Source: FLORIDA A&M UNIVERSITY submitted to
STRENGTHENING DEVELOPMENTAL BIOLOGY RESEARCH AT FAMU TO ENHANCE MUSCADINE GRAPE BERRY QUALITY AND VALUE-ADDED CHARACTERISTICS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0222991
Grant No.
2010-38821-21562
Project No.
FLAX-SHEIKH
Proposal No.
2010-02424
Multistate No.
(N/A)
Program Code
EQ
Project Start Date
Sep 1, 2010
Project End Date
Aug 31, 2014
Grant Year
2010
Project Director
Sheikh, M.
Recipient Organization
FLORIDA A&M UNIVERSITY
(N/A)
TALLAHASSEE,FL 32307
Performing Department
AGRICULTURAL RESEARCH
Non Technical Summary
Muscadines are most popular grapes in the southeastern United States possessing potential nutritional/health benefits and economic value. Unlike the bunch grape, they have limited acceptance by the consumer because of poor berry quality. Developmental biology of muscadine berry is poorly understood for use in improving its quality. Our research has revealed significant differences in sugar and phenolics content of muscadine and bunch grapes. To sustain commercial grape production in SE US it is essential to improve muscadine berry quality. Understanding the molecular and cellular events associated with muscadine berry development and ripening will help achieve this goal. This research employs high throughput sequencing to obtain a functional catalog of genes and proteins expressed during muscadine berry development and ripening. Through comparative analysis with bunch grape, genes and proteins associated with superior nutraceutical, enological and disease tolerance characteristics will be identified. Results of this research will aid in enhancing berry quality, consumer acceptance and economic sustainability of muscadine grape in southeast. Further, it will diversify viticulture research and aid in developing new value-added products to increase farmer's profits and product marketability. This research will provide hands-on training to African American students and help build capacity in developmental biology at FAMU. This project will perform transcription profiling to understand the developmental biology of muscadine berry and determine the progression of genes related to berry quality during maturation and ripening. The obtained data will enhance understanding of the genetic mechanisms that control ripening and plant responses to the biotic and abiotic stresses. The functional catalog developed through this project seeks to link genes and pathways, and provide a list of features that would aid in understanding the genes associated with carbohydrate and protein metabolism, disease resistance, nutraceuticals and value added traits, and their regulation. Using the molecular approach genes induced/ suppressed or newly expressed during berry maturation will be studied to understand the physiology of ripening in muscadine grape which is essential for developing high quality muscadine grape products. This will improve the market value of muscadine grapes and their acceptance which is essential to sustain grape production in SE USA. Outcome from the proposed research will not only benefit the grape breeders or researcher involved in developing better quality grapes but will also help other scientists. The information, material and expertise gained from this project will be extended to identify, isolate and characterize important plant and animal genes. This would also add immense sequence information to the database. The outcome of this research will be disseminated to the scientific community, grape growers and consumer groups through presentations at the conferences and publications in peer reviewed journals.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011139100025%
2011139104025%
2011139105050%
Goals / Objectives
The proposed project addresses USDA-CSREES Strategic Plan 2005-2010 goals 2.0; Enhance the competitiveness and sustainability of rural and farm economies and 5.0; Improve the Nation's Nutrition and Health. The project has five key objectives: (1) Develop a comprehensive gene expression catalog of muscadine grape to determine progression of genes related to berry and juice quality during berry development and ripening, (2) Construct a master proteome reference map of muscadine berry to better understand the role of proteins expressed during berry maturation, and determine interrelationship between expressed genes and proteins, 3) Develop a microarray chip containing genes encoding for desirable berry components associated with superior berry and juice characteristics, (4) Determine the synthetic and accumulation pattern of viticulturally-important berry metabolites for correlating metabolite and gene/protein expression profiles and to determine genetic variation between muscadine and bunch genotypes, (5) Strengthen cooperative linkages between on-campus and off-campus researchers among 1890 institutions, and provide hands-on experiential learning to strengthen student expertise in cutting edge technologies for promoting their career. Outcome from the proposed research will not only benefit the grape breeders or researcher involved in developing better quality grapes but will also help other scientists, specifically plant physiologists and biotechnologists to understand the mechanism of berry/fruit ripening. The information, material and expertise gained from this project will be extended to identify, isolate and characterize important plant and animal genes to promote collaborative research and to enhance developmental biology research at other 1890 institutions there by strengthening ongoing multi-institutional collaborative projects. This would also add immense sequence information to the database. This project will also provide opportunity for student experiential learning where they will learn experimental design, sample collection and analysis, and core bio-techniques such as preparation of buffers, RNA and protein isolation, PCR, cDNA library construction, Microarray chip hybridization and chip analysis, bioinformatics, metabolite analysis, etc. Apart from this they will also learn primer designing, database searching, sequencing, microarray data analysis, cloning, various analytical techniques, report writing and research presentation. A series of workshops and presentations will be conducted during the field days and consumer meetings, and brochures and pamphlets will be developed to increase awareness among the consumers about the health benefits of muscadine grape. The outcome of this research will be disseminated to the scientific community, grape growers and consumer groups through presentations at the conferences and publications in peer reviewed journals.
