Contents Summary1018I.Introduction1018II.Guard cell photosynthesis1019III.Guard cell central metabolism1022IV.Guard cell starch metabolism differs from that of mesophyll cells and plays a key role in stomatal movement1025V.Connectors between mesophyll and guard cells1026VI.Challenges and perspectives in understanding and modelling guard cell metabolism1029Acknowledgements1030References1030 Summary Stomata are leaf epidermal structures consisting of two guard cells surrounding a pore. Changes in the aperture of this pore regulate plant water‐use efficiency, defined as gain of C by photosynthesis per leaf water transpired. Stomatal aperture is actively regulated by reversible changes in guard cell osmolyte content. Despite the fact that guard cells can photosynthesize on their own, the accumulation of mesophyll‐derived metabolites can seemingly act as signals which contribute to the regulation of stomatal movement. It has been shown that malate can act as a signalling molecule and a counter‐ion of potassium, a well‐established osmolyte that accumulates in the vacuole of guard cells during stomatal opening. By contrast, their efflux from guard cells is an important mechanism during stomatal closure. It has been hypothesized that the breakdown of starch, sucrose and lipids is an important mechanism during stomatal opening, which may be related to ATP production through glycolysis and mitochondrial metabolism, and/or accumulation of osmolytes such as sugars and malate. However, experimental evidence supporting this theory is lacking. Here we highlight the particularities of guard cell metabolism and discuss this in the context of the guard cells themselves and their interaction with the mesophyll cells.

SummaryThe control of stomatal aperture involves reversible changes in the concentration of osmolytes in guard cells. Sucrose has long been proposed to have an osmolytic role in guard cells. However, direct evidence for such a role is lacking. Furthermore, recent evidence suggests that sucrose may perform additional roles in guard cells. Here, we provide an update covering the multiple roles of sucrose in guard cell regulation, highlighting the knowledge accumulated regarding spatiotemporal differences in the synthesis, accumulation, and degradation of sucrose as well as reviewing the role of sucrose as a metabolic connector between mesophyll and guard cells. Analysis of transcriptomic data from previous studies reveals that several genes encoding sucrose and hexose transporters and genes involved in gluconeogenesis, sucrose and trehalose metabolism are highly expressed in guard cells compared with mesophyll cells. Interestingly, this analysis also showed that guard cells have considerably higher expression of C 4 -marker genes than mesophyll cells. We discuss the possible roles of these genes in guard cell function and the role of sucrose in stomatal opening and closure. Finally, we provide a perspective for future experiments which are required to fill gaps in our understanding of both guard cell metabolism and stomatal regulation.

Stomatal responses to environmental signals differ substantially between ferns and angiosperms. However, the mechanisms that lead to such different responses remain unclear.Here we investigated the extent to which leaf metabolism contributes to coordinate the differential stomatal behaviour among ferns and angiosperms.Stomata from all species were responsive to light and CO 2 transitions. However, fern stomatal responses were slower and minor in both absolute and relative terms. Angiosperms have higher stomatal density, but this is not correlated with speed of stomatal closure. The metabolic responses throughout the diel course and under different CO 2 conditions differ substantially among ferns and angiosperms. Higher sucrose content and an increased sucroseto-malate ratio during high CO 2 -induced stomatal closure was observed in angiosperms compared to ferns. Furthermore, the speed of stomatal closure was positively and negatively correlated with sugars and organic acids, respectively, suggesting that the balance between sugars and organic acids aids in explaining the faster stomatal responses of angiosperms.Our results suggest that mesophyll-derived metabolic signals, especially those associated with sucrose and malate, may also be important to modulate the differential stomatal behaviour between ferns and angiosperms, providing important new information that helps in understanding the metabolism-mediated mechanisms regulating stomatal movements across land plant evolution.

Trehalose 6-phosphate (Tre6P), a sucrose signaling metabolite, inhibits transitory starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves and potentially links starch turnover to leaf sucrose status and demand from sink organs (Plant Physiology, 163, 2013, 1142. To investigate this relationship further, we compared diel patterns of starch turnover in ethanol-inducible Tre6P synthase (iTPS) lines, which have high Tre6P and low sucrose after induction, with those in sweet11;12 sucrose export mutants, which accumulate sucrose in their leaves and were predicted to have high Tre6P. Short-term changes in irradiance were used to investigate whether the strength of inhibition by Tre6P depends on starch levels. sweet11;12 mutants had twofold higher levels of Tre6P and restricted starch mobilization. The relationship between Tre6P and starch mobilization was recapitulated in iTPS lines, pointing to a dominant role for Tre6P in feedback regulation of starch mobilization. Tre6P restricted mobilization across a wide range of conditions. However, there was no correlation between the level of Tre6P and the absolute rate of starch mobilization.Rather, Tre6P depressed the rate of mobilization below that required to exhaust starch at dawn, leading to incomplete use of starch. It is discussed how Tre6P interacts with the clock to set the rate of starch mobilization. K E Y W O R D SArabidopsis, circadian clock, diel, starch, trehalose 6-phosphate | INTRODUCTIONPlants use light energy to drive photosynthetic carbon (C) gain, metabolism, and growth, but at night depend on C reserves accumulated in previous light periods. In many species, including Arabidopsis, foliar starch is the major C reserve (Smith & Stitt, 2007). Diel regulation of starch turnover may depend on the conditions (Paul & Foyer, 2001). In source-limited plants, C is in short supply and it is crucial to manage C reserves to insure rapid investment in growth while avoiding C starvation at night (Scialdone and Howard, 2015;Smith & Stitt, 2007;Stitt & Zeeman, 2012). In sink-limited conditions, C regulation of metabolism and growth is relaxed (Baerenfaller et al., 2015;Sulpice et al., 2014) and starch often accumulates in leaves and other parts of the plant. This incomplete utilization of starch may be at least partly due to feedback inhibition of starch mobilization by the sucrose signal trehalose 6-phosphate (Figueroa Lunn, Delorge, Figueroa, Van Dijck, & Stitt, 2014;Martins et al., 2013). The following experiments provide further evidence that Tre6P plays a key role in the feedback regulation of starch mobilization. In particular, we ask whether feedback inhibition by Tre6P is minimized to allow full use of starch in conditions where C is in short supply, but operates effectively when C is in excess.When Arabidopsis plants grow in conditions where less C is available per 24 hr cycle, they accumulate a larger proportion of their fixed C to starch in the daytime and slow down mobilization of starch during the night, compared to plants growing with a larg...