INVESTIGATING MECHANISMS THAT INFLUENCE FRUIT RIPENING AND POSTHARVEST STORAGE IN BLUEBERRIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010226
• Project Start Date: Jul 27, 2016
• Project End Date: Jul 25, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
Horticulture

Non Technical Summary
The current issue that this proposal addresses is to understand the mechanisms that regulate ripening and postharvest shelf-life attributes in blueberries. Blueberry is a rapidly growing industry in Georgia. However there is very little information available about the factors that regulate ripening and storage in blueberries. We plan to use transcriptomic sequencing and application of growth regulators to determine key hormonal pathways that regulate blueberry ripening. Data generated from these studies will help to understand factors that regulate ripening in blueberries. The long term goal is to generate tools for the blueberry industry to manipulate ripening.Once harvested, blueberries are a highly perishable commodity having shelf-life of only about 2-3 weeks. This proposal will initially identify varieties that store well compared to other varieties with inferior shelf-life attributes using fruit quality attributes such as texture, weight, acidity and soluble solids. Using RNA-Seg and metabolite analysis, genes and metabolites will identified from varieties that store well. This study will allow us to identify mechanisms that contribute to improved postharvest shelf-life and quality attributes. In the future knowledge form this study should help to develop markers that would help to select for varieties that have superior shelf-life and quality attributes.

Animal Health Component

20%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1120	1040	70%
204	1120	1020	30%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1120 - Blueberry;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;

Keywords

blueberries

postharvest

rna-seq

metabolite analysis

plant growth regulators

Goals / Objectives
The major goals of this project are to determine factors that regulate ripening and postharvest shelf-life attributes in blueberries. This project aimsto investigate the role of key hormone-associated pathways in regulating ripening using transcriptomic and physiological approaches. We plan to identify key genes and metabolic pathways that regulate ripening using transcriptomic analysis. Using a more physiological approach we plan to identify growth regulators that can either accelerate or inhibit ripening. Together thesestudies will help to understand mechanisms that regulate ripening and develop tools to manipulate ripening and improve harvesting practices. Another objective of this proposal is to understand mechanisms that contribute to extension of postharvest shelf-life and quality attributes. This study will involve identifying key genes and metabolites that are associated with varieties that have superior postharvest shelf-life and quality attributes using transcriptomic and metabolic approaches. Data from this objective will help to develop practices to extend the shelf-life attributes in blueberries.

Project Methods
Plant materials will include fruits from multiple cultivars will be harvested at different ripening stages. After harvest, berries will be stored at 4 °C and 95 % relative humidity to represent current conditions of postharvest storage of blueberry. Some of these fruit will be immediately frozen in liquid N2 and stored at -80 °C until needed for further analysis.For measurement of physical fruit quality attributes, measurements will be performed at harvest and at different stages during postharvest (PH). Samples will be collected at intervals that represents a typical 3-week time period during which decline in several physical properties of the fruit are thought to occur during commercial blueberry storage. The physical attributes that will be measured include fruit weight, diameter, and textural characteristics such as fruit firmness. Additional fruit quality characteristics such as total soluble solids content and titratable acidity will be determined.Treatment with plant growth regulators (PGRs) will evaluate the effects of ABA and MeJA on blueberry fruit development and ripening. These PGRs will be applied at a stage when 50% of the berries within individual clusters are ripe. The plants will be treated with ABA (600 ppm) and MeJA (1 μM) along with an adjuvant (0.15% Latron B-1956). Control plants will be treated with water and the adjuvant. Treatments will be replicated on four plants and three clusters from each plant will be evaluated for a given treatment. At the time of application, three clusters within a plant will be tagged and the number of green and ripe berries will be counted. These clusters will be evaluated for ripening every week for two weeks by counting the number of ripe berries within a given cluster. The clusters will be harvested when 80% of the berries within a cluster are ripe and fruit quality will be determined as described above. Fruit samples will be collected at regular intervals after treatment, frozen and stored. These fruit will be used for subsequent RNA-Seq analyses to determine how transcriptomic changes associated with the applications of these plant growth regulators may mediate alterations in fruit ripening.For identification and quantification of metabolites, we plan to use a gas chromatography-mass spectrometry (GC-MS) system. Metabolite profiling using GC-MS will be performed as described in Cevallos-Cevallos et al., (2012). Briefly, samples will be weighed, ground to a fine powder and extracted using methanol/water/chloroform. L-threonine will be used as an internal standard. The filtered extract will be dried and re-suspended in methoxyamine (MOX). Finally after silylation with N-methyl-N trifluoroacetamide (MSTFA), samples will be analyzed on the GC-MS. Peak identification will be performed using the NIST11 mass spectrum library. For analysis of anthocyanins, samples will be lyophilized, ground, and dissolved in an acetonitrile/water/formic acid solvent. This extract will be centrifuged and filtered, and Rhodamine B will be added as an internal standard. Characterization of individual anthocyanins will be performed using HPLC-UV/Vis (Müller et al., 2012).For transcriptome sequencing during ripening and postharvest storage, fruit samples from multiple cultivars at different stages during storage will be used for RNA-Seq analysis. RNA will be extracted from the fruit tissue, and their quality analyzed using a bio-analyzer. Libraries for RNA-Seq will be developed in-house and sequenced at the Georgia Genomics Facility at UGA using the Illumina Next-Seq platform. Three biological replications will be used in this study. Subsequent bio-informatics analysis of these data will be performed by the PI who has previous experience with the analysis of similar large scale next generation sequencing data sets.All the information generated using these methods will be shared with extension agents and the blueberry industry. Scientific presentations will be held at county grower meetings and at national horticulture conferences to disseminate the key findings from this study.All studies will repeated atleast over two years to measure the success of the project. Various demonstrations at the growers' farms will be performed to evaluate and strengthen the findings of the study.

