Leaf starch metabolism sets the phase of stomatal rhythm

ABSTRACT In leaves of C 3 and C 4 plants, stomata open during the day to favour CO 2 entry for photosynthesis, and close at night to prevent inefficient transpiration of water vapour. The circadian clock paces rhythmic stomatal movements throughout the diel (24-h) cycle. Leaf transitory starch is also thought to regulate the diel stomatal movements, yet the underlying mechanisms across time (key moments) and space (relevant leaf tissues) remains elusive. Here, we developed PhenoLeaks, a pipeline to analyse the diel dynamics of transpiration, and used it to screen a series of Arabidopsis mutants impaired in starch metabolism. We detected a sinusoidal, endogenous rhythm of transpiration that overarches days and nights. We uncovered that a number of severe mutations in starch metabolism affect the endogenous rhythm through a phase shift, resulting in delayed stomatal movements throughout the daytime and reduced stomatal preopening during the night. Nevertheless, analysis of tissue-specific mutations revealed that neither guard-cell nor mesophyll-cell starch metabolism are strictly required for normal diel patterns of transpiration. We propose that leaf starch influences the timing of transpiration rhythm through an interplay between the clock and sugars across tissues, while the energetic effect of starch-derived sugars is usually non-limiting for endogenous stomatal movements. One-sentence summary The PhenoLeaks pipeline for monitoring diel transpiration dynamics reveals that leaf starch metabolism sets the timing of the endogenous stomatal rhythm.