After 6 days and 15 days of storage, the kale disks showed lighter green coloration than the kale disks stored for 3 days . However, the kale leaf disks stored under constant light were lighter green than the kale disks stored under light/dark cycles and showed some brown or yellow discoloration after 3 and 6 days . By 15 days, the kale leaf disks stored under constant light lost nearly all green coloration and showed light and dark shades of browning with shape changes resulting from leaf folding and shrinkage . The kale leaf disks stored under constant darkness resembled those stored under constant light, except that the 3-day kale samples were darker green than the 3-day constant light-stored kale leaf disks , suggesting that the constant light may have constituted a greater stress on the kale leaves than constant darkness. These results indicate that post harvest storage with daily cycling of light and darkness improved the appearance of the kale leaf tissue compared to storage under either constant light or constant darkness. However, the preservation benefit obtained from post harvest storage under light/dark cycles at 22°C appeared to be less than that provided by refrigeration; kale leaf disks stored at 4°C with constant darkness, dutch bucket for tomatoes were comparable in their dark green coloration whether stored for 3, 6 or 15 days . Cabbage leaf disks stored under cycles of 12-hour light/ 12-hour darkness showed brown spots along the disk edges that increased in intensity over the storage period of 7, 14, and 21 days .
However, although the 7-day cabbage leaf samples were light green in coloration, the 14- and 21-day cabbage leaf disks stored under light/dark cycles had darker green coloration , suggesting increased photosynthetic activity over storage time. In contrast, although the cabbage leaf disks stored under constant light were also light green after 7 days of storage, the 14- and 21-day cabbage leaf disks were more yellow and included more brown discolorations . Remarkably, the absence of light exposure during post-harvest storage had a dramatic effect on the cabbage leaf disk coloration. Cabbage leaf disks stored under constant darkness at either 22°C or 4°C were pale tan or yellow after 3 days of storage . The constant darkness-exposed cabbage leaf disks stored at 22°C appeared nearly white in color by 14 and 21 days; those at 4°C had a yellowish appearance after 2 or 3 weeks of storage . Lettuce and spinach leaf disks tissue were nearly uniformly green, with little difference in color intensity between 3 and 6 days of storage under cycles of 12-hour light/12-hour darkness . By 9 days of storage under light/dark cycles, however, both lettuce and spinach leaf disks looked slightly less green, and most of the spinach leaf disks had distinct patches of yellow . In contrast, the loss of green coloration and increased yellowing over time was much more apparent in the lettuce and spinach leaf disks stored under constant light; the lettuce leaf disks were pale green by 9 days , and all the spinach disks had large yellow patches . Lettuce and spinach leaf disks stored under constant darkness displayed small brown patches by 3 days . After 6 and 9 days of storage under constant darkness, the lettuce disks had large wet patches of darkened tissue .
However, the spinach leaf disks stored under constant darkness at 22°C for 6 days completely disintegrated and therefore could not be moved for photographic imaging. Lettuce and spinach leaf disks stored in constant darkness at 4°C largely maintained dark green coloration at 6 days and were lighter green at 9 days, similar to that of the disks stored under light/dark cycles at 22°C . However, after 6 days of storage at 4°C, the lettuce leaf disks also displayed browning around the vascular tissues . Overall, the image analysis shown in Figure 1 suggests that post harvest storage under cycles of 12-hour light and 12-hour darkness may enable kale, cabbage, lettuce, and spinach leaf tissues to maintain physiological functioning for longer durations after harvest. The reduction in green color and appearance of brown discoloration suggests that post harvest storage in constant light or constant darkness may accelerate loss of tissue viability.To further characterize kale, cabbage, lettuce and spinach leaf health and viability during post harvest storage, we quantified chlorophyll content in leaf samples after storage under cycles of 12-hour light/12-hour darkness to leaf tissues stored under constant light or constant darkness. Three sets of comparative data are shown . Because our primary focus was to determine whether light/dark cycles were advantageous relative to constant light or constant dark storage conditions, we first conducted two-way comparative statistical analyses between data derived from the samples stored under light/dark cycles relative to comparable samples stored under the alternative condition. Figure 2 presents statistical analysis of storage-dependent differences in chlorophyll levels relative to dry weight at each time point. Additional file 1: Figure S3 shows similar analyses but of storage-dependent differences in chlorophyll levels relative to fresh weight. Finally, to evaluate whether there were significant changes in chlorophyll levels of each plant type over time, statistical analyses of differences in chlorophyll content at the beginning and end of the experiments for kale, cabbage, lettuce, and spinach are shown in Additional file 1: Figure S4.
