Progress report for GNE22-304
Higher frequency of temperature extremes under climate change poses great challenges to grape growing in Northeastern U.S. Among these temperature extremes, late spring frost directly threatens the survival of fragile but most fruitful primary buds, leading to significant crop losses when damage occurs. However, current management methods are either not applicable or not economically friendly to the growers with established smaller vineyards, compromising their use in Northeastern U.S. Some budbreak delaying products were recently evaluated for their potentials to mitigate spring frost damage in grapevine. Among these products, abscisic acid (ABA) analogs exhibit both ease of application and efficacy of budbreak delay in preliminary testing; two characteristics favored by growers. In this proposed project, we will evaluate an ABA analog, tetralone ABA, for its feasibility as a budbreak delaying product for grapevine through 1) monitoring its impact on grapevine dormancy and freezing tolerance in dormant season; 2) monitoring its impact on grapevine growth and physiology in the growing season; 3) evaluating its impact on yield components and fruit composition; and 4) uncovering the mechanism of its budbreak delaying effect using comparative transcriptomics. The findings from this study will be introduced to NY grape and wine community through collaborating with Cornell viticulture extension programs and to nation-wide grape and wine industry through presenting at the 2023 American Society of Enology and Viticulture Annual Conference. The result from this project will directly benefit grape growers and contribute to the long-term sustainability of the grapevine and wine industry in the Northeastern U.S.
The overall goal of the proposed project is to determine whether an ABA analog, tetralone-ABA, can be used as a budbreak delaying product in vineyards to effectively manage late spring frost without negative impacts in the dormant and growing seasons, and the underlying mechanism of its delaying effect through a systematic approach by long-term monitoring of grapevine physiology and RNA-seq. The specific objectives are:
- Monitor the impact of tetralone-ABA on dormant season physiology, including freezing tolerance dynamics and the transition between endodormancy and ecodormancy.
- Identify the underlying mechanism of tetralone-ABA’s delaying effect on budbreak using comparative transcriptomics
- Monitor the impact of tetralone-ABA on grapevine phenology and physiology during the growing season
- Evaluate the impact of tetralone-ABA on yield component and fruit composition
The purpose of this project is to evaluate the feasibility of using an abscisic acid analog, tetralone ABA, as a budbreak delaying product in vineyards to mitigate spring frost damage. Cold-related damage is estimated to result in 5%-15% of crop loss worldwide, and this number is likely to increase due to more weather extremes induced by climate change 1–3. Apart from the direct impact of extreme cold on the survival of grapevine in winter, late spring frosts directly threaten the growth and productivity of grapevines, especially the fragile and most fruitful primary buds. For example, the 2007 Easter freeze led to 50-100% crop loss in vineyards in the Eastern and Midwestern U.S. when an unusually warm and early spring in March was followed by devastating freezing events in April. 4,5 Climate change predictions suggest that while winter temperatures will become less severe on average, there is potential for increased frequency of temperature extremes and damaging spring frosts due to earlier budbreak 6–8. Major crop losses caused by spring frost have been reported with higher frequency in major grape and wine regions in the U.S. and the world 9–11. For the Northeastern U.S., the occurrence of spring frost damage may increase in severity due in part to a high adoption rate of hybrid varieties with Vitis riparia background in this region. These cultivars were selected for their supreme cold hardiness in midwinter; however, they also tend to initiate budbreak earlier in the season, increasing the risk of spring frost damage. With these factors combined, methods for the management of spring frost are urgently needed for the sustainability of the grape and wine industry in Northeastern U.S.
Plant materials and treatments:
The experiment will be conducted on three grapevine cultivars: Vitis hybrid ‘Cayuga White’, Vitis vinifera ‘Riesling’ and their offspring 'NY 81'. Vines are currently maintained within a teaching and experimental vineyard housed at Anthony Road Wine Co. in Penn Yan, NY. These cultivars are early budbreak types with increased frost risk. The experimental vines was maintained by standard vineyards management practices in 2022 and 2023 growing seasons. The treatments will be applied as a foliar spray to the canopy a week after harvest.
Preliminary results generated by Brock University indicate that postharvest foliar application of 0.33 g per vine of (+)-tetralone ABA (1 g·L-1 for 0.33 L per vine) delayed the budbreak of Vitis vinifera ‘Merlot’ cultivars for four to six days in the following spring 13. In this proposed project, the tetralone ABA treatment will be applied at 1 g·L-1 of (+)-tetralone ABA (University of Saskatchewan, Saskatoon, Canada) for 0.5 L per vine to induce longer delay. Control treatment will be water. Tween-20 (Acros Organic, Hampton, NH), at a rate of 0.05 % (v/v) will be added as a surfactant to both treatments. For each cultivar, each treatment will be applied to 12 individuals.