Project Methods
mRNA will be recovered from total RNA isolated from developing berries using mRNA purification kit. cDNA will be synthesized using MMLV reverse transcriptase and 5` and 3` adapter primers using the SMART c-DNA library construction kit. For second strand synthesis, PCR will be carried out on an aliquot of the primary template using Advantage 2 polymerase Mix. High throughput transcript sequencing will be done using Roche 454 sequencer GS FLX System. Approximately 5 μg of each cDNA sample will be sheared via nebulization into small fragments and sequenced in a single 454 run. The sequence data thus obtained will be electronically assembled into a set of unique non-overlapping continuous sequences using the MIRA freeware or the Lasergene V8.1 with SeqMan NGen v2.0 tool. For each contig, a homology search will be performed using BLASTX and BLASTN against NCBI database for functional annotation of the obtained gene. Vitis vinifera microarray chip will be procured from Affymetrix and a comparative study will carried out between and across muscadine and bunch grape genotypes to determine expression pattern of viticulturally important genes. Fluorescent labeled cDNA probes will be prepared by reverse transcription of poly (A+) RNA in the presence of Cy3 or Cy5 coupled aminoallyl-dUTP. Hybridization will be carried out and total number of genes expressed and significantly altered will be identified. DNA Microarray Scanner will be used to determine variation in the expression levels of genes between muscadine and bunch grapes. Genes whose expression is altered will be identified. Specific primers will be designed to the selected developmentally-regulated genes of muscadine berries. One-step Realtime-PCR will be performed from total RNA using the QuantiTect SYBR green Realtime-PCR kit. PCR assay will be carried out with SYBR Green system in the DNA Engine Opticon. Melt curves will be run immediately after the last PCR cycle to examine if the measurements were influenced by primer-dimer pairs. Dissociation curve analysis will be performed after PCR to look for the expression levels of different transcripts during the course of ripening. Identification and characterization of differentially expressed proteins among muscadine genotypes will be done using 2-D electrophoresis (2-DE). The proteins will be quantified using the Gel Doc System. Comparative analysis of 2-DE gel profiles of muscadine and bunch grape genotypes will be carried out using PD Quest software to reveal variation in protein expression during berry maturation and ripening. Target proteins will be excised from gel and subjected to sequencing to determine their identity and function. Glucose, fructose and sucrose levels in berry extracts will be determined by HPLC. Invertase and sucrose synthase activities will be assayed to determine correlation between sugar content and enzyme activity among various grape genotypes. Hands-on experiential training will be provided to students and faculty in developmental biology by providing hands-on experiential learning.

