Progress 10/01/00 to 09/30/06
Outputs
Overall goal of this research is to develop grape cultivars with tolerance to anthracnose and Pierce's diseases and high nutraceutical value. We have identified several polypeptides that showed signigicant differences in their expression levels to Elsinoe infection. We have studied expression levels of some of known PR proteins upon Elsinoe infection. The data showed suppression of chitinase and osmotin in anthracnose-susceptible cultivars and no change in expression of stilbene synthase compared to the tolerant cultivars. Differential Display Reverse Transcriptase-PCR (DDRT-PCR) was employed to study uniquely expressed genes and gene that trigger expression of defense related proteins in response to Elsinoe infestation. DDRT-PCR analysis revealed similar results where there was a drastic reduction in the expression of several cDNA transcripts upon infection in susceptible cultivar compared to tolerant cultivar. Uniquely expressed transcripts in tolerant cultivars were
eluted and cloned to pGEM T-Easy vector. Characterization of these cDNAs revealed that they are related to signal transduction, gene regulation, defense, pathogenesis-related proteins. These data suggest that expression of these genes in tolerant cultivars may be responsible for their tolerance to anthracnose. Above experiments revealed that tolerant cultivars express novel genes upon pathogen infection while in the susceptible cultivars these genes are not expressed. Pierces disease in grape is caused by xylem-limited bacterium, Xylella fastidiosa. DDRT-PCR and Subtractive Hybridization (SSH) methods were used to isolate uniquely expressed transcripts induced upon challenging grape plants with bacterium, X. fastidiosa. DDRT-PCR results revealed significant up-regulation of cDNA transcripts. These cDNAs were re-amplified and cloned to pGEM-T Easy Vector for further sequencing. Unlike the bunch grape genotypes, the muscadine cultivar, Carlos did not show much difference in the cDNA
profile indicating that infection did not have much impact on its gene expression. SSH yielded several partial cDNAs uniquely expressed in moderately tolerant V. Vinifera compared to tolerant Florida bunch hybrid genotype. The uniquely expressed transcripts obtained through DDRT-PCR and SSH analyses have been successfully cloned, sequenced and characterized. Cloning and sequencing reactions were standardized using different concentrations of the template. Expression of adenosine kinase was noticed only in tolerant cultivar studied, which is suggested to play a role in plant defense. Further study of uniquely expressed genes is in progress, which would aid in understanding the mechanism of induced resistance. We are attempting to derive full-length gene/s, responsible for PD tolerance. This research has helped strengthen ongoing viticulture research and served as a means to recruit and train minority students in agriculture. We have also evaluated grape tissue extracts and found them
to show anticancer activity against lung and breast cancer lines. Studies are in progress to determine the nature and identity of these components.
Impacts
This research is aimed at identifying cDNA transcripts and metabolites associated with disease and drought tolerance characteristics of grape for improving value-added traits of muscadine and bunch grape genotypes. Most of the muscadine and Florida hybrid bunch grape genotypes posses various degree tolerance to diseases and nutraceutical value. Understanding the defense mechanism of these genotypes and identifying components responsible for the desirable traits is essential in effectively solving the problems associated with grape production in this region. Results of this research would help identify genetic and cellular components associated with the above issues and help formulate strategies to develop grape cultivars suitable for southeastern USA. Identification of phenolic compounds with anticancer activity would help expand grape production and develop value-added products for increasing farmer's profits and product marketability. Furthermore, this project has
provided hands-on training to several students in biotechnology and help build on-campus capacity in biotechnology.
