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INVESTIGATOR: Tharayil, N.

PERFORMING INSTITUTION:
CLEMSON UNIVERSITY
CLEMSON, SOUTH CAROLINA 29634

STRESS INDUCED MODIFICATION OF THE QUANTITY AND COMPOSITION OF PHYTOCHEMICALS IN FRUITS: OPTIMIZING THE NITROGEN SUPPLY FOR ENHANCING THE NU

NON-TECHNICAL SUMMARY: In the present day, where human health often drives the consumer's food choices, increased attention is given to the quality of fruits and vegetables due to their abundance in phytochemicals. Although the production of these protective compounds are generally higher in plants exposed to resource limited conditions, the current production practices of fruits and vegetables are optimized for higher fruit yield. As a consequence, crops grown under optimal conditions to achieve maximum yield may not necessarily develop their full potential for phytochemicals. Using strawberries as a model crop, the main objectives of the proposed research are to optimize production practices i) that balance between fruit yield and phytochemical content and ii) that would alter the composition of phytochemicals in the fruit for a better overall biological nutritive value. Based on this physiology of stress mitigation in plants we hypothesize that i) the total phytochemical content of strawberries will respond non-linearly to decreasing nitrogen (N) supply and ii) along with the changes in their total quantity, the composition and efficiency of phytochemicals would also change with the level of N supply. We will conduct both greenhouse and field experiments to test the effect of different levels of N treatment on the total content and composition of phenolic compounds in different strawberry cultivars. A metabolomics approach will be used to quantify the major metabolites and stable intermediaries in fruits that originate from Calvin cycle, glycolysis, pentose phosphate pathway, urea cycle, shikimate pathway and phenylpropanoid pathway. The nutritional quality of berries will be quantified through antioxidant assays. We further aim to develop a field based assay that will provide information on the phytochemical content of fruit for grower use in marketing programs.

OBJECTIVES: In the present day, where human health often drives the consumer's food choices, increased attention is given to the quality of fruits and vegetables due to their abundance in phytochemicals. Despite the production of these protective compounds being higher in plants exposed to resource limited conditions, the current production practices of fruits and vegetables are optimized for higher fruit yield. The production of phytochemicals in fruits and vegetables are strongly regulated by the environment, as these compounds protect the plants from biotic and abiotic stresses. As a consequence, crops grown under optimal conditions for maximum yield may not necessarily develop their full potential for phytochemicals. The above contrasting association between quantitative yield and nutritive value of fruits and vegetables call for a paradigm shift in cultivation practices that currently are focused on maximizing yield.The overall goal of the project is develop management recommendations whereby growers will be able to enhance and promote the nutritional qualities of strawberries in order to take advantage of increasing markets for healthy foods. Using strawberries as a model crop, the main objectives of the proposed research are to optimize production practices i) that balance between the fruit yield and phytochemical content and ii) that would alter the composition of phytochemicals in the fruit for a better overall biological nutritive value. Further, the project aim is to develop a field-based assay that would robustly capture the phytochemical content of the fruits, which would enable farmers to determine and advertise the overall nutritive value of the produce for quality assessment and marketability.

APPROACH: Greenhouse and field experiments will be done to test the effect of different levels of N treatment on the total content and composition of phenolic compounds in different strawberry cultivars. A non-targeted metabolomics approach will be adopted for the comprehensive analysis of compounds in fruits to robustly capture the overall changes in fruit quality. The samples will be screened for metabolites belonging to different metabolic classes including sugars, sugar alcohols, phenolic compounds, amino acids, and organic acids. This will identify the major metabolites and stable intermediaries that originate from the Calvin cycle, glycolysis, pentose phosphate pathway, urea cycle, shikimate pathway and the phenylpropanoid pathway. Catalytic efficiencies of enzymes involved in flavonoid biosynthesis, and the molecular composition and content of tannins will be measured. The nutritional quality of berries will be quantified through antioxidant assays. A robust and reproducible colorimetric assay to measure the antioxidant potential of compounds in a fruit samples will be developed for grower use.

