Progress 12/01/09 to 11/30/13
Outputs
Target Audience: Although the importance of aroma in the determination of food quality is well recognized, little is known in the area of molecular biology and biochemistry of aroma biosynthesis. The goal of my research program is to establish the knowledge base and tools that are necessary to improve fruit flavor quality. As such, this research is of interest to scientists who study plant volatile metabolism and the role of volatile compounds in plant biology. Moreover, this research has direct implications for breeders, geneticists, and the fresh produce industry by providing the basic knowledge required for enhancing fruit flavor quality. The PI regularly talks to stakeholders in these industry segments (seed companies, postharvest industry players…) to disseminate the results of this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Project leader Zakharov coordinated the work and the dissemination of results. A graduate student (Minmin Wang) and two Postdoctoral Researchers (Dr. Lei Zhang and Dr. Kyung Hwan Boo) carried out the experiments described in the project and analyzed results along with Zakharov. We are committed to training undergraduate students as part of the project, which has a great potential to expose students to a wide array of research methods. Over the course of this project, seven undergraduate students (Setong Mavong, Elise Croda, Jean Crilly, Khanh Hien Tran, Sharon Wei, Kelvin Ho and Saengking Saechao) have been involved in the research and trained by the graduate student and postdoctoral researchers. This project on amino acid metabolism toward volatile biosynthesis also allowed us to develop a collaboration with the laboratory of Dr. Dudareva at Purdue University. How have the results been disseminated to communities of interest? Our results were presented at various scientific conferences throughout the course of the project: - Banff Conference on Metabolism, 2010 and 2012 (Banff, Canada); poster presentations. - Gordon Research Conference on Plant Metabolic Engineering, 2011 (Waterville Valley, NH); poster presentations. - SOL&ICuGI 2011 conference (Kobe, Japan); invited talk. - Gordon Research Conference on Plant Volatiles, 2012 (Ventura, CA); invited talk and poster presentations. Zakharov also regularly talked to stakeholders in the produce industry (seed companies, postharvest industry players…) to disseminate the results of this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are known to play a key role in energy supply during hypoxic or anoxic conditions in plants. While numerous plant PDC genes have been identified, the enzymes encoded by these genes have rarely been characterized at the biochemical level. Plant PDCs have also been hypothesized to be involved in fruit volatile biosynthesis, particularly in branched-chain amino acid (BCAA)-derived ester production. To date, however, there was no direct biochemical evidence for the decarboxylation of α-keto acids in fruit. Since BCAA-derived volatiles are abundant in melon fruit aroma, we investigated the role of melon PDCs in fruit volatile biosynthesis. We identified two melon pyruvate decarboxylases, PDC1 and PDC2, which were expressed in fruit tissues. The expression of PDC1 was highest in ripe melon while PDC2 was more highly expressed in tissues other than fruit. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could carry out the efficient decarboxylation of pyruvate and that it also had significant activity toward a range of straight- and branched-chain α-keto acid substrates. On the other hand, PDC2 had very low or no activity toward all ?-keto acids tested. RNAi-mediated silencing of PDC1 and/or PDC2 in melon showed that PDC1 is involved in acetaldehyde, propanal and pentanal production, while PDC2 does not contribute significantly to volatile biosynthesis in melon fruit. Importantly, our results also demonstrate that neither of these decarboxylases is involved in BCAA-derived aldehyde formation in melon. In addition, precursor feeding experiments showed that branched chain amino acids could aldo be metabolized into aldehydes via an amine oxidation step or a transamination step. Candidate genes involved in the pathway were cloned, including one amine oxidase (AO) and one transaminase (BCAT). The expression of these genes increase throughout the development of the fruit, reaching maximum expression when fruits are fully ripe. The putative amine oxidase was expressed in E. coli and we could demonstrate activity toward 3-methyl butyl amine and 2-methylpropyl amine to form 3-methylbutanal and 2-methylpropanal, respectively. Transgenic RNAi knockouts of the amine oxidase and the transaminase have been generated and fruit volatile profiles from these transgenic lines were analyzed. Our results show that both AO and BCAT participate in volatile production in melon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Gutensohn M, Klempien A, Kaminaga Y, Nagegowda DA, Negre-Zakharov F, Huh JH, Luo H, Weizbauer R, Mengiste T, Tholl D and Dudareva N (2011). Role of Aromatic Aldehyde Synthase in Wounding/Herbivory Response and Flower Scent Production in Different Arabidopsis Ecotypes. Plant Journal 66(4): 591-602
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Wang M. (2013) Investigation of Volatile Ester Biosynthesis in Melon Fruit (Cucumis melo L., botanical group reticulatus). Ph.D. Dissertation.