Publications
- Vasanthaiah H KN, Ramesh Katam, and Sheikh M Basha. (2007) Efficient Protocol for Isolation of Functional RNA from Different Grape Tissue Rich in Polyphenols and Polysaccharides for Gene Expression Studies. Australian Journal of Enology and Viticulture, In Review
- Vasanthaiah, H. K. N. and Basha, S. M, (2007) Transcripts induced upon Xylella fastidiosa infection in Grapes -25 sequences submitted to NCBI database
- Ramesh Katam, H KN Vasanthaiah and SM Basha (2006) Differential expression of mRNA transcripts and proteins in leaf tissues of peanut genotypes to water stress. Plant and Animal Genomes XIV Conference, Jan 14-18, 2006 San Diego, CA
- Sheikh M Basha, Ramesh Katam, and Naik, KSS (2006). Identification of Drought responsive proteins in peanut. Association of Research Directors, Inc. 14th Biennial Research Symposium April 1-4, 2006, Atlanta US p 82
- Ramesh Katam, Hemanth KNV and Sheikh M Basha (2006) Water Stress Induced compositional changes in peanut leaf. Association of Research Directors, Inc. 14th Biennial Research Symposium April 1-4, 2006, Atlanta US p 83
- Hemanth KNV, Ramesh Katam, and Sheikh M Basha (2006) Identification of Differentially expressed gene/s in Anthracnose-tolerant grape genotype. Association of Research Directors, Inc. 14th Biennial Research Symposium April 1-4, 2006, Atlanta US p 84-85 Ramesh Katam, Hemanth KNV and Sheikh M Basha (2006) Differential Response of Grape genotypes to water stress, ARD Inc.14th Biennial Research Symposium April 1-4, 2006, Atlanta US p. 237
- Ramesh Katam, Hemanth KNV and Sheikh M Basha (2006) Differences in Leaf Protein Expression among Peanut Genotypes in Response to Water Stress. Annual Meeting of American Peanut research and Education Society, Savannah, GA Record 2465
- Ramesh Katam, Hemanth KNV and Sheikh M Basha (2006) Identification of water deficit responsive polypeptides in Florida Hybrid Grapes. Southern section American Society of Plant Biologists March 3-5, University of South Alabama, Mobile, AL Vasanthaiah H KN Ramesh Katam, and Sheikh M Basha (2006) Water Stress Induced Differential Gene Expression in Peanut. Proceedings of American Peanut Research and Education Society, Savannah, GA. Record 2392
- Sheikh M Basha, Ramesh Katam, and Naik KSS.(2006)Identification of Drought Responsive Proteins in Peanut. Peanut Science- In Review
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Progress 01/01/05 to 12/31/05
Outputs
Anthracnose and Pierce disease (PD) are the major diseases in grape causing severe damage to the crop. Determination of biochemical and molecular differences between anthracnose-susceptible and anthracnose-resistant Florida bunch grape would provide the basis of resistance for Anthracnose. A multiparallel analysis of mRNA and proteins were carried out to determine differential expression of mRNA transcripts and proteins in muscadine and Florida bunch hybrid grape genotypes. Muscadine genotypes are included in the study as they are known to have significant tolerance to both biotic and abiotic stresses. The results showed that, there is a significant up-regulation of transcripts (500-800 bp) in anthracnose-tolerant Florida bunch hybrids (Lake Emerald and Blue Lake) compared to anthracnose-susceptible genotypes (Blanc du Bois and Suwannee). Similarly, leaf protein analysis showed significant suppression of several polypeptides (MW 14 to 50 kDa) in anthracnose infected
leaf. Research was also carried out to identify PD associated biochemical and molecular components. The data showed that several high molecular weight polypeptides (>70 kDa) are more pronounced in muscadine genotypes signifying their association with tolerance/resistance to Pierce Disease. The results were confirmed with the molecular studies where differential display RTPCR (DDRT) revealed that gene expression is significantly decreased with increasing periods of PD infection as compared to the uninfected plants. Moreover, gene expression in disease-tolerant genotype (cv. Zinfandel) is least affected compared to the disease-susceptible genotype (cv. Merlot). Water stress is known enhance susceptibility of plants to various diseases. Studies on the effect of water stress on leaf composition showed that water deficit progressively suppressed protein expression in grape leaf. Relationship between water deficit and disease infestation is being studied. Hazing of wine is a major problem
affecting shelf life of muscadine wines. To identify and characterize the haze components, berry and juice samples were collected at different periods before and after juice fermentation. The precipitate and the supernatant resulting during different periods of wine raking were collected and stored. Protocols are being developed to analyze various components of these samples. Berry tissue is being evaluated for their nutraceutical value, especially anticancer activity. Skin, pulp and seed tissue were extracted with various solvents, concentrated and evaluated against human breast and lung cancer cells in vitro. Preliminary data showed that grape tissue suppressed cancer cell growth by 48 to 60%. Protocols are being developed to obtain more concentrated potent compounds for use as food supplement or drug to control various cancers.