PROGRESS: 2015/12 TO 2018/12
Target Audience:Scientists, students, and farmers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training to one postdoctoral fellow, one graduate student and three undergraduate student in developing and optimizing the methods for analyzing metabolome of strawberry. Also, the sample cleanup, chromatography and mass spectrometry method developed for this project was used in a graduate level analytical techniques class to quantify the pytonutrients and pesticides in the strawberries (26 participants). How have the results been disseminated to communities of interest?The results of the study was presented at two scientific society meetings. The outcome of the study was conveyed to organic growers. The analytical method developed during the course of this project was incorporated into a graduate course which trained 26 graduate students. One graduate students and three undergraduates were tained on various aspects of this project. One student (female) successfully completed her MS research and graduated, after publishing two journal articles and presenting the results in 3 meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2015/12 TO 2018/12
What was accomplished under these goals? Phenolic metabolites have a myriad of role in plant physiology with the key function of defense against biotic and abiotic stress. Number of studies have demonstrated that resource availability has a major influence on the total content of phenolics; however, the change in the structural variants within phenolics and their localization is largely limited. Nitrogen (N) reduction predominantly enhances phenolic biosynthesis but we hypothesize that there could N dose-dependent selective upregulation of phenolic metabolites of higher bioactivity. In addition, N dose could also regulate the selective distribution of the biosynthesized phenolics between the cellular compartments. The foliar phytochemistry not only plays a key role in determining the overall defense status of the plant but also regulate the transportation of the metabolites between the source and sink tissues of the plant. Thus, in the present study, we evaluated the response both among and within the different phenolic classes of Fragaria ananassa (cv. Camarosa and cv. Albion) leaves to the applied nitrogen (N) fertilizer treatment (control, 8mM N, 16mM N and 30mM N) using a non-targeted metabolomics approach. The primary metabolites exhibited a non-linear response in both the cultivars with similar, increase or decrease in the content of different sugars, sugar alcohols and organic acids with the decrease in N supply. Using UHPLC-ESI-Orbitrap-MS/MS, 149 non-anthocyanin phenolic compounds were tentatively identified that covered 54% of the total ion chromatogram. Both the cultivars exhibited a similar trend to the applied N supply but the magnitude of response with different classes of phenolics varied. Irrespective of the N treatments, ellagitannins constituted the most abundant class of phenolics in strawberry leaves followed by flavonols and proanthocyanidins. The proanthocyanidins content exhibited a much higher fold change variation with a decrease in N treatment whereas the ellagitannins were the least responsive to the applied N supply. The hydroxycinnamates were more abundant but showed lesser fold change than the hydroxybenzoates with the decrease in N supply. Across all the treatments, the hydroxylated flavonols showed higher abundances than the flavones, with preferential accumulation of dihydroxylated flavonol, quercetin, at lower N treatment. Furthermore, a higher percentage of the flavonols were present in glycosylated form than the acylated form. The percentage of tissue-bound form of the different phenolic classes did not show significant variation to the applied N treatments. A higher percentage of total phenolic observed in the soluble form with the reduction in N suggest their possible availability in transport across the plant. The study concludes that the variation among different phenolics is linked to the biosynthetic origin and physiological role of the metabolite. The results highlight the potential application of decreased nitrogen in increasing the bioactive compounds of the foliage, along with developing eco-friendly and economical crop cultivation practice. Complex biomolecules present in their natural sources have been difficult to analyze using traditional analytical approaches. Ultrahigh-performance liquid chromatography (UHPLC-MS/MS) methods have the potential to enhance the discovery of a less well characterized and challenging class of biomolecules in plants, the ellagitannins. We present an approach that allows for the screening of ellagitannins by employing higher energy collision dissociation (HCD) to generate reporter ions for classification and collision-induced dissociation (CID) to generate unique fragmentation spectra for isomeric variants of previously unreported species. Ellagitannin anions efficiently form three characteristic reporter ions after HCD fragmentation that allows for the classification of unknown precursors that we call targeted reporter ion triggering (TRT). We emonstrate how a tandem HCDCID experiment might be used to screen natural sources using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) emerged. The method was verified not to yield false-positive results in complex plant matrices. We were able to identify 154 non-isomeric ellagitannins from strawberry leaves, which is 17 times higher than previously reported in the same matrix. The systematic inclusion of CID spectra for isomers of each species classified as an ellagitannin has never been possible before the development of this approach.

