ASHS 2025 Conference

The mechanism controlling dormancy in buds of woody perennial plants remains largely unknown. However, it is known that exposure to cold temperatures (chilling) promotes the transition from a non-responsive to a responsive status to growth-conducive temperatures (i.e., endo- to ecodormancy transition). In horticulture, hydrogen cyanamide (HC) has been used for decades to overcome chilling accumulation deficiencies for temperate fruit crops grown in subtropical climates. Given the connection between cold hardiness loss and budbreak, we hypothesized that HC would increase the rate of cold hardiness loss (deacclimation rate). To test this, we collected grapevine (Vitis hybrid ‘Petite Pearl’) cuttings from field conditions in Madison, WI in approximately bi-weekly intervals from December to April. Single node cuttings were prepared and randomly separated into two treatments: a control group [0.5% surfactant (Regulaid®, KALO, Inc.)], and an HC group [5% hydrogen cyanamide (Dormex®, Alzchem Group AG) and 0.5% surfactant]. Both groups were treated by submersion for 10s. Cuttings were then placed in cups of water, and under forcing conditions. The forcing conditions used for all collections were 22ºC and 16h light. In later collections, two additional forcing temperatures were used: 8ºC and 16ºC. Cold hardiness was measured using differential thermal analysis on the day of treatment application, from field collected buds (n>10), and in semi-regular intervals from cuttings under forcing conditions, with interval length depending on temperature [e.g., quasi-daily at 22ºC (T0 1d, T0 2d, …); about every two days for 16ºC (T0 2d, T0 4d, …), about every five days for 8ºC]. At each collection time and for each temperature and treatment, 10 cuttings were set apart to observe time to budbreak. As expected, budbreak occurred earlier in HC treated buds compared to control. Using measurements of cold hardiness over time under forcing, we determined deacclimation rates as the slope of linear regressions. The rate of deacclimation in the control group increased progressively with each collection, as chilling accumulated in the field (1.0ºC/d in December to 1.4ºC/d in March). However, the deacclimation rate of the HC-treated group was always greater than the control (1.6ºC/d in December and 1.7ºC/d in March). In March, at 16ºC, there was also a difference between control and HC group in deacclimation rate (1.2ºC/d and 1.4ºC/d), while there were no differences at 8ºC. HC increases the rate of deacclimation in grapevines. We anticipate that understanding the interplay between cold hardiness, deacclimation, and budbreak will be helpful in uncovering the dormancy mechanism.

Dormancy remains a poorly understood process in temperate woody perennial plants. These plants require cumulative exposure to low temperatures (chilling accumulation) during winter to respond to warm temperatures in spring (forcing) and properly break bud. For successful establishment of temperate woody perennial fruit crops, it is important to understand chilling accumulation and dormancy requirements of species and cultivars. Our recent work indicates cold hardiness is an important co-variate in the analyses related to timing of budbreak, and thus chilling accumulation models and dormancy progression studies. Here we set out to understand aspects of chilling accumulation in different conditions by evaluating two measures of dormancy progression, (i) a classic forcing assay, where time to budbreak is evaluated; and (ii) a newer phenotyping of cold hardiness deacclimation rates using grapevine (Vitis spp.). For a comprehensive analysis, we used grapevine canes from V. vinifera cvs. Cabernet Sauvignon and Riesling, and V. hybrid cvs. Concord, Frontenac, Itasca, Marquette, and Petite Pearl. Canes were collected in several states across the continental United States (CO, IA, MN, NY, PA, SD, TX, WI), and in two locations for two states (NY, WI), representing approximately eight different USDA Cold Hardiness Zones (4a-7b), over the course of two winter seasons (2023-2025). Collections occurred in December, January, February, and March of each season. Upon collection or receipt of shipments, initial cold hardiness of buds was measured using differential thermal analysis (DTA). Following, canes were prepared into single node cuttings, and placed in cups of water and in a growth chamber for forcing (22ºC, 16h day/8h night). A subsample of 15 cuttings was used to evaluate time to budbreak, while the remaining cuttings were used for cold hardiness measurements in semi-regular intervals. We used simple linear regression with cold hardiness measurements to determine deacclimation rates (loss of cold hardiness over time; ºC/day). In general, buds from warmer locations (IA, TX, and Long Island, NY) had less initial cold hardiness (field cold hardiness) than colder locations. Dormancy progression was faster in colder locations than warmer locations, observed in both budbreak assays and evaluation of deacclimation rates. Based on our data, time to budbreak is a function of initial cold hardiness and deacclimation rate. Future work will examine the response of deacclimation rates to chilling accumulation models to determine chilling models that best describe dormancy responses across climates, which will then be incorporated into models that predict field cold hardiness and field budbreak.

