The irrigation frequency was increased to 5 times per day with 200 mL at full bloom . It was then increased to 8 times per day the week after pollination . Irrigation started 1 h before sunrise and finished 1 h after sunset. Three days before harvest, the irrigation frequency was adjusted to one time per day with 4800 mL water to further enhance cracking. Plants were topped on 28 February when they had 2 clusters of flowers and were pollinated on 1, 4, 8, and 11 March. On 18 March, the spray treatments with water or ABA began, applied 3 times per week for 7 weeks, until 22 April. Tomato cracking rate, firmness, total soluble solids and titratable acidity were analyzed on 30 April. The fruit materials for other analyses were preserved until the next day and then analyzed.Previous research indicates that heritable resistance to cracking can be identified in some tomato breeding lines. However, no single genetic locus seems to be responsible for inheritance of the fruit cracking trait and many genes may contribute to the phenotype. Many studies point to the involvement of cell wall structure and possibly the cuticle layer in fruit cracking. As cell wall networks weaken with fruit ripening, even as cell turgor falls, resistance to stresses at the fruit surface may require a greater contribution from wax and cuticle layer structures than they can provide; and cell turgor pushing the plasmalemma against the cell wall also creates some stress on the cell wall polysaccharide networks that may be accommodated by the elasticity of the wall “fabric”; this then leading to cracking. Polysaccharides make up more than 90% of the mass of the plant cell wall. The pectins are relatively uronic acidrich polymers that are the most structurally complex polysaccharides in plant primary cell walls.
PG is believed to be responsible for a large part of the HG pectin depolymerization in ripening tomatoes; PG mRNA, protein, dutch buckets and activity accumulate to very high levels late in the ripening of tomato fruit. Brummell reported that suppression of the ripening-related EXP-encoding gene slowed tomato fruit softening early in ripening, and theyhypothesized that EXP1-mediated relaxation of the wall structure is necessary to allow PG or other enzymes access to polyuronide or other wall substrates. To investigate how PG and EXP may work collaboratively to affect the susceptibility of tomato fruit to cracking, we investigated differences in cell wall composition as influenced by the pg/exp genotype in fruit stressed by increased water uptake following treatment with ABA. Ripening in tomato is accompanied by a shift in pectins from the CSP and SSP to the WSP. The clearest impact of simultaneous suppression of PG and EXP was that the pg/exp fruit displayed a substantially reduced breakdown of the cell wall pectin network as they proceed through ripening and the fruit soften less than WT fruit. The pectin polymers in ripe fruit of the pg/exp genotype are bigger than those in ripe WT fruit, and there was more SSP in pg/exp fruit compared with WT. In our study, the cracking-resistant pg/exp genotype, had more CSP in the mesocarp portion, and more SSP in the exocarp portion of the fruit. In contrast, there was more WSP in fruit of the WT genotype. This observation suggests that both the exocarp and mesocarp cell walls of pg/exp fruit were more intact and thus better able to resist internal stresses that are presumed to promote ripe fruit cracking. The calcium content of the fruit and their cell walls can affect cell wall strength.
Pectins with low levels of methoxyl-esterification can form gels; calcium-ion bridging of unesterified GalA residue carboxyl groups on neighboring HG pectins has been proposed to form “eggbox” structures in the primary cell wall matrix. And the strength of the Ca2+-promoted-gels increases with increasing Ca2+ concentration. In this experiment, the higher Ca2+ level in the AIR from WT fruit than in the AIR from the firmer and less-cracked pg/exp fruit is somewhat surprising. It is not clear how the AIR’s Ca2+ content corresponds with the relative distributions of Ca2+ in the cell wall/apoplast, the cytoplasm and the vacuolar compartments. However, our immuno- fluorescence microscopy with JIM5 and JIM7 antibodies revealed that pg/exp fruit had more highly esterified pectins than WT fruit, indicating less capacity for cell wall binding of Ca2+. In some cases, there is also less Ca2+ in cracking-resistant varieties. We conclude that a ripe fruit with more intact pectins in its primary walls is likely to resist cracking more effectively, as long as a reasonable degree of pectin-pectin bonding is retained. The correlation analysis demonstrated that crack rate was associated most significantly with the protopectin and cellulose rather than Ca2+, which confirms this view. In our study, we used whole-plant sprays of ABA to increase the tendency of tomato fruit to crack. ABA application can decrease stomatal conductance and leaf transpiration, and increases plant water potential, which results in significantly increased xylemic flow into tomato fruit. This xylemic flow also carries more Ca2+ into the fruit as has been reported previously and is evident in the higher Ca2+ levels in both whole fruit and cell walls of ABA treated fruit.