|
Progress 12/01/11 to 11/30/12
Outputs
OUTPUTS: The goal of this project is to investigate the biochemical pathways leading to the formation of volatile esters that are derived from the branched chain amino acids isoleucine, leucine and valine in cantaloupe melon fruit. One graduate student, one undergraduate student intern and one postdoctoral scientist worked on this project during this review period. Through a combination of biochemical, genomic and biotechnological approaches, we have characterized candidate genes potentially involved in the formation of volatile esters. Our results were presented at the 2012 Banff Conference on Metabolism, where the PI and the graduate student working on this project presented a poster. PARTICIPANTS: Project leader Florence Zakharov coordinates the work and the dissemination of results. A graduate student (Miss Minmin Wang), one undergraduate student intern (Mr. Luke Hedrick) and one Postdoctoral Researcher (Dr. Lei Zhang) carried out the experiments described in the project and analyzed results along with Zakharov. TARGET AUDIENCES: This research has direct implications for breeders, geneticists, and the fresh produce industry by providing the basic knowledge required for enhancing fruit flavor quality. The PI regularly talks to stakeholders in these industry segments (seed companies, postharvest industry player) to disseminate the results of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Precursor feeding experiments showed that branched chain amino acids can be metabolized into aldehydes via either an amine oxidation step, or a transamination step. Candidate genes involved in the pathway were cloned, including two putative decarboxylases, one amine oxidase and one transaminase. Transgenic RNAi knockouts of the candidate genes were generated and confirmed the involvement of one candidate pyruvate decarboxylase in straight-chain volatile biosynthesis. Our results indicated that the candidate genes cloned so far are not involved in branched chain amino acid-derived volatile biosynthesis in melon, prompting us to formulate new hypotheses concerning the biochemical pathways involved in branched chain ester formation in melon.
Publications
- No publications reported this period
|
Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: The goal of this project is to investigate the biochemical pathways leading to the formation of volatile esters that are derived from the branched chain amino acids isoleucine, leucine and valine in cantaloupe melon fruit. One graduate student, two undergraduate student interns and one postdoctoral scientist worked on this project during this review period. Through a combination of biochemical, genomic and biotechnological approaches, we have characterized candidate genes potentially involved in the formation of volatile esters. Our results were presented at the 2011 Gordon Research Conference on Plant Metabolic Engineering (Waterville Valley, NH) where both the PI and the postdoctoral scholar presented a poster, at the SOL&ICuGI 2011 conference (Kobe, Japan) where the PI presented a talk, and the 2012 Gordon Research Conference on Plant Volatiles (Ventura, CA) where the graduate student involved in the project presented a poster. PARTICIPANTS: Project leader Florence Zakharov coordinates the work and the dissemination of results. A graduate student (Miss Minmin Wang), two undergraduate student interns (Miss Jean Crilly and Miss Khanh Hien Tran) and one Postdoctoral Researcher (Dr. Lei Zhang) carried out the experiments described in the project and analyzed results along with Zakharov. TARGET AUDIENCES: This research has direct implications for breeders, geneticists, and the fresh produce industry by providing the basic knowledge required for enhancing fruit flavor quality. The PI regularly talks to stakeholders in these industry segments (seed companies, postharvest industry player) to disseminate the results of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Precursor feeding experiments showed that branched chain amino acids can be metabolized into aldehydes via either an amine oxidation step, or a transamination step. Candidate genes involved in the pathway were cloned, including two putative decarboxylases, one amine oxidase and one transaminase. The expression of these genes increase throughout the development of the fruit, reaching maximum expression when fruits are fully ripe. The putative amine oxidase was expressed in E. coli and we could demonstrate activity toward 3-methyl butyl amine and 2-methylpropyl amine to form 3-methylbutanal and 2-methylpropanal, respectively. One of the putative decarboxylases was also expressed in E. coli and the purified enzyme exhibits activity toward keto-leucine, keto-isoleucine and keto-valine. The biochemical characterization of the remaining putative decarboxylase is underway. Transgenic RNAi knockouts of the candidate genes have been generated and are currently being grown in the greenhouse for later analysis of fruit volatile profiles.