Progress 07/27/16 to 07/25/21

Outputs
Target Audience:Target audience include the scientific community, the blueberry growers and the industry. Efforts include publication of scientific research articles, blueberry news letters, presentations in international, national and regional conferences. Changes/Problems:For Objective 2, metabolite analysis in cultivars that differ in shelf-life attributes was not completed and is currently under progress. What opportunities for training and professional development has the project provided?A graduate student was trained in the area of molecular physiology of blueberry ripening and postharvest physiology. The information generated led to professional development by delivering presentations in regional, national and international meetings. How have the results been disseminated to communities of interest?The results have been disseminated by giving oral and poster presentations at conferences with regional, national and international scope. Further results have been presented at grower workshops and information has been presented in newsletter articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under Objective 1, we tested the effect of three plant growth regulators, methyl jasmonate, abscisic acid and ethephon on blueberry fruit ripening. Of these only ethephon, the PGR that release ethylene accelerated fruit ripening. We performed transcriptomic analysis that indicated ethephon can accelerate the rate of ripening by down-regulating photosynthesis related genes. We also evaluated several southern highbush and rabbiteye blueberry cultivars that differ in postharvest storage. For Obejctive 2, two cultivars that differed in storage were used for transcriptome analysis that identified genes involved in water transport, amino acid transport and cell wall modification. These data suggest water loss and fruit softening may play an important role in blueberry fruit quality.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: 3. Wang, Y-W., and S.U. Nambeesan. 2020. Investigate the mechanisms of ethylene-regulated ripening in blueberry fruit by transcriptome analysis. American Society for Horticultural Science Annual Meeting-virtual

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? See final report for details.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audience include the scientific community, the blueberry growers and the industry. Efforts include publication of scientific research articles, blueberry news letters,presentations in international,national and regional conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? See final report for details

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, Y-W., A. Malladi, J.W. Doyle, H. Scherm, and S.U. Nambeesan. 2018. The effect of ethephon, abscisic acid, and methyl jasmonate on fruit ripening in rabbiteye blueberry (Vaccinium virgatum). Horticulturae 4: 24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Nambeesan, S.U. 2019. Optimizing strategies for manipulating ripening and improving postharvest shelf-life in blueberries. Southeast Regional Fruit and Vegetable Conference. Savannah, GA

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Fruit grower and industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of one graduate and undergraduate student. How have the results been disseminated to communities of interest?Conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?Test effect of 1-MCP and AVG on multiple cultivars. Complete analysis of RNASeq data. Develop a method to identify key metabolties in ripening and postharvest fruit qulaity.

Impacts
What was accomplished under these goals? PGRs, 1-MCP and AVG did not affect fruit ripening and postharvest fruit quality in one rabbiteye blueberry cultivar that was tested. Data from RNASeq results is being currently analyzed to detrmine key genes that influnece ripening and postharvest fruit quality.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Wang Y-W, Malladi A, Doyle JW, Scherm H, Nambeesan, SU (2018) The effect of ethephon, abscisic acid, and methyl jasmonate on fruit ripening in rabbiteye blueberry (Vaccinium virgatum). Horticulturae 4: 24.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Nambeesan SU, Doyle JW, Capps HD, Starns C, Scherm, H (2018) Effect of electronic cold-pasteurizationTM (ECPTM) on fruit quality and postharvest diseases during blueberry storage. Horticulturae 4: 25.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Fruit growers and industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One gradaute student and undergraduate student was trained. How have the results been disseminated to communities of interest?Attending conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?Test the effect of ethylene inhibitors at a higher concenteration. Perform RNA seq data analysis on fruits that differ in postharvest fruit qulaity traits.

Impacts
What was accomplished under these goals? We confirmed the effect of ethephon in blueberry ripening and studied the effect of AVG, an ethylene inhibitor on ethylene ripening. We did not see any effect of AVG on ripening and postharvest shelf life attributes. We also identified cultivars that show a difference in the amounts of ethylene producedduring ripening. Further we identified certainacids that may be importantin fruit shelf-life and qulaity.

Publications

Progress 07/27/16 to 09/30/16

Outputs
Target Audience:Fruit and vegetable industry and growers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student and an undergraduate student was trained How have the results been disseminated to communities of interest?Results from thsi work has been preseneted in various conferences. What do you plan to do during the next reporting period to accomplish the goals?Look at effect of PGRs that inhibit ethylene production on bluberry ripening and postharvest fruit qulaity and identify metabolties that are present in cultivars that store well after harvest.

Impacts
What was accomplished under these goals? Plant growth regulator (PGR) ethephon acceleratedripening whereas there were no significant differences with abscisic acidor methyl jasmonate on rate of ripening in blueberries. We did not see any significant differenceswith the three PGRsin various fruit quality attributes and disease incidence measured at various times during postharvest storage.We also identified fruit qulaity attibutes and shelf-life of several emerging and poplular blubeberry cultivars and identified cultivars with good storage attributes.

Publications