analysis of xylem proteome showed that highest amount of protein is present in muscadine, moderate amount in Florida hybrid bunch and lowest in bunch grape, and are associated with disease resistance, energy metabolism, protein processing and degradation, biosynthesis, stress related functions, cell wall biogenesis, signal transduction, and ROS detoxification. Further, β-1, 3-glucanase, 10-deacetyl baccatin III-10-O-acetyl transferase-like, COP9, aspartyl protease nepenthesin precursor were found to be uniquely expressed in muscadine grape while they are absent in PD-susceptible bunch grape.
Publications
- Katam,R.,Basha,S.M., Parks, C., Sakata, K. and Latinwo, L.M. (2012). Identification and characterization of differentially expressed leaf proteins among Vitis species. Annals of Applied Biology, 160, 273-290.
- Basha,S.M., Vasanthaiah HKN and Devaiah Kambiranda. 2012. Genetic variation in sugar accumulation among Vitis species. International Journal of Wine Research 201,4,15-23
- Katam,R., Chibanguza,K., Basha,S.M., Latinwo, L.M. and Gottschalk, V. 2012. Analysis of Xylem Proteins Differentially Expressed in Vitis Species and Their Possible Association with Peirces Disease Tolerance. Frontiers of Plant Proteomics (In Review).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Grapes are a rich source of polyphenolics which show anti-hypertensive, anti-cancer and anti-arthritic activities. In this regard we have isolated several aqueous and non-aqueous soluble compounds from grape tissue. Testing of these fractions against breast, colon and lung cancer cells has shown major differences in their anticancer activity. Analysis of tissue extracts showed significant genetic differences in bioactive compounds level among muscadine genotypes. These differences in anticancer activity are being correlated with the content, composition and potency of phenolics in berry. The primary compounds responsible for nutraceutical value of grape are phenolics including resveratrol. In this regard we have isolated and characterized six isoforms of stilbene synthase gene that codes for resveratrol. We have found that unlike other phenolics present in the grape berry resveratrol does not accumulate during berry development but is induced only under stress as a defense response which results in inconsistent resveratrol levels in the berry. Current research is addressing this uncertainty by studying resveratrol gene expression and developing technologies to enable consistent resveratrol expression in grape berry. Muscadine wines are gaining popularity because of their unique fruity flavor, full body and perceived health benefits. However, sustaining muscadine product quality is a problem because of low berry sugar content and composition which affects its wine quality and flavor. Photosynthate produced in the leaf is converted to sucrose and transported to berry where it is broken into glucose and fructose by invertase. Our data has shown significant variation in invertase activity levels, and sucrose, glucose and fructose content among grape species. Studies are in progress to understand sucrose metabolism for determining the role of sucrose synthase and invertase enzymes, and their regulation at molecular and cellular level for producing superior muscadine products. In southern USA only two types of grape can be grown, Muscadinia rotundifolia (muscadines) and Vitis spp. (Florida hybrid bunch) grapes because of the bacterial disease known as Pierce's disease. Our studies have successfully identified several genes and proteins that are uniquely expressed following infestation of grape plants with E. ampelina (anthracnose) and Xylella fastidiosa (PD). Electrophoresis showed drastic reduction in expression of several proteins in susceptible cultivars compared to tolerant cultivars upon challenging with E. ampelina and Xylella. Comparative analysis of gels showed expression of 33 polypeptides in tolerant genotypes following Elsinoe infection. We have deposited their sequences in the EMBL UniProtKB database and their accession numbers are P85085, P85087 and P85088. We have also found several differentially-expressed pathogenesis-related proteins and genes between susceptible and tolerant grape cultivars following anthracnose infection. Currently we are characterizing pathogen-responsive proteins and transcripts using a large range of genotypes to confirm association of these genes, proteins and metabolites with disease tolerance. PARTICIPANTS: Devaiah Kambiranda Hemanth Vasanthaiah, Jarvis Jackson TARGET AUDIENCES: Grape growers, grape industry, scientists, researchers, students PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Fungal and bacterial diseases are serious problem which hamper bunch grape cultivation in southern United States. Resistant vines have evolved an active defense response system in which they express specific genes and synthesize pathogenesis-related proteins/metabolites that act directly on pathogens, inhibiting their growth. Therefore, understanding the molecular and cellular basis of disease tolerance is essential for developing disease tolerant grape cultivars. Our results have shown significant up-regulation of transcripts and proteins