Performing Department
(N/A)
Non Technical Summary
It is not understood how tree fruit/nut yield alternately fluctuates from one year to the next, hindering the development of effective approaches for mitigating the alternate bearing phenomenon for consistent production of tree crops. This project aims to understand how sugar concentrations affect alternate bearing of pecan. It is known that carbohydrate levels are positively correlated with alternate bearing of nuts and the hormone gibberellin (GA) inhibits female bud formation in pecan. It is also known that the SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET) transport both sugar and GA in multiple plant species and the expression of some SWEET genes are induced by sugar. Therefore, we hypothesize that high sugar concentrations in an on-year of pecan nut production upregulate the expression of specific SWEET genes, which increases the transport of GA that suppresses the development of female flowers in developing buds, leading to a low female bloom return in the following year. To test this hypothesis, we propose the following research objectives: 1. Determining how sugar concentration, CiSWEET expression, GA concentration, and floral gene expression are correlated in pecan trees undergoing alternate bearing. 2. Determining how changes in sugar concentration affect CiSWEET gene expression, gibberellin concentration, and floral gene expression in stem cuttings. 3. Characterizing sugar-induced transcriptomic changes in first-year developing buds on pecan stem cuttings and validating the reproductive roles of selected genes in pecan and Arabidopsis. The information garnered will serve as a foundation for developing strategies to address alternate bearing of pecan and other tree species.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this project is to understand how alternate bearing occurs at the molecular level in pecan with the hypothesis: high sugar concentrations in phloem in a high-yield year of pecan upregulates the expression of specific CiSWEET genes, which causes the accumulation of GA in first-year developing buds that suppresses the development of female flowers, leading to a low female bloom return in the following year. There are three objectives:Objective 1. Determining how sugar concentration, CiSWEET expression, GA concentration, and floral gene expression are correlated in pecan trees undergoing alternate bearing.Objective 2. Determining how changes in sugar concentration affect CiSWEET gene expression, gibberellin concentration, and floral gene expression in stem cuttings.Objective 3. Characterizing sugar-induced transcriptomic changes in first-year developing buds on pecan stem cuttings and validating the reproductive roles of selected genes in pecan and Arabidopsis.
Project Methods
Objective 1. This project will use two main Oklahoma commercial cultivars, 'Pawnee' and 'Kanza'; all trees are 30 years old and planted at OSU Cimarron Valley Research Station (Perkins, OK).Manage tree production: Nut thinning is applied in the OSU experimental pecan orchard in mid-August every year as a regular management process. In year 1, all the trees are expected at a moderate 'On' production year. We will manage tree production with four fruit thinning treatments in August to produce both 'On' year and 'Off' year trees for year 2 and 3. In each treatment, 20 trees will be used, and 80 trees of each cultivar will be used for all the experiments in the three objectives. In Table 1, 'On' is a high production year; 'Off' is a low production year. 'On (heavy)' means the production could be overloaded. 'Expected production' refers to the nuts amount in July, not the final yield, as some treatments will remove nuts in August. 'Moderate thinning' is a 40-60% thinning level depending on cultivars (Smith et al., 1993), and 'heavy thinning' will thin off 80-100% nuts.Starting from early May, when the female buds initiate, current year (latest) stem samples will be collected once a week from the field. Four trees at a similar growth condition of each treatment will be selected, and on each tree, four current-year stems will be sampled weekly until the end of August in year 2 and 3. In year 1, current-year stem samples will be collected once a month in June, July, and August to test the carbohydrate and GA levels of each stem-sampling tree (8tree/treatment/cultivar) before fruit thinning treatment; the data will be used for comparisons with the data from year 2&3. Each stem's top section (10 cm in length from the terminal bud) will be used for sugar and starch tests. Four mixed buds at the top section of each stem will be collected in liquid nitrogen and stored at -80 °C for gibberellin and CiSWEET and floral gene transcriptional analysis.Objective 2. The investigation in Objective 1 relies on the plant materials from the field, which does not allow manipulation of the parameters for deeper mechanistic understanding. To overcome this shortcoming, we will use the stem cutting system described earlier to investigate the response times between the consecutive steps of the signaling process, namely sugar increase to the CiSWEET expression to GA increase to expressional changes of the floral genes and GA-responsive genes. Specifically, we will determine how soon the stem near the apex will exhibit an increased sugar concentration and how long it takes to reach the peak sugar concentration after the sucrose treatment in comparison with the control. We will sample enough stem cuttings with both low and high sugar concentrations before the sucrose treatment, likely more than 20, at each of multiple time points to find the shortest response time and peak-reaching time. To reduce experimental noises, the test and control stem cuttings will be collected from the same trees in pairs. Our experience in determining stem sugar concentrations is that stems collected from the same three at a time have similar sugar concentrations. This sample collecting strategy will be used for all subsequent quantitative comparisons involving cultivated stem cuttings. Based on these two time points, we will similarly determine the shortest and peak response times for the CiSWEET expression changes following the sucrose treatment. Our hypothesis calls for a lag time in the initial CiSWEET expressional increase compared with the initial sugar increase and possibly its peak level also occurs after the peak sugar concentration. The durations of these lag times will provide clues to whether the response is posttranslational (short response times, e.g., within a few minutes) or transcriptional/translational (long response times, e.g., 10s of minutes). Lag times in the subsequent step--GA increase to floral and GA-responsive gene expression changes will also be determined.Objective 3: Results from the investigation described in Objective 2 are expected to reveal the initial response time in each of the three steps of the signaling process when the stem cuttings are treated with a sucrose solution. However, which genes that mediate the expression of the target genes such as the selected CiSWEETs, CiAP3, CiAG, and possibly CiLFY and CiAP1 are unknown. To identify the additional genes involved in the signaling process, we will use RNA samples isolated shortly before the initial response times of the first and third steps to generate RNAseq data. We will have five sucrose-treated and three control (treated with water) RNA samples (> 5 µg RNA/sample) for each of the two points to statistically determine the differentially expressed genes (DEGs) between the test and control samples. A total of ten samples will be sent to LC Sciences (Houston, Texas; https://lcsciences.com/) for sequencing and analysis. LC Sciences will prepare the libraries from the RNA samples for 150-bp paired-end sequencing to generate > 80 million reads per sample. Sequence analysis will be conducted at LC Science using their in-house bioinformatics pipeline to determine the DEGs. Based on the functions of the identified DEGs and published studies regarding sugar signaling, GA signaling, and floral gene expression in plants, Candidate genes mediating the responses in the first and third steps of the signaling process will be chosen. These candidate genes will be studied by RT-PCR and RT-qPCR in the cDNA samples used in the investigation in Objective 1. Because the cDNA samples in Objective 1 will represent one on-year and two off-year of pecan nut production, how the expression levels of these candidate genes in the first-year buds are associated with the on- and off-year will further test our alternate bearing hypothesis.