Progress 09/01/10 to 08/31/14

Outputs
Target Audience: The target audiences of the project are the grape growers, wineries and scientists working on grape and other small fruit crops. This research outcome was presented during FAMU Grape Harvest Festival and field days, FAMU-CAFS Open house, Florida Grape Growers Association Meeting, and Plant and Animal Genome Conference where growers, wineries, consumers and scientific community were made aware of the research findings and its benefits. Outcome of this research, for the first time, has provided overview of transcriptional changes during muscadine berry development and ripening. The project data aided grape breeders and other researchers to screen grape populations for selecting desirable genotypes to develop elite grape cultivars and superior products. The project also helped train two minority undergraduate students in laboratory techniques and research methods, and currently, one African American graduate student is conducting thesis research in our laboratory. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided hands-on experiential learning to three Florida STEM scholar students, seven undergraduate and two graduate students as well as six REU students in various molecular and cellular laboratory techniques, data analysis, preparing reports and platform presentations. Further, it increased collaboration and cooperative research among FAMU faculty across the Colleges. The following is the list of presentations made by faculty and students across the Colleges. Oral Presentations Scientists Ramesh Katam, Devaiah M. Kambiranda, Katsumi Sakata, Tiratharaj Singh, Steve V. Sluyter, Paul A. Haynes, Mehboob B. Sheikh, Lekan M. Latinwo. 2013. Characterization of Muscadine Berry Proteome Using Label and Label Free Mass Spectrometry Approaches. HUPO 12th Annual World Congress, Yokohama, Japan. Devaiah Kambiranda, Sheikh M Basha, 2013. Proteome Analysis of Muscadine Berry Ripening. Southern Association of Agricultural Scientists Meeting, Orlando, FL. Devaiah Kambiranda, Sheikh M Basha, 2013. Quantitative Proteome Analysis of Developing and Ripening Muscadine Grape Berry. Plant and Animal Genome conference, San Diego, CA. Undergraduate Student Remy Babich, 2013. Genetic Variation of Leaf Protein Composition among Florida Hybrid Bunch Grape. Presented at NSF-REU workshop, FAMU. Poster Presentation 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. Katam R, Sridhar V, Smith S, Chinthakuntla P, Badisa LV, Kambiranda DM, Musa M, Sluyter SV, Haynes PB, Gottschalk V, Latinwo LM, Basha SM, Cramer G. 2013. Integrative Metabolomics and Proteomics Approach to Identify Anticancer Compounds in Muscadine Grape. Poster presented at Plant and Animal Genome conference, San Diego, CA. How have the results been disseminated to communities of interest? The results of this research werepresentedat the FAMU Grape Harvest Festival, community events, Florida Horticultural Society and Florida Grape Grower Association meetings and International Plant and Animal Genome Conference as well as during laboratory visits by off-campus researchers, Alumni Association, students, grape growers, hobbyist wine makers and public interested in growing grapes. What do you plan to do during the next reporting period to accomplish the goals? This project ended on August 31, 2014 and a new project is being developed to advance the current findings.

Impacts
What was accomplished under these goals? In southeastern United States muscadine grape industry is growing rapidly as interest in grapes and wine increases nationally. Berry composition plays a major role in determining its acceptance for fresh fruit consumption as well as wine making. Because of poor understanding of muscadine berry developmental physiology limited progress has been made in addressing the deficiencies associated with muscadine berry composition and its product quality. Hence, this project was focused on studying muscadine berry components during berry ripening that govern its enological and food functional traits to identify the components that influence its characteristics. In this regard proteome, transcriptome and biochemical changes were monitored at different berry developmental stages to better understand berry ripening physiology of muscadine grapes. Sugars produced as a result of photosynthesis are transferred to the berry in the form of sucrose where it is converted to glucose and fructose by the action of invertase and sucrose synthase during veraison. Any limitations in these steps can alter sugar content and composition, and adversely affect product quality and value. To identify the problems associated with sugar metabolism, sugar composition and sugar metabolizing enzyme activities were studied during grape berry development and ripening. Variation in sugar content and composition was monitored among forty eight muscadine genotypes during berry development and ripening. Sugar analysis revealed that sucrose accumulation in muscadine berry starts at the onset of berry maturation and continues up to ripening, while in Florida hybrid bunch and Bunch grape genotypes, glucose and fructose are the primary sugars and present during all the stages of berry development and ripening. Our studies have revealed that invertase enzyme required for breakdown of sucrose is low in muscadine grapes compared to bunch grapes. Additional studies on identifying factors responsible for low invertase activity of muscadine grapes revealed presence of invertase-inhibitor in muscadine berry. These data suggested that low Invertase activity during veraison may be the primary reason for sucrose buildup in muscadine berry while high levels of invertase present in Florida hybrid bunch and Bunch grapes hydrolyzes sucrose resulting in glucose and sucrose accumulation. Furthermore, to investigate gene and proteome changes during berry development and characterize traits important for superior enological characteristics, sugar metabolism, disease tolerance and secondary metabolism, global transcript and protein profile of muscadine grapes (cv. Noble) at key stages of development were determined. Analysis of developing and ripening muscadine berries revealed presence of highest amounts of differentially expressed transcripts between pre-veraison and veraison