in tolerant genotypes compared to susceptible genotypes. Induction of these novel genes/proteins upon pathogen infection in tolerant genotypes indicates that they are well adapted to resist pathogen infestation. Our data has clearly demonstrated that tolerant genotypes were able to express several unique genes and metabolites upon pathogen infection to prevent their entry and growth while susceptible cultivars fail to express them. These genes appear to play an important role in inducing disease tolerance in grape and additional studies would help determine the molecular and cellular basis of disease tolerance as well as antimicrobial characteristics of pathogen-induced proteins. Nutraceutical research has strengthened ongoing research on value added characteristics of grape and small fruits. It also enhanced ongoing grape and small fruit research programs which involve monitoring accumulation pattern of berry components with nutraceutical and enological significance. Since the phenolics present in berry also play an important role in disease tolerance, findings of this study would help develop disease tolerant grape cultivars with value added characteristics. The outcome of this research will help growers obtain high yield of disease free crops with value added characteristics which will increase grower profit and food safety. This research also combined metabolomics, genomics and bioinformatics areas for computationally comparing data on gene, protein and metabolite expression in grape. Functional genomics approach taken in this study has enabled in effectively addressing physiological and developmental problems and correlating these findings with plant systems. The outcome of this project will help improve berry quality, enological and nutraceutical characteristics of muscadine grape berry to increase Florida grape grower profit, expand grape cultivation and promote grape product consumption. The project not only will aid in understanding the molecular and cellular basis of ripening in Vitis species but also help extend ongoing research in insect, biological and pharmaceutical sciences at FAMU. It will also enable application of metabolomics for studying ripening physiology of other fruits such as raspberry, black berry, citrus, etc. In addition this research would help bring together scientific talent and student resources from agriculture, biology, chemistry, environmental sciences, pharmacy and extension programs as well as provide an added advantage to the grower and student training. This research has the potential for transferring laboratory based technology to commercial application.
Publications
- Devaiah Kambiranda, Hemanth KN. Vasanthaiah., Sheikh M. Basha 2011. Relationship between acid invertase activity and sugar content in grape species. Journal of Food Biochemistry; DOI: 10.1111/j.1745-4514.2010.00483.x.
- Sheikh MB, Vasanthaiah HKN, Kambiranda DM. 2011. Genetic Variation in Sugar Composition among Muscadine, Florida Hybrid Bunch and Bunch Grape Genotypes (Accepted in International Journal of Wine Research).
- Vasanthaiah HKN, Kambiranda DM, Sheikh MB. 2011. Variation in Molecular Responses of Grape Genotypes Differing in Pierces Disease Tolerance (Submitted to Applied biochemistry and biotechnology journal)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In Florida, Pierce's Disease (PD) has precluded commercial production of European grapes but PD-tolerant muscadine and Florida hybrid bunch [hybrids of American wild grape species with European grapes (V. vinifera)] grapes are very popular. With the emphasis to grow high quality and disease-free grapes in Florida, this research was conducted to determine the molecular and cellular basis of disease tolerance and value-added characteristics for enhancing grape product value, grower profit and consumer acceptance. One of the reasons for poor quality of muscadine wines is believed to be their low sugar content and undesirable sugar composition. To better understand sugar metabolism in muscadine berry, changes in sugar accumulation and sugar metabolizing enzyme activities were studied during berry development and ripening. Analysis of berry extracts revealed that glucose and fructose were present in equal ratio prior to maturation and subsequently sucrose accumulation increased during veraison. In ripened berry, sucrose content ranged between 19-30%, glucose 30-35% and fructose between 31-36% among the muscadine genotypes. In hybrid bunch and bunch grapes, glucose and fructose percentage ranged between 48-50% and 49-51%, respectively throughout berry maturation and ripening. Sugar metabolizing enzyme analysis revealed that muscadine grapes contain relatively low levels of acid invertase and sucrose synthase activities compared to bunch grape. These data suggest that high sucrose content, low sugar metabolizing enzyme activities and lower amounts of glucose and fructose in berry appear to be the reasons for inferior quality of muscadine wines. Muscadine grapes are known to posse's high nutraceutical value. Resveratrol is one of the important phenolic compounds found in muscadine grape and has been linked to its versatile characteristics. Isolation and characterization of