Consistent with the loss of green coloration in the representative leaf disk samples shown in Figure 1A, post harvest storage of kale leaf disks in either constant light or constant dark led to significantly greater losses in kale chlorophyll content within 3 or 6 days of post harvest storage compared to storage under 12-hour light/12-hour dark cycles . Kale leaf disks stored for 15 days under constant light lost 97% and 93% of their original chlorophyll content relative to dry and fresh weight, respectively ; kale leaf disks stored under constant darkness lost 88% and 89% of their chlorophyll content relative to total dry and fresh weight, respectively . In contrast, 15 days of storage under cycles of 12-hour light/12-hour darkness led to loss of only 36% and 9% of the kale leaf disk chlorophyll relative to dry and fresh weight, respectively . Kale leaf disks stored at 4°C under constant darkness, however, performed statistically better than those stored under cycles of light/dark , with no significant decreases in chlorophyll content relative to total dry or fresh weight over the full 15 days of the experiment . Post harvest storage of cabbage leaf disks under light/ dark cycles resulted in significantly higher chlorophyll levels than storage under constant dark either at 22°C or 4°C at all time points examined . Indeed, cabbage leaf disks began with only modest chlorophyll levels . However, when the cabbage leaf disks were stored under either constant light or light/dark cycles, chlorophyll content increased over time with significantly higher levels remaining even after three weeks of storage . Light-induced synthesis is likely responsible for the elevated chlorophyll levels and the enhanced green coloration observed in the cabbage leaf images of the samples stored under light/dark cycles or constant light but absent in the cabbage leaf disks stored under constant darkness either at 22°C or 4°C . Storage under light/dark cycles was also more successful than constant light exposure in maintaining higher chlorophyll levels after long-term storage of 3 weeks . Over time, storage under constant light may be counterproductive; whereas cabbage leaf disks stored for 7 days under constant light had significantly higher chlorophyll content than leaf disks stored under light/dark cycles , by three weeks of storage, the leaf disks stored under light/dark cycles retained at least 2-fold more chlorophyll than samples stored under constant light . These results indicate that light during post harvest storage can have a profound effect on chlorophyll levels in cabbage, consistent with previous reports, blueberry grow pot and that diurnal cycling of light and darkness prolongs this benefit during longer term storage. Storage under cycles of 12-hour light/12-hour darkness also promoted chlorophyll retention in lettuce leaf disks, comparable to that of lettuce leaf disks stored under refrigeration; chlorophyll levels were statistically indistinguishable between lettuce leaf disks stored under light/dark cycles versus those refrigerated under constant darkness conditions after 3, 6 or 9 days of storage . Postharvest storage of lettuce leaf disks either at 22°C under light/dark cycles or under refrigeration resulted in no significant change in chlorophyll content over the course of the 9-day experiment, whereas the lettuce leaf disks stored under either constant light or constant darkness, lost more than 50% of their starting chlorophyll content . Chlorophyll content of spinach leaf disks was not significantly affected by treatment conditions for the first 3 days of post harvest storage . However, the spinach leaf disks stored at 22°C in constant darkness disintegrated by 6 days and were therefore unable to be further analyzed. Spinach leaf disks stored under light/dark cycles had similar chlorophyll content to those stored under constant light with relatively stable chlorophyll retention until day 9 when chlorophyll levels in both samples decreased significantly from initial levels . In contrast, refrigeration led to stable chlorophyll levels in the spinach leaf disks over the course of the experiment .
These results indicate that chlorophyll content of post harvest green leafy vegetables varies depending upon the storage conditions and suggests that storage under 12-hour cycles of light and darkness, known to maintain the plant circadian clock, can improve kale, cabbage and lettuce chlorophyll content maintenance relative to storage in constant light or constant dark. Perhaps surprisingly light/dark cycles during post harvest storage may be at least as beneficial as refrigeration with respect to chlorophyll content for cabbage and lettuce.Over time during post harvest storage, plant tissues typically show visible signs of tissue disintegration . To determine if maintaining light/dark cycles during storage of post harvest leafy vegetables could prolong tissue integrity, we compared electrolyte leakage from kale, cabbage, lettuce, and spinach leaf disks stored over time under cycles of 12-hour light/12-hour darkness to leaf disks stored under constant light or constant darkness at 22°C or constant darkness at 4°C. Figure 3 shows that post harvest storage under light/dark cycles and refrigeration were comparable, with respect to leaf tissue integrity maintenance of kale, cabbage and lettuce, as measured by electrolyte leakage. When directly comparing light/dark storage to other conditions, a statistically significant benefit to diurnal stimuli during storage was apparent relative to constant light for kale , constant darkness for cabbage and lettuce , and constant darkness and constant light for spinach . Post harvest storage under constant dark was detrimental to kale, cabbage, and lettuce tissue integrity, with at least 4-fold increases in electrolyte leakage, whereas storage under light/dark cycles at 22°C or refrigeration resulted in no significant increase in electrolyte leakage over the course of the experiment . Constant light treatment also led to significant increases in electrolyte leakage from kale and lettuce leaf disks, but not cabbage leaf disks, over the storage periods examined . Overall the results shown in Figure 3 and Additional file 1: Figure S5 provide evidence that daily cycles of light and darkness during post harvest storage resulted in superior leaf tissue integrity maintenance largely comparable to refrigeration, whereas either constant light or constant dark storage conditions were detrimental.Our results indicate that storage of kale, cabbage, lettuce, and spinach in light/dark cycles can improve the post harvest longevity of chlorophyll levels and tissue integrity. Next we were interested in determining whether plant maintenance under daily cycles of light and darkness affects human-health relevant metabolite content. In particular, we sought to examine whether kale and cabbage stored under light/dark cycles maintain their glucosinolate content longer than when stored under constant light, constant darkness, or refrigeration. Figure 4A shows total glucosinolate levels in kale leaf disks after 0, 3, 6 and 15 days of post harvest storage under different conditions. Individual glucosinolate levels are shown in Additional file 1: Figure S1. Total glucosinolate levels were comparable between kale leaf disks stored at 22°C under light/dark cycles and leaf disks stored at 4°C in the dark ; after 15 days of post harvest storage under these conditions, total glucosinolate levels decreased by less than 35% . In comparison to light/dark storage conditions, constant light or constant darkness exposure during storage resulted in significantly reduced glucosinolate content in the kale leaf disks . By 15 days of post harvest storage under constant light or constant darkness at 22°C, the kale leaf disks lost over 80% and 99% of initial levels, respectively.