Publications
- No publications reported this period
|
Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: One graduate student and two postdoctoral scientists were trained to carry out the experiments described in this project. These included precursor (1) feeding experiments on melon fruit tissues to investigate the biochemical pathways leading to branched chain amino acid-derived aldehydes, (2) quantitative Real-Time PCR to assess expression levels of target genes during fruit development and ripening, (3) establishment of a transformation procedure for melon in order to down-regulate the expression of target genes in planta. Our results were presented at the 2010 Banff Conference on Plant Metabolism. Our activities on the investigation of amino acid metabolism in melon lead to the development of a collaboration with the group of Dr. Dudareva at Purdue University on the investigation of aromatic amino acid decarboxylases involved in volatile formation in plants. PARTICIPANTS: The PI of the project (Dr. Florence Zakharov) oversees the development of experiments, methods and analyzes results along with other individuals involved in the project, and the dissemination of the results. A graduate student (Miss Minmin Wang) and two Postdoctoral Researchers (Dr. Lei Zhang and Dr. Kyung Hwan Boo) carry out the experiments described in the project and analyze results along with the PI. We are committed to training undergraduate students as part of the project, which has a great potential to expose students to a wide array of research methods. In the past year, 3 undergraduate students (Kelvin Ho, Saengking Saechao and Khanh Hien Tran) have been involved in this project and trained by the graduate student and postdoctoral researchers. We collaborate on other aspects of amino acid metabolism with the laboratory of Dr. Dudareva at Purdue University. TARGET AUDIENCES: This research has direct implications for breeders, geneticists, and the fresh produce industry by providing the basic knowledge required for enhancing fruit flavor quality. The PI regularly talks to stakeholders in these industry segments (seed companies, postharvest industry players..) to disseminate the results of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results demonstrate that the biochemical mechanisms of aldehyde formation in melon fruit are tissue-specific and differ between the pericarp and mesocarp of the fruit. Using precursor feeding experiments, we were able to show that branched chain amino acids are metabolized into aldehydes via an amine oxidation step, in addition to a transamination step. Candidate genes involved in the pathway were cloned and their expression during fruit development and ripening has been assessed. These include two putative decarboxylases, one amine oxidase and one transaminase. The expression of these genes increase throughout the development of the fruit, reaching maximum expression when fruits are fully ripe. A collaborative investigation of aromatic amino acid metabolism in Arabidopsis showed that the production of phenylalanine-derived aldehyde, phenylacetaldehyde, serves as a defense mechanism against attacking herbivores in one ecotype, whereas it is involved in pollinator attraction in another ecotype.
Publications
- Gutensohn M, Klempien A, Kaminaga Y, Nagegowda DA, Negre-Zakharov F, Huh JH, Luo H, Weizbauer R, Mengiste T, Tholl D and Dudareva N (2011). Role of Aromatic Aldehyde Synthase in Wounding/Herbivory Response and Flower Scent Production in Different Arabidopsis Ecotypes. Plant Journal, in press, DOI: 10.1111/j.1365-313X.2011.04515.x
|
|