these phenolic compounds has confirmed presence of resveratrol in muscadine grape. Molecular analysis of muscadine berry has revealed six isoforms of stilbenes in muscadine berry that encode resveratrol. Of these, four isoforms were found to be unique to muscadine and express differentially during berry development. Protein modeling of this gene showed unique folding at certain amino acids which would indicate the potential for increasing resveratrol biosynthesis in the berry. Pierce's disease (PD) research has revealed expression of several defense and signal transduction related genes exclusively in PD-tolerant cultivars. Amino acid sequences were deduced based on their full-length nucleotide sequence. Protein modeling revealed presence of signal peptides unique to PD-tolerant cultivars which may possibly play an important role in their ability to resist Xylella infection. Amino acid sequence was also found to vary at position 234(L/R) for Chalcone synthase and at 204(G/S), 249(G/E), 260(V/L) and 347(T/S) for Chitinase. PR10 gene also showed changes at the amino acid numbers 14(V/I), 123(C/S) and 127(V/I) while PR4 gene showed changes at the amino acid numbers 37 (E/A), 57 (Q/K), 119 (K/Q), 128(N/A), 129(Q/K), 133(I/T), 137(E/Q) and 140 (D/N). PARTICIPANTS: Stephen Leong and Mehboob Sheikh, PI and Co-PI; Research Associates- Hemanth Vasanthaiah, Katam, R and Devaiah, K; Undergraduate Students - Saleem Kufar and Jarvis Jackson TARGET AUDIENCES: Grape Growers; Grape Consumers; Grape, Fresh fruit and Juice Industry; Health and Natural Product Industry PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Proteomics and genomics study has enabled identification of novel proteins and transcripts in PD tolerant cultivars that are associated with PD tolerance. Xylella fastidiosa infestation appeared to significantly affect both protein and transcript expression in PD-susceptible genotypes but had minimal impact on PD-tolerant genotypes suggesting that PD-tolerant genotypes were able to resist bacterial infection and maintained their normal physiological activities. Full length gene isolation and protein modeling studies revealed changes at the specific amino acid location in tolerant cultivars, signifying their possible role in resisting Xylella infection. Further studies on these genes will help enhance the tolerance level of commercially important PD-susceptible bunch (Vitis vinifera) cultivars for their successful cultivation in southeastern United States. The identification of molecular and cellular components that are positively and negatively associated with PD would aid in developing protein/molecular markers for screening grape germplasm to identify PD-tolerant genotypes. This will also pave way to understand the defense mechanism of the PD-tolerant genotypes which will aid in addressing bacterial disease problems in other crops. The nutraceutical research yielded a unique muscadine stilbene synthase (STS) cDNA (MSB1) sequence showing sequence variation with V. vinifera STS. Full-length of this unique stilbene synthase gene has been amplified and further in silico analysis revealed that it may have the potential to synthesize higher amount of resveratrol and the critical stilbene phytoalexins to defend against pathogen attack. Additionally, this study will enable to better understand resveratrol regulation in developing berry and possible genetic variation in its content and composition among the cultivars for improving nutraceutical value of grape products. This would further increase their market value, grower's profits and product marketability. Research on enhancing nutraceutically important compounds of muscadine grapes revealed presence of endophytic community in muscadine grapes. Studying the proteins and metabolites (the products of cellular processes) generated by these microbial communities will help isolate and identify these novel compounds with nutraceutical importance to promote grape product value. This research will demonstrate the importance of endophytic organisms persistent in muscadine grapes and its effect on improving their nutraceutical value. Research on physiology of berry development and ripening revealed that sugar composition of Vitis species varies widely, and the enzymes involved in sugar metabolism such as invertase and sucrose synthase are lower in muscadine compared to bunch grape indicating that muscadines have lower capability to breakdown sucrose. Comparative study of the enzymes involved in sugar metabolism among different Vitis species will reveal interrelationship between sucrose content and enzyme activity. These data will be useful in identifying desirable genes/enzymes missing or underexpressed in muscadine grape for use in improving the flavor characteristics of muscadine grape products.
Publications
- Vasanthaiah HKN, Basha SM and Katam R. 2010. Differential Expression of Chitinase and Stilbene Synthase Genes in Florida Hybrid Bunch Grapes to Elsinoe ampelina Infection. Plant Growth Regulation, 61(2): 127-134.
- Basha SM, Mazhar H. and Vasanthaiah HKN. 2010. Proteomics approach to identify unique xylem sap proteins in PD-tolerant Vitis species. Applied Biochemistry and Biotechnology 160(30).
