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 , hydroponic dutch buckets 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 otherparameters 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 gasexchange 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 .
Phloem area has also been linked to fruit growth and sugar accumulation in other crop species. 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 . Phloem anatomy is influenced by both the climate that plants have adapted to and the climate plants experience during the growing season . This suggests that more work is needed to evaluate how plastic responses to interannual or geographic variability to climate influence cultivar differences in phloem anatomy and sugar transport capacity.
In Arabidopsis, the effects of growing conditions on phloem anatomy depended strongly on the climate the genotypes evolved in. Comparisons between cool and hot growing conditions showed that high temperatures reduced the proportion of phloem area in the minor veins, and that these reductions were larger in Arabidopsis genotypes that evolved in cool than hot climates . These results suggested that phloem plasticity in response to growing conditions outside evolved temperature ranges was greater ingenotypes adapted to cool climates, increasing genotypic differences in phloem anatomy under hot growing conditions. Interestingly, we found the opposite pattern in grape, that phloem area in the pedicel and petiole was significantly larger in the cultivars typically grown in warm than hot climates, even though our common garden experiment was in a hot growing region . Comparisons in different regions or in years with different climatic conditions are needed to determine how strongly the cultivar differences in anatomy observed here depend on the conditions during phloem development.Xylem and phloem area scaled in the midvein, petiole, and pedicel, which produced similar relationships in xylem and phloem areas with maximum °Brix accumulation rates and climate groupings . The relationships with xylem area could simply reflect selection for phloem traits and developmental constraints that make xylem and phloem differentiation proportional, or both xylem and phloem area could impact °Brix accumulation rates. °Brix is a concentration and determined by water and sugar contents. The phloem supplies most of the water to the berries after veraison . The total volume of phloem water influx is generally much larger than the volume of the berries, forcing the berries to export water to the canopy through the xylem to avoid cracking or splitting . A larger phloem area would increase the water influx into the berries, bato bucket which could require a larger xylem area to compensate for water export. Further, the xylem accounted for most of the vascular area in each organ, and the ratio of xylem to phloem area increased with stem cross sectional area, which also made this ratio significantly larger in warm- than hot-climate cultivars . This larger xylem:phloem ratio could accelerate °Brix accumulation by increasing the capacity for water export relative to influx. Thus, selecting for a lower xylem:phloem ratio could slow berry sugar accumulation. Xylem and phloem areas also scale in other species, including ash , Pelargonium , fir , poplar, and ginkgo , and, notably, xylem: phloem ratios were smaller in species with larger fruit . However, some grape cultivars produce blockages in the pedicel xylem during ripening that reduce conductivity and water efflux, which could make the ratio of xylem to phloem area less important to °Brix accumulation rates. Overall, more work is needed to clarify the effects of individual tissue areas and xylem: phloem area ratios on ripening.Another interesting findings from the current study was that sieve element area was a significant predictor of brix accumulation rate , while porosity of the elements , most of the smaller sieve element area/ lower brix accumulation rate cultivars were from the warm climate category. These findings suggest that grapevines have primarily adapted to control sugar accumulation rate by changing the number and width of sieve elements, although these two traits were not correlated . Conversely, phloem cross sectional area and mean sieve element area were correlated in the pedicels for other species, including pumpkin and tomato . However, similar to our findings, variation in sieve element area was small for pumpkin, and the differences between cultivars were not significant .
Cooler growing regions are typically more humid and prone to disease pressure , and less porous sieve plates can facilitate the faster formation of callose blockages to more quickly restrict pathogen spread through the phloem . Future work may consider the transcriptional abundance of sugar unloading proteins , and how this relates with phloem anatomical characteristics related to pathway resistance.Phloem anatomy was a stronger predictor of berry sugar accumulation rates than vegetative physiology parameters capturing vine carbon gain and water status. This was unexpected, since photosynthesis determines the carbon available for ripening, and water stress has been shown to strongly impact sugar accumulation rates in many of the cultivars tested here . However, our experimental vines were irrigated during the ripening period to maintain leaf water potentials in a relatively narrow range . This irrigation regime follows standard commercial practices for California, which could have limited cultivar differences in vine water stress and photosynthesis and thus, their impacts on sugar accumulation. These findings suggest that measuring phloem anatomy could provide more insight into plant capacity for berry sugar accumulation under standard, irrigated conditions than conventional vegetative physiology traits. Alternatively, leaf-level photosynthesis could have been decoupled from °Brix accumulation by variation in vine balance , which would impact the ratio of whole-plant carbon supply to demand. A larger ratio of canopy area to fruit mass would increase maximum °Brix accumulation rates. Future work should estimate leaf area per cultivar to ensure that relationships between phloem anatomy and maximum °Brix accumulation rates scale with variation in vine balance.Overall, we found that phloem cross-sectional area in the petioles and pedicels was the most predictive trait for the maximum rate of sugar accumulation in the berries across wine grape cultivars tested. Carbon dioxide in the atmosphere is expected to double by centuries’ end, and the dual effects on plant carbon availability and growing season temperature are projected to strongly accelerate sugar accumulation and exacerbate the detrimental impacts on wine quality . We suggest that reduced phloem areas could be a useful and novel phenotype to screen for in existing cultivars to slow carbon transport rates in hotter growing regions, allowing more time for flavor development. If petiole phloem area is well-conserved across life stages and under different growing conditions, this would be an especially useful trait to accelerate phenotyping since grapevines must mature for several years to begin producing fruit. However, future work is still needed to clarify how xylem area and vine balance influence sugar concentrations, and how interannual and site-specific environmental variability influence anatomical traits and sugar accumulation.Age-related macular degeneration is the leading cause of blindness among seniors in developed countries, and third worldwide after uncorrected refractive errors and cataracts. In early stages, the disease is characterized by small to intermediate drusen with pigmentary changes that may progress rapidly to more advanced forms such as choroidal neovascularization or central geographic atrophy with loss of central vision. Lutein , zeaxanthin , and the isomer meso-zeaxanthin are macular pigments that filter damaging blue light and provide oxidative defense in the macula. These pigments are found in plants as xanthophylls, with increased dietary intake proposed to reduce the development and progression of AMD. The relative concentration of xanthophyll carotenoids in the retina can be measured non-invasively by psychophysical and objective methods, expressed as macular pigment optical density.