Reduced starch availability and degradation reduces growth during the night.
Even in control conditions, the growth of sh2 was impaired (Table 1, Figure 8d). Starch accumulates gradually during the photoperiod in the mature zone of the leaf and is remobilized at night by amylase activity providing the energy supply for growth processes in the growth zome (Smith and Stitt, 2007; Stitt and Zeeman, 2012; Thalmann et al., 2016). Consequently, it is not the starch content itself, but the ability to sustain a steady supply of soluble sugar during the night which is crucial for plant growth (Rasse and Tocquin 2006). Starch degradation during the day is induced under stress conditions (Yano et al., 2005, Thalmann et al., 2016). The sh 2 mutant had reduced starch levels in the mature leaf tissues (Figures 4c, and 8a) and accumulated higher soluble sugar levels during the day (Figure 5a and 8a), but these concentrations decreased rapidly to very low levels early in the night (Figure 8c). Caspar et al. (1985) linked the rapid declining sugar levels in the early night observed in another starchless mutant to nonproductive respiration and suggested that respiration during the night is proportional to the availability of soluble sugars rather than the energy demand for growth. We showed that the growth impairment during the night is due to the low soluble sugar concentrations in sh2 and could be fully restored to WT rates by sucrose feeding (Figure 8e and f).
Overall, we showed a strong effect of altered energy and antioxidant metabolism in the maize leaf proteome in response to drought. Subsequent functional analysis of sh2 allowed us to unravel the central role of starch synthesis in growth regulation and stress defense.

Acknowledgments

We would like to thank Karin Schildermans, Tim Willems, and Azmi Abdelkrim for their help during optimizing and performing the proteome analysis, as well as Els Prinsen for the abscisic acid measurements and Danny Huybrecht and Sevgi Oden for the technical support in the lab work.