Dormant season data collection:
After treatment application, grapevine bud freezing tolerance of each treatment will be determined weekly using differential thermal analysis based on standardized protocol, until full budbreak of all vines 15. Briefly, grapevine buds will be loaded in sample plates and treated with a decreasing of temperature with 4 °C·h-1 from 0 °C to -50 °C in a programmable freezer. Thermocouples on samples plates will detect low-temperature exotherm (LTE), which corresponds to the freezing tolerance of the bud. LTE will be examined in triplicate with five buds per replicate. The purpose of freezing tolerance measurement is to evaluate tetralone ABA’s impact on the dynamic of freezing tolerance in dormant season 16. In addition to LTE, bud samples from both cultivars will be collected three times from the field when conditions reach 500, 1000, and 1300 chilling units (NC model, https://products.climate.ncsu.edu/ag/chill-models/). Collected buds will be subjected to deacclimation assays to examine tetralone ABA’s impact on the deacclimation (losing of freezing tolerance). These three time points correspond to the stages of endodormancy (unable to deacclimate and budbreak under growth permissive condition), transition from endodormancy to ecodormancy (able to deacclimate and budbreak under growth permissive condition) and ecodormancy.
During each deacclimation assay, canes collected from the field will be chopped into single bud cuttings, and the cuttings will be incubated with the cut end dipped in water and placed in a growth chamber at 20 °C. At 0-day, 2-day, 4-day, 7-day, and 14-day intervals in the growth chamber, LTE of 10 replicate buds will be measured. Deacclimation rate, the coefficient of the linear regression between LTE and days in chamber, will be computed during each deacclimation assay 17. In parallel to LTE, bud samples of 'Riesling' will also be collected for RNA-seq to investigate tetralone ABA-induced divergence of transcriptome during deacclimation. RNA-seq will be conducted in triplicate with five buds per replicate.
Budbreak data collection:
In March 2023, a 1-m cordon will be identified on all treated and control grapevines as an observation zone, and the canes within the observation zones will be pre-pruned to retain 30 buds to monitor the progression of budbreak. The canes out of observation zones will be subjected to standard pruning. From the start of budbreak (EL-5 in grapevine phenology) 18, the number of broken buds will be recorded every two days until all vines reach full budbreak. It is expected that the whole course of budbreak will take about one month depending on treatments’ effect.
Growing season and harvest data collection:
After budbreak, the development of each experimental grapevine will be recorded weekly according to E-L phenology system to monitor tetralone ABA’s impact on grapevine vegetative growth over the growing season 18. Besides, leaf stomatal conductance and chlorophyll a fluorescence of each experimental grapevine will be measured weekly using LI-600 porometer/fluorometer (LI-COR Biosciences, Lincoln, NE) to reveal treatments’ impact on the overall grapevine physiological state during the growing season. As both parameters exhibit diurnal dynamics, all measurements will be conducted between 10:30am to 12:00pm.
RNA-seq library preparation and data analysis:
Spectrum Plant Total RNA Kit (Sigma Aldrich, St Louis, MO) will be used to extract total RNA from ground bud tissues. Three biological replicates will be extracted and processed for each genotype x treatment combination. The Cornell University Institute of Genomic Diversity (Ithaca, NY, USA) will provide technical support for library construction using Lexogen QuantSeq 3’mRNA-Seq Prep Kit (Lexogen, Greenland, NH). Sequencing of libraries will be conducted at Cornell University Institute of Biotechnology (Ithaca, NY) using NextSeq500 (Illumina, Inc., San Diego, CA, USA) with 95 samples per lane.
RNA-seq data will be analyzed following a previously generated pipeline in the Londo lab 19. Briefly, standardized library QC (FastQC), trimming (BBDuk) and transcript alignment (STAR) will be conducted using a workstation hosted in Cornell BioHPC (https://biohpc.cornell.edu/). The resulting gene count matrix will be analyzed in DESeq2 20. DESeq2 model will include ‘chilling units’, ‘days in chamber’, and ‘treatment’ to determine DEGs (differentially expressed genes) between two treatments during deacclimation assays. Weighed gene co-expression network analysis (WGCNA) will be used to generate gene co-expression network 21. Pathway enrichment analysis will be conducted on DEGs list through GSEA using VitisNet function pathways 22,23. Significantly enriched pathways will be correlated for their known biological functions, and hub players will be determined based on the synergy of statistical significance and biological functionality.