Freeze-desiccation due to exosmosis to extracellular ice is considered as the major stress during equilibrium freezing. This causes structural / functional perturbations in the plasma membrane which leads to leakage of cellular contents. To gain further insight into the cellular mechanism of freeze-thaw injury, four cations (K , Ca2 , Mg2 , Fe2 ), known for their critical roles in plant growth and development, were measured in the leachate from injured spinach (Spinacia oleracea L. ‘Reflect’) leaves exposed to four freezing-durations (FDs) (0.5, 3.0, 5.5, 10.5 h) at a fixed temperature. In general, leakage of K , Ca2 , Mg2 increased incrementally at longer FDs and leaves sustained greater water-soaking after prolonged freezing. Data indicated a higher abundance of reactive oxygen species (O2− and H2O2) in leaves with greater injury at longer FDs. PSII efficiency was incrementally compromised at longer FDs as determined by chlorophyll fluorescence (Fv/Fm). Total electrolyte leakage from tissues right-after-thaw versus those allowed to recover for 6-d revealed that injury at 0.5 or 3 h FDs was recoverable, but leaves were irreparably injured at 5.5 or 10.5 h FDs. K was the most abundant cation in leachate. Data suggests that K -leakage can be used as proxy for total electrolyte-leakage in determining LT50 and can serve as an ionic marker to delineate moderate (recoverable) versus severe (non-recoverable) freeze-injury. Ca2 - and K -leakage data, together, are compatible with an earlier conjecture that leaked K ions replace membrane-associated Ca2 during post-thaw. It is proposed that thus structurally weakened plasma membrane, together with inhibited active transport functions of plasma membrane (noted in previous studies) lead to enhanced K -leakage from more severely freeze-injured leaves. Unlike other cations, Fe2 -leakage was indeed lower in the injured (0.5 FD) leaves compared to unfrozen control. Moreover, Fe2 was undetectable in the leachate at longer FDs. It is hypothesized that such lack of Fe2 in the leachate could result from Fenton reaction in injured tissues which converts soluble Fe2 into insoluble Fe3 . Enhanced Mg2 -leakage at greater freeze-injury suggests structural/functional impairment of chlorophyll / chloroplast complex, resulting in reduced quantum yield of PSII.

Drought stress poses significant environmental challenges to agricultural plants, especially tomatoes, by hindering their growth and reducing yields. Biostimulants like fulvic acids (FA) have emerged promising strategies for mitigating drought effects and enhancing water-use efficiency. However, the regulatory mechanisms of FA-induced drought tolerance are not yet fully understood. This study aimed to characterize FA-induced drought tolerance mechanism in tomatoes. Four-week-old plants were treated with FA at 240 mg per plant, and drought conditions were imposed by withholding 75% of the water supplied to well-watered plants. The plant growth performance and the physiological responses were evaluated. Leaf samples were collected at two stages: the early drought stage (3 days after treatment) and the later stage (7 days after treatment), for untargeted hormonomics and metabolomics analysis using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Under drought conditions, control plants exhibited significant stress symptoms, including reduced height and leaf wilting during the later phase. In contrast, FA-treated plants developed less drought symptoms and improved stomatal conductance. The hormonomics and metabolomics analysis identified 114 hormones and 243 metabolites in ESI . Using orthogonal partial least-squares discriminant analysis (OPLS-DA), we determined that 39 hormones and 162 metabolites (with a VIP score > 1.0) were significant discriminants among the different treatments. Under drought conditions, 2-hydroxy melatonin and abscisic acid (ABA) levels were significantly increased in FA-treated plants, along with higher concentrations of amino acids such as glycine and threonine. These findings suggest that fulvic acids modulate the phytohormones ABA and melatonin to induce drought tolerances, orchestrating a response that enhances drought tolerance by sustaining elevated levels of osmoprotective amino acids.

