br Materials and methods br Results
Materials and methods
Results To examine the time and organ specific effect of normal photoperiod and dark environment on the Senegenin sale of different SlHXKs and on the enzyme activities, these parameters were determined in different leaf positions of tomato plants. Under control conditions, transcript levels of HXK coding genes, especially those of SlHXK1 and SlHXK4 exhibited diurnal fluctuations in the mature leaves of tomato with a maximum in the late light period and a subsequent decrease in the dark (Fig. 1A, G). Similar tendency can be found in the case of SlHXK1 in the old leaves (Fig. 1A). Moreover, the expression of SlHXK3 was the highest in the early dark period in the mature leaves (Fig. 1E). SlHXK1 expression showed similar tendency but lower transcript abundance during the first 24 h of dark exposure in the mature leaves, however transcript levels of SlHXK1 exhibited a transient peak in the old leaves after 6 h followed by a second maximum at 24 h in the dark (Fig. 1B). Similar tendencies can be detected in the expression pattern of SlHXK2 in the old leaves while the transcript abundance of SlHXK2 increased only in the mature leaves after 6 h. The mitochondrial SlHXK3 responded very early to the absence of light, because the transcripts of SlHXK3 accumulated 8-fold from 3 to 12 h upon the dark treatment in the mature and old leaves (Fig. 1F). Surprisingly, the expression of SlHXK3 increased significantly at all leaf positions under long-term darkness and the highest expression was found in the old leaves, while the expression of the other genes was inhibited after 7 days of dark treatment (Fig. 2). The specific activity of HXK with glucose substrate showed also leaf age-specific and diurnal regulation in the leaves of tomato plants (Fig. 3). During the normal photoperiod, the most characteristic changes were observed in the mature leaves, where HXK activity showed the highest levels compared to other leaf stages (Fig. 3A). Similar but not so pronounced tendencies can be found in the other leaf positions during the normal photoperiod. Interestingly, HXK activity increased after 3 h in the mature leaves, but then decreased continuously in all leaves of tomato under prolonged darkness (Fig. 3B). After 7 days of dark treatment HXK activities decreased significantly at all leaf positions of plants and the most significant decrease was found in the old leaves (Fig. 4). The activity of HXKs depends on a number of physiological processes during dark-induced senescence, which determine the substrate concentration available for enzymes. After all, it is photosynthetic activity that is responsible for the optimal glucose concentration for HXKs. The stomatal conductance can determine the net CO2 assimilation rate of leaves. Under normal photoperiod the lowest stomatal conductance was observed in the old leaves and the highest in the mature leaves (Table 2). The stomatal opening was inhibited and stomatal conductance decreased if the plants were kept in darkness for 24 h or 7 days, but there were no significant differences between the two time points. In parallel, CO2 assimilation showed similar tendencies as the changes in stomatal conductance in the different leaf positions under normal photoperiod, while photosynthetic activity ceased in the dark (Table 2). Fv/Fm, the maximal quantum efficiency of PSII, is an important stress marker of PSII damage. This parameter was lower in the old leaves but did not change after 24 h in the dark. However, Fv/Fm decreased significantly after 7 days under darkness in all leaf positions (Table 2). Similar changes were observed in chlorophyll a + b contents. The amount of these photosynthetic pigments was lower in the old leaves and it was maintained at constant level after a 24 h dark period. Chlorophyll a + b content decreased further in all leaves of tomato plants, which proved to be significant in old leaves after a 7-day-long dark treatment (Table 2). Glucose content, a product of photosynthesis was the highest in the young leaves and lower in the other leaf positions. Interestingly, the 24-hour-long dark treatment already decreased the glucose content significantly in all leaves of tomato and this tendency was much more pronounced after 7 days (Table 2.).