Long term effects of prolonged darkness

Long-term effects of prolonged darkness on specific physiological activities were also monitored at different leaf positions. Basically, stomatal conductance and CO2 assimilation were different at various leaf stages and were decreased or inhibited significantly within 24 h in all leaf positions upon prolonged dark treatment. Similar effect of long-term darkness on these parameters was observed after 7 days. In addition, prolonged dark treatment caused a significant decline in Fv/Fm and CHC a + b content, especially in the old leaves of plants suggesting that the functional and morphological disorganisation of chloroplasts was in progressed stage by this time. Based on these results, old leaves were the most sensitive to long-term effect of darkness and exhibited the most pronounced symptoms of senescence. Similar results were found in Arabidopsis, where stomatal conductance, Fv/Fm and chlorophyll content decreased differently during senescence of younger or older leaves (Mohapatra et al., 2010). Moreover, Fv/Fm and chlorophyll content decreased also significantly after 24 h in darkened Arabidopsis (Buchanan-Wollaston et al., 2005). Since the absence of light inhibited CO2 fixation and synthesis of carbohydrates, the major source of chemical energy for plants, the leaves had to consume stored organic molecules such as polysaccharides. Sugar starvation, which can be observed in our experiments, can also modulate the activity of HXKs (Cho et al., 2010, Granot et al., 2013, Sheen, 2014). Glucose is not only the main reduced carbon and energy source but also a signalling molecule, which regulates more than 2000 plant genes. Thus glucose and its derivatives may interact with phytohormones through the glycolytic and metabolite sensing pathways that rely on the hexose-phosphorylating function of HXK (Li and Sheen, 2016, Aguilera-Alvarado and Sánchez-Nieto, 2017). Basically, the accumulation of glucose, the potential substrate for HXK was the highest at the end of the light period and the lowest at the end of the dark period similarly to HXK activity in the leaves of Arabidopsis (Kunz et al., 2015) and tobacco plants (Häusler et al., 2000). Based on our results, the glucose content was the highest in the young developing leaves and lower in the mature and/or old leaves, but HXK activity and gene expression were the most pronounced in the mature leaves. Significant decrease was found in glucose content and HXK activity in all intact leaves within 24 h under darkness. These changes were detected in parallel with the inhibition of CO2 assimilation but it occurred before the damage of photosynthetic apparatus or decrease in photosynthetic pigments. Except for the mitochondrial SlHXK3, the expression of all other HXK genes in tomato was down-regulated and the HXK activity decreased significantly in all leaf positions under dark starvation, which led to the initiation of chlorophyll breakdown and senescence. Thus it can be supposed, that increased expression of chloroplastic (SlHXK4) and mitochondrial HXK genes (SlHXK1 and SlHXK2) contributes to the maintenance of enzyme activity under natural day–night cycles, and the hexokinase activity is necessary for the maintenance of steady state status of primary metabolic processes. The most interesting change is the increased expression of SlHXK3 in both sink and source leaves after 7 days of dark exposure. In contrast to other HXKs, this gene was highly up-regulated by starvation and can be considered as a famine gene. We can also speculate that these changes are the last efforts of tissues to maintain minimal metabolic activity and mitochondrial integrity or this enzyme, similarly to plants overexpressing AtSLHXK1 under the control of 35S promoter, enhances senescence (Granot et al., 2013). However, it can be concluded that higher total HXK activity in younger leaves is connected with slower rate of dark-induced senescence and chlorophyll loss, in which SlHXK3 may have a central role.