stages (2,409-group1). Differentially-expressed transcripts in post-veraison and ripe berries were 550 (group-2) and 60 (group-3), respectively. Among the group-1 and group-2, highest number of transcripts belonged to signaling, transport and regulation of gene expression, where as in group-3, cellular processes and transport were the highest number of transcripts. Our results also indicated that as ripening progressed, photosynthesis, protein synthesis and nucleotide metabolism were turned off and transcripts involved in glycolysis were active. From the phenylpropanoid/stilbene biosynthetic pathway at least 46 transcripts were up-regulated in ripe berries when compared to veraison and immature berries, and 12 terpene synthases were predominantly detected only in a single sample. Prominent expression of sucrose synthase enzyme was detected in ripe berries, and decreased level of invertase enzyme gene expression was noticed during ripening. Proteome analysis using iTRAQ (isobaric tags for relative and absolute quantitative measurement) revealed changes in sugars, amino acids and organic acids metabolism. New proteins associated with antioxidant activity, biotic and abiotic stress tolerance related proteins such as nulceredoxin, polygalacturance protein, vicilin-like antimicrobial peptides and major latex proteins that were not previously reported during berry development were identified. Several flavor and aroma related proteins unique to muscadine grape were also identified. These findings indicate that muscadine grapes uniquely express proteins related to the production of various enologically-important components that are not expressed in other grape species. A switch from invertase to sucrose synthase was also observed in muscadine grape berry that may play an active role in sugar metabolism. Overall, the proteome research revealed expression of 674 proteins, of which 76 proteins showed a 1.5 fold change in maturing and ripening berry. In conclusion, our biochemical, gene and proteome research revealed that key enzymes required for active sugar breakdown were down-regulated in muscadine grape berry compared to bunch grape, which produce superior wines. This work has also provided an overview of the cellular process and metabolic pathways active in muscadine berry, for the first time, and identified the functional role of novel genes and proteins associated with unique muscadine berry characteristics which will be useful for understanding and manipulating organoleptic properties of muscadine grape and other fruit crops, in general.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Devaiah Kambiranda, Ramesh Katam, Sheikh M Basha, Shalom Seibert (2013). iTRAQ Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry. Journal of Proteome Research: DOI 10.1021/pr400731p.


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: The target audiences of the project are the grape growers, wineries and scientists working on grape and other small fruit crops. This research outcome was presented at FAMU field days, Florida Grape Growers Association Meeting, Plant and Animal Genome Conference and Southern Association of Agriculture Scientist meeting where growers, wineries and scientific community were made aware of the research findings and its benefits. The research outcome provided understanding of muscadine grape berry physiology and helped identify genes and proteins coding for value-added traits to study genetic variation in grape. The project data aided grape breeders and other researchers to screen grape populations for selecting desirable genotypes to develop elite grape cultivars. The project also helped train six underserved minority undergraduate students in laboratory techniques and research methods, and currently two African American graduate students are conducting thesis research in our laboratory. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided hands-on experiential learning to several undergraduate and graduate students in various molecular and cellular laboratory techniques, data analysis, preparing reports and platform presentations. Further it increased collaboration and cooperative research among FAMU faculty across the Colleges. The following is the list of presentations made by faculty and students across the Colleges. Oral Presentations Scientists Devaiah Kambiranda, Sheikh M Basha, 2013. Proteome Analysis of Muscadine Berry Ripening. Southern Association of Agricultural Scientists Meeting, Orlando, FL. Devaiah Kambiranda, Sheikh M Basha, 2013. Quantitative Proteome Analysis of Developing and Ripening Muscadine Grape Berry. Plant and Animal Genome conference, San Diego, CA. Undergraduate Student Vescio K, Kambiranda D, Reitzel M, Potlakayala SD. 2012. Proteome profiles of American and Chinese chestnut in response to Salicylic Acid treatment. Presented at the American Chestnut Annual Conference. High School Student 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. Poster Presentation 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. Katam R, Sridhar V, Smith S, Chinthakuntla P, Badisa LV, Kambiranda DM, Musa M, Sluyter SV, Haynes PB, Gottschalk V, Latinwo LM, Basha SM, Cramer G. 2013. Integrative Metabolomics and Proteomics Approach to Identify Anticancer Compounds in Muscadine Grape. Poster presented at Plant and Animal Genome conference, San Diego, CA. How have the results been disseminated to communities of interest? The results of this research werepresentedat the Florida Horticultural Society and Florida Grape growers association meeting as well as during laboratory visits by off-campus researchers, students, grape growers, hobbyist wine makers and public interested in growing grapes. What do you plan to do during the next reporting period to accomplish the goals? Enzyme analysis will be carried outin 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. Global transcriptome analysis (RNA-Seq) at key stages of development will be undertaken to perform a comprehensive analysis of gene families contributing to commercially important berry characteristics. By performing RNA deep sequencing we propose to compare the obtained transcriptome data with the Vitis vinifera mRNA reference sequence and also perform de novo sequence assembly to identify private genes present in muscadine grapes.