PUBLICATIONS (not previously reported): 2015/12 TO 2018/12
1. Type: Journal Articles Status: Published Year Published: 2018 Citation: Bowers JJ, Gunawardena HP, Cornu A, Narvekar AS, Richieu A, Deffieux D, Quideau S, Tharayil N. Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry. Scientific Reports.8, Article number: 10399
2. Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Narvekar, A. Tharayil, N. Nitrogen limitation induces non âlinear variation in the content, composition and localization among different foliar phenolic classes in strawberry. Frontiers in Plant Sciences.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bowers JJ, Gunawardena HP, Cornu A, Narvekar AS, Richieu A, Deffieux D, Quideau S, Tharayil N. Systematic Evaluation of Polyphenolic Natural Products by Mass Spectrometry. American Society of Mass Spectrometry. Annual Meeting San Diego. May 21, 2018
4. Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bowers JJ, Gunawardena HP, Cornu A, Narvekar AS, Richieu A, Deffieux D, Quideau S, Tharayil N. Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry. Metabolomics 2018. Annual Meeting of the Metabolomics Society. Seattle, Washington, June 24-28, 2018.
5. Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Narvekar, A. Tharayil, N. 2018. Nitrogen stress induced modification of the foliar phytochemical composition in strawberries. Graduate Research And Discovery Symposium (GRADS) 2018, Clemson University.
6. Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Narvekar, A. 2018. INFLUENCE OF ALTERED NITROGEN REGIMES ON THE DYNAMICS OF FOLIAR PHENOLIC OF STRAWBERRY. MS. Thesis, Clemson University, Clemson SC

PROGRESS: 2016/12/15 TO 2017/12/14
Target Audience:The resuls of the study, on how plant sress could be modulated to increase the phytochemical content, was presented at the Crop Science Society of America Annual meeting. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training to one graduate student (female). The project fostered interanational collaboration with scientist at University of Bordeaux, France, in developing mass spectrometric methods for an in-depth profiling of phytochemicals in plants. How have the results been disseminated to communities of interest?The results of the study was presented at the annual meeting of the Crop Science Society of America. What do you plan to do during the next reporting period to accomplish the goals?1) Complete and publish the mass spectral library of ellagitannins. 2) Continue the research on stress response in strawberry, focusing on the influence of N stress on aroma comompounds.