Impacts
Our studies are focused at identifying gene/s and gene products uniquely associated with PD, Anthracnose and drought tolerance for developing bunch grape genotypes suitable for southeastern United States. Muscadine grapes are known to be a good source for nutraceutical compounds as well as resistance source for disease and drought tolerance traits. Therefore, identification and characterization of the cellular and molecular components associated with these traits would help enhance the disease tolerance, nutraceutical value and stress tolerance characteristics of grape genotypes. Outcome of this research would aid in cloning the genes for incorporating into commercial grape cultivars and develop value added grape products for improving farmers profits and consumer health. Determination of biochemical and molecular differences between anthracnose-susceptible and anthracnose-resistant Florida bunch grape genotypes would provide understanding of the molecular and
biochemical basis of resistance to these diseases. Identification of berry proteins responsible for wine hazing would help standardize or modify fermentation conditions to improve shelf life of muscadine wines. It is important to investigate these components as they are known to contribute to the quality of wines such as color, flavor, stability, etc. Research on evaluating various Muscadine berry extracts for their biological activity against human cancer cells will provide us the alternate source of utilizing the grape byproducts for controlling human cancer. This will increase product value and farmers benefit.
Publications
- Basha SM, Mazhar H, Katam R and HKN Vasanthaiah. (2006) Improving grape disease tolerance and nutraceutical values via biotechnology. Annual Meeting of the Florida Grape Association, Ocala, FL Basha, SM, Katam R, Mazhar, H and KSS Naik. (2005). Differential Response of Peanut Genotypes to Water Stress. Prospects and Emerging Opportunities for peanut Quality and Utilization Technology, Kasetsart University, Bangkok, Thailand. Basha, SM, Katam R, KSS Naik (2005) Variation in Drought induced protein Expression among peanut genotypes. 37th Annual Meeting of American Peanut Research and Education Society, Portsmouth, Virginia, July 11-15, 2005
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Progress 01/01/04 to 12/31/04
Outputs
Phenolics are of great importance for grape-derived food and drink products because they are responsible for their organoleptic properties. Compositional changes in phenolics compounds of grape berry during development are of interest because of their importance in wine and juice quality. Phenolics compounds contribute substantially to the quality of wines affecting color, flavor, stability and aging behavior. Grape phenolics and proteins have been implicated as contributors of haze formation in grape wine and juice, a characteristic that is not acceptable by consumer. In addition, phenolics compounds possess antioxidant properties because of their free radical trapping behavior. Research was conducted to determine accumulation pattern of phenolics during berry development and to determine genetic variation in total phenolics compounds of muscadine grape genotypes. Total phenolics were extracted from seed, skin and pulp tissue of 15 muscadine and five Florida hybrid
grape genotypes and quantified. The results showed that the seed contained significantly higher levels of phenolics followed by skin and pulp. The phenolics content of the tissue varied between 0.02% and 8% (% Gallic acid equivalent /g tissue). The phenolics content was higher at the onset of berry development. Phenolics content of the pulp decreased while that of the skin remained similar during berry maturation. In contrast, phenolics content of the seed increased with increasing berry maturity up to 95 days post-flowering suggesting that accumulation of phenolics continued during seed maturation. However, phenolics content of the seed from over-ripe berries decreased. Antioxidant activity of the berries varied widely among the genotypes during berry maturation. Among the muscadine genotypes, cv.Summit showed the highest (222 mg TEAC/g) antioxidant activity while Farrar showed the lowest (45.81 mg TEAC/g) activity.
Impacts
Muscadine grape genotypes are tolerant to most of the diseases including feared Pierce's disease. Identification and isolation of gene/s and gene products responsible for disease tolerance and nutraceutical value for incorporation into popular grape genotypes is desirable. Our studies are aimed at identifying gene/s and gene products uniquely associated with PD tolerance for developing bunch grape genotypes suitable for cultivation in the southeastern United States. In addition, Muscadine grapes are rich in nutraceutical compounds such as phenolics, and contain high antioxidant activity. Identification and characterization of the cellular and molecular components associated with the phenolics would help enhance the neutraceutical value of grape. This would aid in the development of value added grape products for improving farmers profits and consumer health.
Publications
- Changes in phenolics and antioxidant activity of muscadine grape genotypes during berry development. 2004. American Society of Enology and Viticulture, San Diego, CA.