- Chinnamuthu CR, Basha SM, Kokiladevi E and Vasanthaiah HKN. 2010. Nanotechnology Application in Viticulture and Wine Making. Grape Times, May - June Newsletter, Florida Grape Growers Association, pp 2-3 & 5.
- Devaiah K, Vasanthaiah HK N and Basha SM. 2010. Identifying Constraints Involved in Sugar Metabolism in Muscadine Grape. Proceedings of the Florida Horticulture Society (In press).
- Umar G, Vasanthaiah HK N, Devaiah K, Basha SM, Phills BR and Hunter W. 2010. Assessment of Genetic Diversity Among Selected Raspberry Cultivars. Proceedings of the Florida Horticulture Society (In Press).
- Kambiranda D, Vasanthaiah HKN and Basha SM. 2010. Relationship between acid invertase activity and sugar content in Vitis species. Journal of Food Biochemistry (In Press).
- Vasanthaiah HKN, Kambiranda D, Umar G, Basha SM and Phills BR. 2010. Raspberry Crop Improvement Through Biotechnology. In: Plant Biotechnology and Transgenic Research, Thangadurai D, Othman RY and Biradar AP (eds), Bentham Science Publishers, USA (Accepted).
- Kambiranda D, Vasanthaiah HKN and Basha SM. 2010. Grape Crop Improvement Through Biotechnology. In: Plant Biotechnology and Transgenic Research, Thangadurai D, Othman RY and Biradar AP (eds), Bentham Science Publishers, USA (Accepted).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Our disease research has revealed differential expression of mitochondrial ATP synthase, glutamine synthetase and ribulose 1-5 bisphosphate-carboxylase, in tolerant genotypes indicating their ability to up-regulate and induce new proteins to defend against the pathogen. Molecular analysis showed up-regulation of chitinase, stilbene synthase, protein/sugar kinases and transcriptional factor in anthracnose-tolerant grape cultivars. The data also showed that these genes were more rapidly expressed in tolerant cultivars within a day after E. ampelina infection and peaked by day 3. Induction of these anti-fungal genes appears to enable tolerant cultivars to withstand fungal infestation compared to susceptible cultivars. Our Pierce's disease research has shown that a set of xylem sap proteins with pI between 4.5 to 4.7 and Mr of 31 kDa were present in abundant amount in PD tolerant muscadine grape, and absent in PD-susceptible bunch grapes. Expression of chalcone synthase, chitinase III, PR 4 and 10, stilbene synthase, transcriptional factor, enolase, adenosine kinase, elongation factor alpha gene, quinone reductase, ribosomal RNA genes, and translationally-controlled tumor protein genes in tolerant genotypes reveals their adaptation mechanism to overcome pathogen infestation. The full-length of chalcone synthase, chitinase, PR 4 and PR 10 genes have been constructed and in silico analysis revealed that they are unique to Florida hybrid bunch grapes. Wine continues to be a major market for muscadine grapes. The lack of uniform ripening within a cluster is a major problem in muscadine grape production. Molecular studies have revealed expression of protein kinase and protein phosphatase 2C (PP2C) genes in muscadine berry which are known to regulate berry ripening process. Expression of PP2C appears to be negatively regulating abscisic acid (ABA) signaling. Suppression of ABA in certain muscadine berries within the cluster may be the possible reason for its uneven ripening. Muscadine grapes contain low levels of sugars which adversely affect their wine quality. Our studies have revealed that muscadine grapes have high sucrose:glucose, sucrose:fructose ratio and low glucose:fructose ratio compared to the bunch grapes. These results have demonstrated that in muscadine grape sucrose is transported to the berry at the onset of ripening but is only partially converted to glucose and fructose. These studies have also revealed that acid invertase required for sucrose breakdown is low in muscadine grape compared to bunch and Florida hybrid bunch grapes indicating a positive correlation between invertase activity and berry sugar content. Our nutraceutical research has shown that resveratrol encoded by stilbene synthase (STS) is one of the important phenolic compounds found in muscadine grape that has antimicrobial activity and inhibits growth of various cancers by up to 50-60%. Cloning and sequencing of muscadine STS gene revealed six isoforms of STS, of which four were found to be unique to muscadine. Expression studies using real-time PCR revealed higher expression of certain STS isoforms in muscadine berry during development. PARTICIPANTS: StephenLeong and Mehboob Sheikh, PI and co-PI; Research Associates- Hemanth Vasanthaiah, Katam, R and Devaiah, K; Undergraduate Students - Saleem Kufar and Curtis Wallace, Valarie Washington; Graduate Student-Yvette Betty O'Conner TARGET AUDIENCES: Grape Growers; Grape Consumers; Grape, Fresh fruit and Juice Industry; Health and Natural Product Industry PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