The irrigation of grasses dominates domestic water use across the globe, and a better understanding of water use and drought resistance in grasses is of undeniable importance for water conservation. Drought resistance is a complex trait composed of three distinct, but complementary, strategies: escape, avoidance, and tolerance. In grasses, drought escape is commonly displayed via summer dormancy, and drought avoidance and tolerance are displayed by grasses experiencing dehydration. Breeding programs have released cultivars with improved drought resistance, but the underlying mechanisms remain unknown. In this study, we used a number of plant physiology methods to characterize the mechanisms driving drought resistance in four zoysiagrass cultivars reported to exhibit contrasting levels of drought resistance. They were Lobo, Zeon, Empire, and Meyer. A dry-down was performed through deficit irrigation until 70% decline in evapotranspiration. No drought escape mechanism was identified in this project. Drought avoidance was characterized by the rate of dehydration over time, and drought tolerance was characterized by the decline in functional traits with increasing dehydration. Through this approach, we were able to separate avoidance from tolerance and demonstrate that drought tolerance governs drought resistance in commercial cultivars of zoysiagrass. Interestingly, we also demonstrated that canopy mortality during drought can only be reliably assessed using image analyses shortly after rehydration. This is because severe leaf rolling occurs during drought, confounding leaf rolling with actual leaf mortality. This study advances our understanding of i) drought resistance across commercial cultivars of zoysiagrass and ii) potential methods to select drought-resistant cultivars in turfgrass breeding programs.

Salinity stress is a growing concern in agriculture, particularly as climate change accelerates soil salinization and limits freshwater availability. Here, we evaluated the effectiveness of classic (low-throughput) versus high-throughput physiological phenotyping methods in detecting early salinity tolerance in Brassica juncea cultivars (‘Carolina Broadleaf’ and ‘Southern Giant Curl’). Traditional phenotyping relies on point measurements such as shoot biomass and leaf gas exchange, which, while valuable, are time-intensive, offer limited temporal resolution, and can be destructive. In contrast, high-throughput phenotyping enables continuous, real-time monitoring of plant physiological responses, providing a dynamic and detailed understanding of stress adaptation mechanisms. We conducted a 42-day experiment in a controlled greenhouse environment, exposing mustard green cultivars to three salinity treatments: control (0.397 dS/m), moderate salinity (10.81 dS/m, ~20% of seawater), and high salinity (24.93 dS/m, ~50% of seawater). The high-throughput PlantArray system was used to measure key physiological parameters, transpiration rates, and net plant weight gain, while traditional phenotyping involved weekly surveys of including stomatal conductance, chlorophyll fluorescence, and biomass accumulation. We found that high-throughput phenotyping allows for earlier and more precise detection of salinity tolerance. Classic methods confirmed significant reductions in biomass, with shoot fresh weight decreasing by up to 80% in high-salinity treatments, but these differences were only detectable at harvest and not before. In contrast, high-throughput phenotyping revealed early signs of osmotic adjustment within the first 20 days, as plants initially maintained transpiration before exhibiting a decline due to ion accumulation. ‘Carolina Broadleaf’ resist moderate salinity, maintaining growth comparable to the control for the first 20 days, suggesting that early harvesting could mitigate yield losses. Overall, this study underscores the advantages of high-throughput phenotyping in improving the precision and efficiency of breeding programs. By integrating continuous physiological measurements, this approach enables earlier and more informed selection of salt-tolerant cultivars, reducing time needed for tolerance screening. Future research should focus on expanding these methods to operational conditions and integrating genomic data to enhance genotype-environment modeling for stress adaptation.