- Differential response of peanut to water stress. 2005 International Peanut Confernece, Bangkok, Thailand.
- Differences in xylem sap composition among grape genotypes. 2005. Annual Meeting of Florida Grape Growers Association. Ocala, FL.
- Interrelationship between xylem sap composition and Pierce's disease tolerance. 2005. MANNARS General Conference, Pittsburgh, PA.
- Immunochemical charactrization of methionine-rich proteins from peanut. 2004. Australian J. Experimental Agriculture 44: 1-4.
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Progress 01/01/03 to 12/31/03
Outputs
Studies on protein composition of xylem and phloem indicated that total protein content of the xylem varied greatly between bunch (Blanc du Bois 0.46%) and muscadine (Noble 0.85%) genotypes. Berries of anthracnose tolerant cultivars (Blue Lake and Lake Emerald) deposited a 21000 Dalton protein very early during berry development while; this protein was seen only in the ripe berries of the anthracnose-susceptible (Suwannee and Blanc du Bois) cultivars. Western Blotting with anti-chitinase antisera failed to react with any of the grape protein bands indicating absence of chitinase-type proteins in grape berry. Seed proteins of Muscadine cultivars were identical and separated into ten distinct bands ranging in molecular weight from 20000 to70000 Daltons. Among Florida bunch hybrids, Lake Emerald showed absence of a 30000 Dalton band in the seed. The skin protein profiles revealed the presence of a 20000 Dalton band in California Bunch and Muscadines genotypes. However,
this band was absent in the Florida Bunch Hybrids. Amino acids and sugars content of the xylem and phloem tissue of resistant and susceptible genotypes differed significantly. Phenolics content and composition varied widely among the grape genotypes. Phenolics data indicated presence of significant levels of phenolics in the skin and seed tissue. In addition, major compositional differences in antioxidant activity and phenolic content were also observed among the grape genotypes.
Impacts
Muscadine grape genotypes are tolerant to most of the diseases including feared Pierces disease. Identification and isolation of PD-tolerant gene/s from Muscadine genotypes for incorporation into popular bunch grape genotypes is desirable. Our studies are aimed at identifying gene products uniquely associated with PD tolerance for possible incorporation into PD-susceptible bunch grape genotypes. In addition, Muscadine genotypes are rich in phenolics and contain high antioxidant activity. Studies on these compounds would help enhance neutraceutical value of grape products.
Publications
- Mazhar, H. and Basha, S.M. 2004. Immunochemical characterization of methionine-rich proteins from peanut. Australian J. Experimental Agriculture (In Press).
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Progress 01/01/02 to 12/31/02
Outputs
For identifying the components of disease resistance in muscadine and Florida bunch hybrids, comparative biochemical studies have been conducted using susceptible bunch and tolerant muscadine genotypes. The studies have revealed significant quantitative and qualitative differences in protein content and composition between the tolerant and susceptible grape genotypes. Muscadine cultivars have higher levels of protein in berry, leaf and xylem tissue as compared to vinifera bunch genotypes. Gel electrophoretic studies have showed that high molecular weight polypeptides (above 70 kDa) were more pronounced in muscadine genotypes suggesting that some of these polypeptides may be involved in disease tolerance/resistance. Capillary Electrophoresis (CE) studies also showed major qualitative and quantitative differences in specific proteins between the tolerant and susceptible genotypes. Studies are in progress to isolate the proteins unique to disease tolerant genotypes for
use in anti-microbial assays to confirm their microbial toxicity. Further studies on differences in protein expression during development and ripening, and pathogen attack between susceptible and tolerant genotypes will help in identifying proteins/polypeptides expressed differentially between the tolerant and susceptible genotypes.
Impacts
Studies aimed at identifying the gene products such as proteins and polypeptides unique to tolerant genotypes will help in determining the biochemical basis of disease resistance, and clone genes corresponding to the proteins involved in resistance. Once the target gene/s is identified they can be incorporated into the established commercial genotypes. Such genotypes will greatly benefit the growers, grape industry and consumers.
Publications
- Mazhar, H. and Basha, S.M. 2002. Effect of desiccation on peanut (Arachis hypogaea L.) seed protein composition. Environmental and Experimental Botany 47: 67-75.
- Jain, A., Basha, S.M., Lu, J., Lorenzo, A. and Leong, S. 2002. Genetic variation and developmental changes in the leaf and fruit sugar content of grape genotypes, Proc. Fla. State Hort. Soc. (In Press).