E. ampelina infection was found to significantly affect the enzymes associated with plant metabolism and defense pathways in susceptible cultivars compared to tolerant cultivars which alter normal physiological processes of the plant. Molecular analysis also showed that Elsinoe infection affected the expression of genes more in anthracnose-susceptible cultivars compared to -tolerant cultivars. Determining antifungal activity of uniquely expressed polypeptides and genes will enable us to determine their role in anthracnose tolerance. This information will aid in developing hybrid bunch cultivars tolerant to anthracnose and to formulate bio-pesticides against anthracnose. Proteome analysis of xylem sap from different Vitis species showed quantitative and qualitative differences in their sap composition suggesting that xylem sap protein composition of these species is different and these variations may have an impact on their tolerance to PD. The molecular data suggested that Xylella infestation affects both the cDNA transcripts and protein expression in susceptible cultivars. Several unique cDNA transcripts associated with PD tolerance were identified. Expression of these novel transcripts in tolerant cultivars showed their ability to recognize the stress. Further studies will pave way to understand the defense mechanism of the tolerant cultivars, which will also aid in addressing disease problems in other crops. The stilbene synthase (STS) research suggested that various isoforms of stilbene synthase gene exist among grape cultivars and that the isoform number and expression level in muscadine cultivars is different than the one in bunch grape. This genetic variation is believed to contribute for PD tolerance of muscadine grape. Further studies using external stimuli such as UV irradiation and inoculation with non-pathogenic endophytes to induce stilbene synthase gene expression should enhance resveratrol production in grape which should increase their value-added characteristics. Further, identifying berry developmental stage/s and genotypes with maximum STS expression would help increase muscadine health and market value. These data will aid in developing full-length muscadine stilbene synthase gene encoding resveratrol for its possible incorporation into commercially-important wine grape cultivars to enhance their nutraceutical value. Isolation and characterization of uniquely expressed genes from uniform and non-uniform ripening grape species will help determine the factors affecting ripening in muscadine berry. Our results have suggested that expression of PP2C in uneven ripening muscadine berry alters ABA signaling thus affecting the ripening process. Additional studies to determine the role of ABA in berry ripening should help understand the mechanism of muscadine berry ripening and develop ways to regulate ABA expression for promoting uniform ripening in muscadine. These efforts will lead to the development of muscadine grape cultivars with uniform ripening characteristics to increase product quality, grower's profits and product marketability.
Publications
- Vasanthaiah HKN, Katam R and Basha SM. 2008. Efficient Protocol for Isolation of Functional RNA from Different Grape Tissue Rich in Polyphenols and Polysaccharides for Gene Expression Studies. Electronic Journal of Biotechnology, 11(3): 1-8.
- Katam R, Sheikh M Basha, Vasanthaiah HKN. 2009. Identification and characterization of xylem components associated with Pierces disease tolerance in grape. Proceedings of the Florida Horticulture Society 112.
- Basha SM, Katam R, Vasanthaiah HKN and Matta F. 2009. Proteome Analysis of Muscadine Grape Leaves. International Journal of Wine Research, 1: 161-173.
- Vasanthaiah HKN, Katam R and Basha SM. 2009. Characterization of Unique and Differentially Expressed Proteins in Anthracnose-Tolerant Florida Hybrid Bunch Grapes. Applied Biochemistry and Biotechnology, 157(3): 395-406.
- Vasanthaiah HKN, Basha SM, Katam R and Kambiranda D. 2009. Investigation into Uneven Ripening Problem in Muscadine Grape for Enhancing their Enological Characteristics. Proceedings of the Florida Horticulture Society 112.
- Basha SM, Katam R. and Vasanthaiah HKN. 2009. Comparative Leaf Proteomics Studies in Vitis Species. Proceedings of the Florida Horticulture Society 112.
- Basha SM, Mazhar H. and Vasanthaiah HKN. 2010. Proteomics approach to identify unique xylem sap proteins in PD-tolerant Vitis species. Applied Biochemistry and Biotechnology 160(30): 932-944.
- Louime C, Vasanthaiah HKN, Basha SM and Lu J. 2010. Perspective of Biotic and Abiotic Stress Research in Grapevines (Vitis sp.). International Journal of Fruit Science 10(1).
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