IMPACT: 2016/12/15 TO 2017/12/14
What was accomplished under these goals? PLANT PHYSIOLOGY: Secondary metabolites play a significant role in acclimatizing plants to various environmental stresses. Though many of these secondary metabolites have direct benefits to human health, the current agricultural practices, by providing optimal growing conditions, compromises the biosynthetic capacity of plants to produce phytochemicals. Our work focuses on the nutrient stress dependent inflection point of carbon partitioning between growth and production of phenylpropanoids in plants, and the occurrence of non-linear responses between nitrogen fertilization and phytochemical content. In the present study, we evaluated the effect of four levels of nitrogen fertilizer (12, 23, 44 and 81mg N) on the foliar content, composition and localization of metabolites in two cultivars (Camarosa and Albion) of Fragaria ananassa. Non-targeted metabolomics approach was used to capture the dynamics of primary and secondary metabolites, along with apparent activities of the rate-limiting enzymes of the key metabolic pathways. In general, across both cultivars, the content of primary and secondary metabolites exhibited a contrasting trend with respect to N treatments, where the content of primary metabolites increased with N-sufficiency, whereas the content of secondary metabolites increased with N-deficiency. Non-targeted metabolomics putatively identified more than forty phenylpropanoids encompassing hydroxycinnmate derivatives, flavones, flavonols and flavan-3-ols. Both the total and extractable proanthocyanidins content decreased with increase in N fertilization, which then remained unchanged beyond 44 mg N, exhibiting a non-linear relationship of phytochemical content with N-treatments. In contrast, ellagitannins, encompassing castalgin and geraniin, initially decreased with increasing N, but increased at highest rate of N fertilization. Flavone content decrease from 12 to 44 mg N, but increased at 81mg N; however, flavonols did not show the increase at higher N-level, elucidating potential role of difference in the hydroxylation as a mode to mitigate N stress. In general, plants receiving lower N rates exhibited highest antioxidant capacity. METHOD DEVELOPMENT: The quality and composition of nutraceuticals derived from fruits and vegetables has been placed under greater scrutiny in recent years in part due to the willingness of health-conscious consumers to spend more for higher quality agricultural products. In addition to bioactive compounds such as vitamins E, C, sterols and carotenoids, the polyphenolic compounds in plants offer various degrees of antioxidant, anticancer, antimicrobial, anti-inflammatory, and anti-neurodegenerative benefits.One specific group of polyphenolic metabolites that is of a higher nutraceutical value and is widely distributed in higher plants are ellagitannins. How The hexahydroxydiphenoyl (HHDP) group of ellagitannins has been leveraged in quantitative methods to measure ellagitannin content since hydrolysis liberates hexahydroxydiphenic acid that rapidly lactonizes into ellagic acid, irrespective of the chemical identity of the ellagitannin species. However, this approach does not provide much insight into the structure of individual ellagitannins, which is critical since the nutraceutical value of ellagitannins are regulated by their molecular identity. Ellagitannins are one of the most diverse groups of plant phenolics and their complexity presents a major hindrance to structural elucidation efforts. By leveraging the architecture of a quadrupole-Orbitrap-ion trap platform we developed a robust method that will identify ellagitannins from complex plant matrix that requires no apriori knowledge about the chemical identity of the molecule. Firstly, using refined and synthesized ellagitannin standards, we leverage the high resolution and accurate mass capabilities of the Orbitrap mass analyzer to classify precursors with specific product ions generated from higher energy collision dissociation (HCD) as ellagitannins. Further, we utilized collision-induced dissociation (CID) to generate unique fragmentation spectra of isomeric variants to differentiate between isomeric forms. We present an approach that allows for the screening of ellagitannins by employing higher energy collision dissociation (HCD) to generate reporter ions for classification and collision-induced dissociation (CID) to generate unique fragmentation spectra for isomeric variants of previously unreported species. Ellagitannin anions efficiently form three characteristic reporter ions after HCD fragmentation that allows for the classification of unknown precursors that we call targeted reporter ion triggering (TRT). We demonstrate how a tandem HCD-CID experiment might be used to screen natural sources using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) emerged. The method was verified not to yield false-positive results in complex plant matrices. We were able to identify 154 non-isomeric ellagitannins from strawberry leaves, which is 17 times higher than previously reported in the same matrix. The systematic inclusion of CID spectra for isomers of each species classified as an ellagitannin has never been possible before the development of this approach.

PUBLICATIONS: 2016/12/15 TO 2017/12/14
1. Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bowers JJ, Gunawardena HP, Cornu A, Narvekar AS, Richieu A, Deffieux D, Quideau S, Tharayil N. Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry. Scientific Reports. In Review.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Narvekar A, Tharayil N. 2017. Nitrogen Stress Induced Modification of the Foliar Phytochemical Composition in Strawberries.Crop Science Society of America Annual Meeting, Oct 22-25, 2017, Tampa, FL.

PROGRESS: 2015/12/15 TO 2016/12/14
Target Audience:The proposed project objective and the associated approaches were discussed with local farms and producers. The discussion has facilitated mutiple subprojects that will be initated dring 2017in association with Happy Berry Farm, which is a local small-fruit farm with several berrieis including balckberries, blueberries and grapes, in which the stress induced enhancement of phytochamicals could be tested. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training to one graduate student and one undergraduate student in developing and optimizing the methods for analyzing metabolome of strawberry. Also, the sample cleanup, chromatography and mass sepctrometry method developed for this project was used in a graduate level analytical techniques class to quantify the pytonutrients and pesticided in the strawberries (13 participants). How have the results been disseminated to communities of interest?The result of the project was presented at the Project Directors meeting. The proposed project objective and the associated approaches were discussed with local farms and producers. The discussion has facilitated mutiple subprojects that will be initated dring 2017in association with Happy Berry Farm, which is a local small-fruit farm with several berrieis including balckberries, blueberries and grapes, in which the stress induced enhancement of phytochamicals could be tested. What do you plan to do during the next reporting period to accomplish the goals?1) Future studies are directed towards evaluation of the fruiting dynamics in terms of sugar accumulation and abundance of phenolic compounds as a response to varied nitrogen rates. 2) Optimize the colorimetric assay to capture the overal antioxidant content and capacity of berry fruits. 3) Complete and publish the metabolome database of strawberry that would include identification of atleast 150 compounds based on mass spectrometry based metabolomics (accurate mass and fragmentation patter)