Impacts
What was accomplished under these goals? Our studies have revealedthat muscadine grapes contain sucrose, glucose and fructose as major sugars, and sucrose accumulation starts at the onset of berry maturation and gradually increases during veraison. Enzyme assays revealed thatunlike bunch grapes Invertase, the enzyme responsible for sucrose hydrolysis is lower andsucrose synthase is not down-regulated during veraison in muscadine grape. In this study proteome analysis of muscadine grape wasundertakento reveal global proteinexpressionof enologically-important traitsduring berry development and ripening. Berry proteins were isolated from four developmental stages, trypsin digested, peptides labeled with different isobaric tags and subjected to LC/MS/MS. Raw data was subjected to bio-informatics analysis to reveal the function, identity and quantity of proteins at each stage of berry development. A total number of 674 unique proteins were detected across four berry developmental stages in four replications. Of these, 76 proteins were determined to be highly expressed across four time points in muscadine berry. Expression of these proteins across berry development was confirmed by real time PCR. The results indicated that glycolytic enzymes, glyceraldehyde phosphate dehydrogenase and enolase were highly active in ripe berries indicating active glycolysis. Proteome and real time PCR analysis confirmed that sucrose synthase was active post-veraison in muscadine berries. Invertase-inhibitor protein was also found to be up-regulated during muscadine berry ripening.Other proteins of interest detected include flavor and aroma proteins, alcohol dehydrogenase, hydroperoxide lyase andisopentynyl diphospahte isomerase which are involved in the production of terpenes. The proteome data indicated that although several cellular process are similar toVitis vinifera the expression pattern of glycolytic enzymes, PR proteins, aroma and flavor proteinsis unique to muscadine suggesting that muscadine grapes have different regulatory mechanism than bunch grapes. Muscadine grape proteome analysis also indicated that sucrose synthase is active in ripe berries and participate in sucrose hydrolysis. These data suggest that higher expression of Invertase-inhibitor during muscadine berry ripening may regulate Invertase enzyme expression and responsible for its lower Invertase activity which leads to high sucrose accumulation in the berry.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: P0876- 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. P0875- Katam R, Sridhar V, Smith S, Chinthakuntla P, Badisa LV, Kambiranda DM, Musa M, Sluyter SV, Haynes PB, Gottschalk V, Latinwo LM, Basha SM, Cramer G. 2013. Integrative Metabolomics and Proteomics Approach to Identify Anticancer Compounds in Muscadine Grape. Poster presented at Plant and Animal Genome conference, San Diego, CA


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: In southeastern United States muscadine grape industry is growing rapidly as interest in grapes and wine increases nationally. However, muscadine grape products are not readily acceptable to the consumers because of its undesirable characteristics. Berry composition plays a major role in determining its acceptance for fresh fruit consumption or wine making. Because of poor understanding of berry developmental physiology limited progress has been made in addressing the problems associated with muscadine berry ripening. Sugars produced as a result of photosynthesis in the leaves are transferred to the berry in the form of sucrose where it is converted to glucose and fructose by the action of invertase and sucrose synthase during veraison. Any limitations at these steps can alter sugar content and composition, and affect berry quality. Our studies using HPLC have showed that Muscadine grapes primarily contain sucrose, glucose and fructose in ripened berries. Studies on changes in sugar accumulation during muscadine berry development and ripening have revealed glucose and fructose as the primary sugars in Bunch and Florida hybrid bunch while Muscadine grapes contain glucose, fructose and sucrose. Developmental studies of muscadine grape berry showed that sucrose accumulation in muscadine berry starts at the onset of berry maturation and continue up to ripening while in Florida hybrid bunch and Bunch grape genotypes glucose and fructose are the primary sugars and present during all the stages of berry development and ripening. To determine reasons for the observed differences in sugar composition among grape genotypes, changes in sucrose accumulation pattern and invertase enzyme activity levels were studied. The results revealed major variation in the acid invertase activity among Vitis species. Highest invertase enzyme activity was observed in bunch genotypes followed