- Jain, A., Basha, S.M., Leong, S and Mbuya, O. 2002. Differential accumulation of free amino acids in the leaf and fruit of muscadine and Florida bunch grape genotypes. Proc. Fla. State Hort. Soc. (In Press).
- Mazhar, H. and Basha, S.M. 2000. Immunochemical characterization of methionine-rich proteins from peanut. African J. Biotechnology. (Submitted).
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Progress 01/01/01 to 12/31/01
Outputs
A. Pierce's Disease. DNA sequence data of known resistant genes such as Xa21, Pto, Rps, N, L6, and Cf9 were retrieved from Gene Bank database. Based on sequence comparison, eight sense and six anti-sense primers were designed and oligo-nucleotide primers were synthesized. Genomic DNA from five wild grape genotypes, five vinifera bunch, five Florida bunch and three-muscadine grape genotypes was isolated and purified. The oligo-nucleotide primers were used in different combinations to amplify homologous DNA sequences from grape lines using the genomic DNA and PCR methods. Several DNA bands ranging between 200 to 600 bp were amplified only in Florida bunch and muscadine genotypes indicating specific amplification in the tolerant/resistant grape genotypes. Ten of these differentially amplified DNA bands were isolated from the gel and DNA fragments were purified. These fragments were cloned in to a sequencing vector (pKS Bluescript+) and sequenced. The DNA sequence of
these fragments was blasted to NCBI Gene Bank for homology search. DNA sequence of these fragments showed that two of these fragments have 23-28 % homology with plant disease resistant (PR) genes. For further characterization, genomic DNA of grape genotypes was digested with Eco RI and blotted on a membrane; and hybridized with radiolabelled probe of these fragments. Both of these fragments showed positive hybridization signals in muscadine genotypes only indicating that these fragments were positively associated with resistance/tolerance. ii)Biochemical studies-To identify the components of disease resistance, comparative biochemical and molecular studies between susceptible bunch and tolerant muscadine were conducted. A positive correlation in the total protein content and disease tolerance was observed. Muscadine cultivars had higher levels of protein in xylem tissue as compared to vinifera bunch. The polypeptide profile showed that high molecular weight polypeptides (> 70 kDa)
were more pronounced in muscadine genotypes indicating that these polypeptides might be associated with tolerance/resistance to PD. B. Anthracnose: Leaf and berry samples corresponding to different developmental stages were collected from anthracnose susceptible and resistant bunch cultivars along with muscadine cultivars. Protein analysis showed presence of a 30kDa protein in the anthracnose-tolerant bunch and muscadine genotypes. This protein however, was absent in the susceptible cultivars. A protein product, viz. Messenger was applied to bunch cultivars at two-week intervals. Following Messenger application the plants were evaluated. The plants that received Messenger were healthy and free from disease while the unsprayed control was heavily infected with anthracnose. Tissue analysis showed compositional differences between sprayed and unsprayed plants. C. Phytochemical s. Liquid/liquid extraction method was performed to obtain various fractions containing different classes of
phytochemicals. TLC analysis of these fractions showed differences in the polar and non-polar constituents of leaves, stem, fruit, juice and wines of bunch and muscadine grapes
Impacts
Studies aimed at identifying the gene/s and gene products unique to PD tolerant genotypes will aid in determining the biochemical and molecular basis of disease tolerance and in the identification of specific gene/s involved in Pierce's Disease resistance. Usage of natural compounds to control fungal disease will provide an environmentally safe means to control grape diseases. In addition, this study will help in identifying unique proteins induced following Messenger application to help identify the genes and gene products involved in disease resistance. Characterization of phytochemicals will help in developing value added grape products to aid consumer health.
Publications
- Mazhar, H. Basha, S.M. and Lu, J. 2001. Variation in the berry protein compositon of muscadine cultivars. American J. Enology and Viticulture 53:266-270.
- Ashok Jain, S. M. Basha and Jiang Lu. 2002. Variation in the sugar accumulation pattern of muscadine grape genotypes. Journal of the American Society for Horticultural Science (In review).
- Ashok Jain and S. M. Basha. 2002. Variation in the amino acid accumulation pattern of muscadine grape genotypes. (In Preparation).
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