The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, hydroponic channel plant health and fruit production, as well as for those concerned with C storage.
The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds . The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios . Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening .
Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown . Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality.
However, the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance . Plants with a greater cross-sectional area dedicated to phloem , sieve tubes with wider lumen areas , and larger and more abundant pores in the sieve plates are expected to have a lower hydraulic resistance . Total phloem cross-sectional area in the shoots has been found to vary between several grape cultivars , and a greater cross-sectional phloem area has been linked to faster sugar accumulation in the fruit in other crop species . However, the variation of phloem structural traits across cultivars adapted to a diverse range of climatic conditions and the relationship of these traits to sugar accumulation is largely unknown for grapevines. Establishing these anatomical links could allow breeders to modify sugar accumulation by selecting for phloem traits, instead of management practices that can negatively impact the fruit zone environment or yield . In this study, hydroponic dutch buckets we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 winegrape cultivars typically grown in climatically diverse grapegrowing regions. We assessed phloem and xylem vascular anatomy in leaf petioles and midveins and berry pedicels, to capture hydraulic resistance along the long-distance transport pathway. We also measured maximum berry sugar accumulation rates in the post-veraison ripening period to capture the greatest capacity for sugar transport . We predicted that traits that reduce hydraulic resistance, including larger total cross-sectional phloem areas, larger mean lumen areas for individual sieve tubes, and more porous sieve plates would increase maximum sugar accumulation rates. We also predicted that cultivars typically grown in hotter wine regions would havetraits that increase hydraulic resistance, as an adaptation to increase wine quality by reducing the rate of sugar accumulation. In addition, we measured photosynthesis and vine water stress to compare the impacts of phloem anatomy, vine carbon supply, and vine water status on sugar accumulation rates. Overall, our goals were to determine the most influential traits for sugar accumulation in grape berries and evaluate the role of phloem anatomy in adapting grape cultivars to a wide range of different climates.Leaves and berries were sampled to measure petiole, midvein, and pedicel anatomy in the morning on three days at the end of the growing season . Two berries and leaves per vine were excised with a razor blade.
Leaf position was standardized as the 6th leaf from the shoot apex, to capture the most photosynthetically active leaves. Two leaf and one berry sample per vine were then prepared for light microscopy, and the other berry sample was prepared for scanning electron microscopy. For light microscopy, a 1-cm segment of leaf petiole and lamina and the entire pedicel of the berry were immediately excised and placed into a vial of chilled Formalin-Acetic Acid . For scanning electron microscopy, pedicels were immediately flash-frozen and immersed in liquid nitrogen for 1-min and placed into a chilled micro-centrifuge tube of 100% ethanol, then the tube was immersed in liquid nitrogen until the ethanol congealed . Samples were then immediately placed on ice and stored in a -20°C freezer for at least 24 hours before further processing.After 7 days in FAA, the light microscopy samples were soaked in 50% ethanol for 5 mins and then stored in 70% ethanol in preparation for paraffin embedding. Samples were first infiltrated with paraffin by using an Autotechnicon Tissue Processor to treat samples with the following sequence of solutions: 70%, 85%, 95%, 100% ethanol, 1 ethanol:1 toluene, 100% toluene , and paraffin wax , each for 1 hour. The infiltrated samples were then embedded into paraffin blocks with a Leica Histo-Embedder , and allowed to cool. A rotary microtome was then used to make 7μm-thick cross-sections for leaf laminas, petioles, and berry pedicels. Pedicel cross-sections were sampled from the receptacle and petiole and midvein cross-sections were sampled near the interface of the lamina and petiole. After the cross-sections were imaged, pedicels for four cultivars were remelted from their wax molds, oriented longitudinally and sectioned again at 7μm to obtain sieve element lengths. Sections were stained using a 1% aniline blue and 1% safranin solution following a modified staining procedure . Sections were then viewed under bright field or florescence microscopy using a Leica DM4000B microscope and a DFC7000T digital camera . Each pedicel , midvein , and petiole section was then measured for total phloem and xylem cross-sectional area using ImageJ software, by manually selecting relevant tissue areas. Vascular tissue in longitudinal sections and cross sections was identified by cell size and/or stain color. Safranin stained the secondary cell walls of the xylem red and phloem cell walls were stained blue by aniline blue. The phloem area measurements included sieve tubes and phloem fibers and parenchyma , and xylem area measurements included xylem vessels, fibers, and parenchyma. Xylem and phloem rays greater than 4 cell layers thick were excluded. The pedicel electron microscopy samples were processed following Mullendore . Briefly, samples were thawed at room temperature, washed in DI water, and cut into 1 mm cross sections with a fresh double-sided razor blade. Sections were then transferred to 1.5 ml of 0.15% Proteinase K solution and mixed at 55°C and 300 RPM rotation for 14 days with an Eppendorf Thermomixer . Samples were then washed in DI water and placed into an 0.1% amylase solution for 24 hours at 50°C. Samples were then washed in DI water again, lyophilized overnight, mounted on aluminum stubs, and viewed under a Field Emission Scanning Electron Microscope . Sieve plates were viewed under low vacuum , 20- KV of accelerating voltage and a spot size of 2.5.Phloem anatomy was a stronger predictor of maximum sugar accumulation rates than vine carbon gain or water stress. Maximum sugar accumulation rates were not significantly correlated with photosynthesis or midday leaf water potentials . Including photosynthesis and midday leaf water potential as additional predictors also did not substantively improve the relationships between maximum sugar accumulation rates and petiole or pedicel cross-sectional phloem areas. Akaike Information Criterion corrected for small sample size values were higher for the larger models than the univariate models predicting maximum accumulation rates from petiole or pedicel phloem area alone, indicating that accounting for vine carbon gain and water stress did not improve predictive capacity for sugar accumulation . In addition, only one correlation was found between phloem petiole area and minimum mid-day water potential , while other average photosynthesis and water potential variables did not correlate with the phloem anatomical parameters. Finally, a previous dataset measuring leaf area for each cultivar did not find any significant correlations with °Brix accumulation, or other parameters measured .