by Florida hybrid bunch and muscadine. Changes in sucrose metabolizing enzyme levels in berry during berry maturation and veraison were monitored to determine interrelationship between sugar content and composition and sugar metabolizing enzyme activity. Results showed that invertase and sucrose synthase enzyme activities varied among the grape cultivars during berry development. Among the six varieties tested, African Queen had the highest invertase and sucrose synthase activities while Carlos had lowest invertase and sucrose synthase activities. The results indicated that sucrose buildup occurs at the onset of berry maturation in muscadine while in Florida hybrids and Bunch grape only glucose and fructose accumulate during berry development and ripening. These data suggest that low invertase activity in muscadines grapes results in sucrose build up in the berry. To investigate differences in muscadine carbohydrate metabolism a quantitative proteome analysis during berry development and ripening is being performed using iTRAQ labeling and Mass spectrometry sequencing. Preliminary data showed presence of more than 745 proteins across various stages of berry development and ripening as well as quantitative variation of these proteins across berry ripening is revealed. PARTICIPANTS: Mehboob Sheikh, Devaiah Kambiranda, Anse Kaplan, Jarvis Jackson TARGET AUDIENCES: Grape Growers, grape researchers, grape consumers PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
One of the main tasks in grape berry research has always been the investigation of berry development and ripening since grape producers are interested in improving berry quality, increasing yield and applying desirable cultural practices. The main focus of our research is to enhance sugar content and composition of muscadine berry to improve its value and consumer acceptance. Hence this research is aimed at identifying the constraints involved in sugar accumulation and conversion in the berry which affects its quality and limits its usefulness and acceptance. In this regard this research determined variation in sugar content and composition and activity of enzymes related to sucrose-metabolism during berry development using grape cultivars differing in sugar content. The results revealed that muscadine berry sugar composition is distinct from that of other Vitis species, and the major sugar constituents of muscadine berry are sucrose, glucose and fructose. This information is important and reveals that unlike bunch grapes muscadine grapes contain a significant amount of sucrose in grape berry which has a negative effect on berry quality. This result will help focus on causes of sucrose buildup in muscadine berry and its effect on berry quality. Further, our data also revealed that in muscadine grapes sucrose accumulation begins at the onset of veraison and that its invertase activity is low compared to bunch grapes. The low invertase activity appears to be primarily responsible for sucrose build up in the berry and may cause negative feedback on sugar synthesis and accumulation limiting overall sugar content in Muscadine berry. These data also provide insight into the existence of genetic variation in grape berry sugar content and composition among Vitis species and will be a major source for understanding the molecular and cellular basis of sugar deficiency in muscadine grape. Further, our results also indicated that muscadine grape cultivars behave differently than the bunch grape with respect to sucrose metabolism in berry. The data suggest that utilization of sucrose breakdown products (hexokinase and respiration) may be different in muscadines. Quantitative proteomics analysis performed during different stages of berry development revealed expression of proteins involved in various metabolic processes during berry ripening. Further analysis of proteome data will reveal proteins responsible for critical cellular processes and will be useful for better understanding of carbohydrate metabolism in muscadine grapes to enhance their enological characteristics. Proteins identified as unique to muscadine grape will be useful for improving disease and abiotic stress tolerance in other grape species as muscadine are tolerant to most common grape diseases. In addition to research, this project is providing opportunity to undergraduate and graduate students for obtaining hands-on experiential learning in biochemical analysis, proteomic technologies and Bioinformatics data analysis which will enable them to pursue higher studies and seek job opportunities in high tech agriculture and biomedical sciences.

Publications

  • Sheikh MB, Vasanthaiah HKN, Kambiranda DM. 2012. Genetic Variation in Sugar Composition among Muscadine, Florida Hybrid Bunch and Bunch Grape Genotypes. International Journal of Wine Research, 4: 15-23.


Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Grape berry composition plays a major role in determining fruit and wine quality. Limited progress is made in addressing problems associated with berry ripening relevant to muscadine product quality. To sustain commercial muscadine production it is essential to understand the physiology of berry development and ripening. Sugars produced as a result of photosynthesis are transferred to the berry in the form of sucrose where it is converted to glucose and fructose by the action of invertase and sucrose synthase during veraison. Any limitations in these steps can alter sugar content and composition, and adversely affect product quality and value. To identify the problems associated with sugar metabolism, sugar metabolizing enzyme activities were studied during grape berry development and ripening. Biochemical analysis of berry extracts revealed that glucose and fructose are the primary sugars and their deposition continued up to berry maturation. Sugar accumulation pattern changed dramatically during veraison and ripening among the Vitis species. In muscadine grape, sucrose accumulation began at the onset of veraison and continually increased during ripening. However, in Florida hybrids and bunch grapes glucose and fructose continued to increase during veraison and ripening and were the only sugars present throughout the berry development and ripening. To determine reasons for the observed differential sugar accumulation between muscadine and bunch genotypes we studied variation in invertase activity which breakdown sucrose into glucose and fructose. The results revealed major differences in invertase activity among the Vitis species. Highest invertase activity was observed in bunch followed by Florida hybrid and muscadine. These data clearly demonstrated that bunch grapes exhibit highest invertase activity while muscadines show lowest invertase activity. Changes in other sucrose metabolizing enzyme activity levels in muscadine berry during maturation and veraison were monitored to determine genetic variation in their activity compared to other Vitis species. The results revealed major differences in invertase and sucrose synthase enzyme activity among the Vitis species during berry development and ripening. Of the six muscadine cultivars tested, African Queen showed highest invertase and sucrose synthase activities while Carlos had the lowest invertase and sucrose synthase activities. These results suggested that in muscadine berry, sucrose buildup is initiated at the onset of berry maturation where as in Florida hybrids and Bunch grapes glucose and fructose were the only sugars deposited during all the stages of berry development and ripening. Low invertase activity during veraison appears to be the primary reason for sucrose buildup in muscadine berry while high levels of invertase present in Florida hybrids and Bunch grapes hydrolyzes sucrose resulting in glucose and sucrose buildup. A quantitative proteome analysis is being carried out in developing and ripening muscadine grapes to reveal variation in the levels of enzymes, sugar transporter proteins and possible enzyme inhibitors and their role in carbohydrate metabolism. PARTICIPANTS: Devaiah Kambiranda - Research Associate. Jarvis Jackson - Student Assistant TARGET AUDIENCES: The data was presented at the Annual Florida Grape Growers, Florida State Horticultural Society and American Society for Plant Biology meetings to obtain feedback on how the results obtained could be used to improve berry quality The results were also shared with collaborators and grape scientists through informal discussions, electronic communication and telephone. Results are published in scientific journals of national and international repute. The achievements were also disseminated through extension activities and presentations at various seminars, field days, workshops for the benefits of farmers, public, industry and other scientists PROJECT MODIFICATIONS: Further research will be carried out to investigate the activity of sugar metabolizing enzymes in Muscadine berry during ripening, and determine possible genetic variation in its content and activity. Low invertase activity in muscadine grapes will be further investigated to explore the presence of inhibitors and activators. Large scale proteomic analysis will be performed to analyze protein expression and metabolism in muscadine grapes.

Impacts
Outcome- 1. Sugar analysis revealed sucrose, glucose and fructose accumulation in muscadine berry during veraison and ripening while only glucose and fructose accumulation in Florida hybrid and bunch grapes 2.Invertase enzyme activity was relatively lower in muscadine grapes compared to Bunch and Florida hybrid bunch. 3.The observed variation in sugar composition appears to be the major reason for poor muscadine product quality compared to bunch grape. 4.These data revealed possible genetic regulation of acid invertase activity to control the amount of sugar build up in the grape berry. Impacts- 1.The results obtained revealed interrelationship between sugar accumulation and invertase enzyme activity patterns and occurrence of genetic variation among the Vitis species. 2.The data will aid in understanding the carbohydrate metabolism in Muscadine berry to develop means for improving berry composition to enhance its enological characteristics. 3.This information will benefit in developing strategies for overcoming sugar composition issues in muscadine grape employing genomic tools. 4.The project would also provide opportunity to undergraduate and graduate students for obtaining hands-on experiential learning in biochemical and genomic techniques.

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).
  • Devaiah Kambiranda, Hemanth KN Vasanthaiah Sheikh M. Basha. 2010. Genetic Variation in Sucrose Metabolizing Enzymes among Six Muscadine Varieties. Proceedings of the Florida state Horticultural society, 123: pp. 32-34.