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, we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 wine grape cultivars typically grown in climatically diverse grape growing 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,25 litre plant pot 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 have traits 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.Berry chemistry, anatomy and physiology were measured in summer 2020 for 18 grape cultivars established in an experimental vineyard on the University of California, Davis campus . There were 13 red-fruited and 5 white-fruited cultivars. Further, 9 cultivars were classified as hot-climate, 7 as warm-climate and 2 as temperate climate, using the definitions from Anderson & Nelgen . Anderson & Nelgen sorted the major wine-growing regions worldwide into climate categories based on mean temperature over the growing season . Cultivars were then placed into their respective climate category based upon the highest proportion of bearing area grown in a particular climate category as of 2020. This proportion of land area devoted to growing a particular cultivar worldwide was taken as a thermal requirement, genotypically driven, to match sugar accumulation with a region’s climate. Plants were growing as mature vines, grafted to the same root stock , and trained to a bilateral, spur-pruned, vertical shoot-positioned trellising system with a North-South row orientation .
Cultivars were divided between two adjacent vineyard blocks . Davis is considered a hot, dry site for wine growing, with campus weather stations reporting a decadal average mean annual precipitation of 436 mm and mean growing season temperature of 19.8°C . Our study period was exceptionally hot, with mean daily and mean maximum daily temperatures ranging from 22.8-24.5°C and 32.6-34.4°C, respectively, partly due to the anomalous August 16 – 18 heatwave . Over the study period, vines were drip-irrigated weekly at 50% replacement of vineyard evapotranspiration, which was estimated from the reference evapotranspiration reported by the campus weather status and published crop coefficients for this trellising system and vine × row spacing .Berries were sampled at regular intervals defined by Brix values from 50% veraison to harvest . For each cultivar, 30 berries per vine were collected from different parts of the cluster and both sides of the vine from 2 – 6 vines . Berries of each replicate were crushed, and the grape juice obtained was centrifuged at 4200 rpm for five minutes. Next, each juice sample was analyzed for TSS using a refractometer Sper Scientific 30051 , pH with an Orion Star A211 pH meter , and titratable acidity by titration with 0.1 N NaOH with an end point at pH 8.2 .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 . Vials were put on ice and refrigerated at 4°C for at least 24 hours before further processing. 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,30 litre plant pots bulk 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 .Overall, we found that total cross-sectional phloem area in the pedicels and the petioles significantly predicted maximum °Brix accumulation rates in the berries , as well as sieve element area in pedicels . Other sieve tube traits, such as sieve plate porosity, were not correlated with sugar accumulation rates, indicating that grapevines mainly increase their maximum capacity for sugar transport by adding more and wider sieve tubes to the transport pathway.
Total cross-sectional areas were significantly lower in cultivars typically grown in hot than warm growing regions, suggesting these cultivars have been inadvertently selected for smaller phloem areas to slow sugar accumulation, delay ripening, and achieve an optimal flavor profile provided by longer grape maturation times prior to harvest . Further, although there wasn’t a significant difference in sieve element area between cultivar climate category in the pedicel phloem, sieve element area did significantly predict brix accumulation rate. Phloem area was also a stronger predictor for sugar accumulation rates than the typical vegetative physiology parameters of gas exchange and water potential . This study points to a new anatomical phenotype that can be used by grape breeders to select for cultivars with smaller petiole or pedicel phloem areas to decrease sugar accumulation rates to berries as an adaptation to increasing temperature.Our phloem area and °Brix accumulation results align with findings from trait comparisons in other crop species and experiments manipulating phloem area in grape and other crops. In grapevine , abscisic acid and gibberellin hormone treatments increased the phloem cross-sectional area in the midveins, pedicels, and stems along with berry sugar concentrations, despite reduced photosynthetic assimilation . The increased phloem area enhances the hydraulic conductivity of the transport pathway , facilitating the transport of sugars from source to sink . For example, modifying the expression of a phloem cell proliferation regulatory gene in tomato increased phloem area, yield, and fruit sugar concentration . Similarly, in giant pumpkin varieties, the phloem area in pedicels and petioles was positively correlated with fruit yield . These findings highlight the potential for optimizing phloem area to enhance plant productivity by matching source production and sink utilization. Additionally, our study suggests that targeting phloem/xylem in petioles could be an efficient approach for plant breeders to improve yield by enhancing hydraulic conductance and carbon export to fruits .One of the goals of this study was to investigate how cultivars adapted to different climate regimes varied in sugar accumulation and vascular anatomy traits under common garden conditions. Approximately half of the variance in berry sugar concentration is attributable to climate , making common garden experiments crucial to isolate the effects of plant traits on sugar accumulation. We found that, for red varieties, total phloem cross sectional area in the petioles and pedicels was significantly larger in the varieties typically grown in warm regions than hot regions . This could be an adaptation unknowingly selected by generations of winemakers to slow sugar accumulation and synchronize sugar and flavor development in hot climates. For white varieties, phloem area did not increase significantly from hot to warm regions . There could have been less selective pressure to increase sugar accumulation in the warm-climate white than red varieties, since white wines are typically made with lower alcohol content, and the absence of anthocyanin production could reduce metabolic demands for sugar .