The higher incidence of cracking in ABA treated RR tomato fruit was likely due to accumulation of water in the fruit when leaf transpiration was reduced by ABA, likely resulting in increases in turgor pressure in the fruit. However, the fruit genotypes showed significant differences in their tendency to crack when cracking was promoted by ABA application. The increase in cracking in response to ABA treatment was not observed in RR pg/exp fruit but was observed in WT and glk2 fruit. And there was no difference in cracking among the three genotypes when plants were treated with water. ABA treatment also had an influence on tomato cell wall composition, resulting in lower amounts of WSP and SSP in mesocarp and blossom end tissues of all three genotypes. This does not appear to be related to influences on fruit ripening as no visible differences in ripening were observed and ABA is generally reported to enhance ripening, not slow ripening. The higher proportion of chelator soluble cell wall material may be a response to the higher Ca2+ levels in the fruit due to the higher xylemic flow, but it is unclear what role if any this played in the increased cracking of ABA treated fruit. Cell walls from pg/exp fruit also appeared thicker and denser than cell walls from WT fruit under electron microscopy, perhaps because of reduced disassembly of the cell wall polysaccharide polymer network. This difference could be another reason for resistance to cracking in this genotype. The thicker and denser cell walls from pg/exp fruit are reflected in the higher levels of CSP, SSP and cellulose in cell wall extracts prepared from the mesocarp of pg/exp fruit. Cantu previously demonstrated that the reduction of both PG and EXP activities resulted in isolated and in situ cell walls that swelled much less than walls from WT fruit that soften significantly at the fully ripe stage; which supports the conclusion that pg/exp fruit has a more intact cell wall than WT fruit. In our experiments, pg/exp had a thicker and denser cell wall that may resist swelling. The cuticular wax layer was thicker in pg/exp fruit, which could also contribute to resistance to cracking. While waxes are part of the overall extracellular matrix, they are not targets of either PG or EXP, which suggests that suppression of the ripening-associated SlPG and SlEXP1 genes may also have impacts on other structures at the fruit surface. It is interesting to note that in addition to changing cell wall network integrity, tomato fruit cuticle chemistry, grow bucket and structure have been identified as fruit factors that influence ripening-associated fruit softening. The correlation analysis also showed that cracking rate was significantly associated with cell wall composition and cell wall-thickness, as well as with wax-thickness. Since increased water uptake by the fruit due to exposure of the plants to ABA promotes cracking, the physical constraints of the more intact cell walls in the pg/exp fruit seem to be the major factor providing resistance to cracking at the later stage of ripening .Approximately one-third of food produced globally is lost or wasted , yet fewer resources are devoted to post harvest research and development than to efforts for improving productivity. The modular design of plants allows plant tissues and organs to remain biologically active even after harvest. Therefore, capitalizing on the ability of harvested vegetables and fruits to continue to sense and respond to diverse stimuli, similarly to intact plants, may be a powerful approach to promote post harvest quality. Research demonstrating the biological advantage of a functional circadian clock in plants led us to investigate whether maintaining diurnal cycles may promote longevity and therefore reduced yield loss during post harvest storage of vegetables.
The circadian clock enables plants to anticipate and prepare for the daily environmental changes that occur as a consequence of the rotation of the earth. Coordination of plant circadian rhythms with the external environment provides growth and reproductive advantages to plants, as well as enhanced resistance to insects and pathogens. The circadian clock also regulates aspects of plant biology that may have human health impact, such as levels of carbohydrates, ascorbic acid, chlorophyll , and glucosinolates in edible plant species. Plants exhibit exquisite sensitivity to light stimuli, and isolated plant leaves maintain responsiveness to light after harvest and can continue light-dependent biological processes, such as photosynthesis. Additionally, the clocks of post harvest fruit and vegetable tissues can been trained with 12-hour light/12-hour darkness cycles producing rhythmic behaviors not observed in tissues stored in constant light or constant dark. For example, light exposure delays broccoli senescence and yellowing but accelerates browning in cauliflower, a close relative of broccoli. Other studies report that light exposure to broccoli during post harvest storage either provides no additional benefits or decreases performance. Post harvest light exposure improves chlorophyll content in cabbage, but leads to increased browning of romaine lettuce leaves. Although exposure of spinach to light during post harvest storage can improve nutritional value, light can also accelerate spinach water loss, leading to wilting. Together, these findings are inconclusive as to whether light exposure during post harvest storage can be generally beneficial, and the variation of the results may be attributable to differences in the plant species examined and the specific conditions used during post harvest storage, such as lighting intensities, temperature, humidity or packaging. Alternatively, light may be advantageous but only if present in its natural context with 24-hour periodicity because of such timing on circadian clock function. This study aimed to examine whether mimicking aspects of the natural environment predicted to maintain circadian biological rhythms during post harvest storage of green leafy vegetables improves performance and longevity compared to post harvest storage under constant light or constant darkness. We focused this work on several popular and nutritionally valuable species, including kale and cabbage , members of the Brassicaceae family with worldwide production of approximately 70 million tons. In addition, we analyzed green leaf lettuce and spinach , which have worldwide production of approximately 25 and 22 million tons, respectively. Here, we report on the promotion of post harvest longevity, including tissue integrity and nutritional value, of green leafy vegetables by provision of 24-hour light/dark cycles during storage compared to storage under constant light or constant darkness.Fruits and vegetables after harvest can respond to repeated cycles of 12-hour light/12-hour dark, resulting in circadian clock function and rhythmic behaviors. Because a functional plant circadian clock is physiologically advantageous we sought to address whether post harvest storage under conditions that simulate day/night cycles, thereby potentially maintaining biological rhythms, would affect post harvest longevity. We chose to address this question using green leafy vegetables, including commonly consumed kale , cabbage , green leaf lettuce and spinach , because we anticipated that the leaf organ would likely maintain light sensitivity and responsiveness even after harvest. To begin to determine whether daily light/dark cycles during post harvest storage affects leaf longevity, we compared the overall appearance of leaf disks that were stored at 22°C under cycles of 12-hour light/12-hour darkness versus leaf disks stored under constant light or constant darkness for various lengths of time . Under cycles of 12-hour light/12-hour darkness, kale leaf disks were dark green after 3 days of storage .