Recipient Organization
Agricultural Research Service
600 E. Mermaid Lane Rm 2023
Glenside,PA 19038-8551
Performing Department
(N/A)
Non Technical Summary
Increase of food production is urgently needed to feed the constantly expanding population. Tomato is one of the most cultivated and consumed fruit crops worldwide, consumed either fresh or in various processed forms (e.g. paste, sauces). Tomato is also the most prominent dietary source of the nutraceutical lycopene, vitamin C and other nutrients in the U.S. and many countries. Development of novel strategies to improve both tomato fruit yield and quality are thus of utmost importance for food and nutritional security. In our previous study, we found that overexpression of the Or gene not only greatly increases tomato fruit carotenoid content, but also enhances fruit set and total fruit yield. To elucidate the roles and mechanisms and to develop breeding tools, three specific objectives are proposed: 1) to generate Or gene mutations and examine the effects on fruit set and yield; 2) to elucidate how it affects on fruit set, and 3) to assess the association of the gene expression with fruit set and heat tolerance in a tomato diversity panel. The proposed research is expected to contribute to our fundamental understanding of reproductive biology and inform breeding programs to generate elite varieties with improved yield along with nutritional quality and heat tolerance in crops beyond tomato.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The major goals of this proposed research are to understand how this ubiquitously presented Or gene in plants affects fruit set and to identify genetic materials that can be potentially utilized to boost tomato crop yield.
Project Methods
Objective 1: To investigate the effects of SlOr family gene mutations on fruit yieldTo generate individual slor and slorl single mutants, two sgRNAs targeting either SlOr or SlOrL were designed and transformed into tomato cv. Ailsa Craig (AC) via A. tumefaciens-mediated transformation. The slor slorl double mutants will be generated by crossing the slor and slorl single mutants to assess any functional redundance. The knockout lines and WT control will be examined for fruit set/yield by counting the first 10 trusses from each plant for flower number and fruit number, followed by measuring fruit size and total fruit weight per plant. Pollen viability, germination, and tube length assays as well as in vivo pollen cross experiments in the slor, slorl, and slor slorl mutant lines and control will be carried out to determine whether the SlOr family gene mutations affect pollen fitness. In addition, we will stain and quantify ROS levels and measure sugar and carotenoid levels in pollen and flower organs in the mutant lines and control to examine whether slor and slorl mutations impact ROS, sugar, and carotenoid levels to affect pollen fitness and fruit set. We will also apply auxin and GA to unpollinated ovaries of mutants and control to assess whether auxin and GA are involved in the Or-regulated fruit set.Objective 2. To elucidate the mechanisms underlying Or-regulated fruit setThe evolutionarily conserved Or family proteins likely exert their function in fruit set via interacting with the key enzymes/proteins critically important for fruit set. To isolate SlOr-interacting proteins, we will use two alternative and complimentary strategies to isolate the interacting candidates, which include comparative proteomics and biotin ligase-based proximity labeling. The best candidates potentially involved in the Or-regulated fruit set in various processes will be verified by protein-protein interactions using BiFC, Y2H, and Co-IP. Following confirmation of physical interactions, we will examine whether Or family proteins regulate their interacting partner activities for functions and characterize them to show their involvement in pollen fitness and associated processes, thus providing novel mechanistic insights into tomato fruit set.?Objective 3. To assess and associate SlOr family gene expression with fruit set and heat tolerance in diverse tomato germplasm.The Co-PD Giovannoni's lab is performing RNA-seq analysis of a diverse tomato germplasm collection that contains 150 sequenced accessions. We will mine expression of SlOr and SlOrL in flowers, leaves, and fruit of the diversity panel. Five plants per accession in the 150 accession collection will be grown in the Freeville field at Cornell University to associate SlOr family gene expression with fruit set in the germplasm collection. The first 5 trusses from each plant will be examined for flower drop incidence and fruit set number. We will perform a second-year field trial for confirmation. In addition, we can develop crosses between several high or low expression lines and a small number of tester lines to examine fruit set in subsequent generations following backcrosses and selection for the high expression alleles. This activity will also generate useful germplasm directly applicable for breeding. In addition, selected accessions that have high SlOr and/or SlOrL transcript levels and accessions that have low SlOr and/or SlOrL expression in their flowers and leaves along with slor knockouts and AtOr OE lines will be grown at optimal temperature for 4-weeks in greenhouses followed by heat stress. When plants start to flower, we will investigate the first 5 trusses from each plant growing under both optimal and heat stress conditions for flower and fruit number to calculate fruit set rate.