Tetralone ABA and control treatments were applied on Oct. 05, 2022. For each cultivar and each treatment, 12 vines were sprayed. After treatment application, the senescence process was quantified through weekly monitoring of relative chlorophyll concentration (SPAD) using an MC-100 Chlorophyll Concentration Meter (Apogee Instruments, UT, USA). During each measurement, three leaves from each vine were randomly selected and measured, and the mean SPAD of the three leaves was identified as the relative chlorophyll concentration of the vine.
As shown in Figure 1, the SPAD of 'NY81' and 'Riesling' was significantly lower in tetralone ABA-treated vines at one or two weeks after treatment. In contrast, the treatment effect was less significant in 'Cayuga White'. The visual assessment also indicates that tetralone ABA treatment accelerated leaf senescence in 'Riesling' (Figures 2 and 3).
Figure1. Weekly measurement of relative chlorophyll concentration
Figure 2. 'Riesling' one week after control treatment
Figure 3. 'Riesling' one week after tetralone ABA treatment
Figure 4. Grapevine bud freezing tolerance monitring
For 'Riesling', the enhancement of freezing tolerance (significantly lower LTEs) of the buds treated with tetralone ABA in the first four collections indicates that the early season cold acclimation was enhanced in response to tetralone ABA. However, the maximum freezing tolerance so far in the season (as shown in the collections on Dec. 28, 2022) was not enhanced. During the prolonged warmth in early January, the buds under control treatment lost some freezing tolerance as an unwanted deacclimation response. This deacclimation response was inhibited in the vines under tetralone ABA treatment. For 'NY81', the promotive effect of tetralone ABA on early-season cold acclimation was also observed, and tetralone ABA significantly enhanced the maximum freezing tolerance so far in the season. However, tetralone ABA showed no effect on the deacclimation response in January. For 'Cayuga White', tetralone ABA showed no effect during the early season cold acclimation. The maximum freezing tolerance on Dec. 28, 2022, was significantly enhanced in the buds treated with tetralone ABA. The deacclimation response in January was more intense in 'Cayuga White', but the buds treated by tetralone ABA exhibited significantly lower LTEs than control in both collections in January. The results indicate that tetralone ABA has different effects on the dormant season physiology of different grapevine genotypes.
Two deacclimation assays were done using field-collected 'Riesling' buds at 552 and 1005 chilling units. The results of the deacclimation assays are shown in Figure 5. In the first deacclimation assay (552 chilling units), the deacclimation rates of the buds in two treatments were minimum, indicating the buds were under endodormancy. The LTEs of buds in tetralone ABA treatment were lower than the control treatment at every collection in the assay. In the second deacclimation assay (1005 chilling units), the deacclimation rate of the buds in tetralone ABA treatment was 0.2 °C•day-1 lower than the control treatment, indicating tetralone ABA treatment slowed deacclimation.
Figure 5. Deacclimation of 'Riesling' buds in two deacclimation assays
Education & Outreach Activities and Participation Summary
In spring 2023, we plan to work together with Cornell viticulture extension specialists through Cornell Recent Advances in Viticulture and Enology (CRAVE) and Business Enology Viticulture NY (BEVNY) to introduce tetralone ABA as a budbreak delaying and translate the result generated from the proposed project to the growers in NY. An on-line survey will be produced to address the potential adoption of budbreak-delaying products for the management of spring frost with NY grape growers. A detailed questionnaire will focus on the specific aspects of the economic impact of frost damage, current mitigation method, the acceptable range of cost for mitigating, willingness to utilize budbreak-delaying products such as tetralone ABA and major concerns about using these products. Upon the completion of the proposed project, an article will be prepared to introduce key findings through Appellation Cornell, a quarterly newsletter facing grapevine growers regarding the most recent viticulture research advances from Cornell University. A manuscript will be prepared to be published in The American Journal of Enology and Viticulture, a niche journal with major audience of grape growers and university viticulture extension specialists. The result will also be presented at the 2023 American Society of Enology and Viticulture National Conference. Through these approaches, the valuable findings from the proposed project will not only be delivered to the grape and wine industry in NY, but also to the nationwide viticulture research and extension community, who will further propagate and use these findings to benefit local grapevine communities.