IMPACT: 2015/12/15 TO 2016/12/14
What was accomplished under these goals? Background: Secondary metabolites facilitate the acclimation of plants to various environmental stresses. These phytochemicals are also of a greater importance for human health due to their ability to quench reactive oxygen/nitrogen species. However, the current crop production practices that are optimized for higher quantitative yield compromises the phytochemical content of the produce. This project focuses on the concept of nutrient-dependent inflection point of carbon partitioning between growth and defense in plants, and tests various hypotheses that link production of phenylpropanoids and stress adaptation at a molecular level. Method:We evaluated phytochemical content and antioxidant capacity oftwo cultivars (Camarosa and Albion, June bearer and Day neutral cv. respectively) of strawberry (Fragaria ananassa) in response tosix different levels of nitrogen fertilizer (12, 23, 34, 44, 54 and 81mg N). The N treatment that supplied 81 mg N represents 100% N supply as per the currently recommended fertilizer regimen. We developed and optimized both targeted and untargeted metabolomics methodology that enabled simultaneous determination of >150 metabolites in strawberries. Results:Using mass spectrometry based non-targeted metabolomics approach we identified 152 unique metabolites from the strawberry leaves.Main classes of the phenylpropanoids,viz.flavonoids, phenolic acids, ellagitannins and proanthocyanidins, were significantly higher under low nitrogen treatments compared to nitrogen sufficiency at full nutrition.Overall, the plants receiving lowest nitrogen had2-4 times higher phytonutrient content as that of plant that received recommended rate of N.However this relationship was non-linear; in both cultivars the phytonutrient content decreased as the percent N supplied increased from 15 to 42% of the field recommended rate, however the 55% of N had lower phytonutrient content than the 100% field-recommended rate of N.The genotype interaction with environmental growth parameter was evident from the principal component analysis (PCA) derived score plot, where the two cultivars showed distinct directional projection. The hierarchical clustering analysis of the metabolite profile of both cultivars under low nitrogen treatment (15 % N) were grouped separately. Hydrolysable (ellagitannins) and condensed tannins content (proanthocyanidins) like geraniin and procyanidin respectively was elevated at 15% N as compared to higher N treatments. In general the ellagitannis were less responsive to N treatments compared to condensed tannins. The monomers which comprises these polymers like gallic acid, protocatechuic acid, di-, tri- and tetragolloyl glucose were found accumulated at higher rates of N. Flavonol compounds, including rutin, quercetin and kaempferol were upregulated at lower N rates at 15 % N and 30% N. Lower N rates exhibited highest antioxidant capacity on DPPH assay. The primary metabolite like α-Ketoglutarate, succinate, sucrose and lyxose were higher at lower N rate, but glutamate was higher at higher N rates (55% N). Conclusion:The results demonstrated foliar accumulation of antioxidant phenolic compounds under nitrogen stress in strawberry plants. The phenolics and the associated antioxidant activity increased non-linearly in both the strawberry cultivars as a function of nitrogen stress. This shows that,irrespective of the genetic identity,the precise management of nitrogen stress could be a valuable tool in managing the phytochemical content of strawberries. Future studies are directed towards evaluation of the fruiting dynamics in terms of sugar accumulation and abundance of phenolic compounds as a response to varied nitrogen rates.

PUBLICATIONS: 2015/12/15 TO 2016/12/14
1. Type: Journal Articles Status: Other Year Published: 2017 Citation: Narvekar, A. Tharayil, N. Content and composition of foliar phytochemicals varies non-linearly with the nitrogen supply. Journal of Agricultural and Food Chemistry (to be submitted).
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Narvekar, A., Suseela, V., Stewart, M., Tharayil, N. 2017. Nitrogen stress induced modification of the foliar phytochemical composition in strawberries. USDA NIFA Physiology of Agricultural Plants PD meeting. San Diego. CA, Jan 12, 2017.