Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)-enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H(+)-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases a-AMYLASE3 (AMY3) and b-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropinmediated blue light signaling downstream of the plasma membrane H 1-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H 1 , K 1 , and Cl 2 transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starchderived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis.

Guard cell membrane ion transport and metabolism are deeply interconnected, and their coordinated regulation is integral to stomatal opening. Whereas ion transport is exceptionally well understood, how guard cell metabolism influences stomatal movements is less well known. Organic metabolites, such as malate and sugars, fulfill several functions in guard cells during stomatal opening as allosteric activators, counter-ions, energy source and osmolytes. However, their origin and exact fate remain debated. Recent work revealed that the guard cell carbon pool regulating stomatal function and plant growth is mostly of mesophyll origin, highlighting a tight correlation between mesophyll and guard cell metabolism. This review discusses latest research into guard cell carbon metabolism and its impact on stomatal function and whole plant physiology.

Summary Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that offers the potential to engineer improved water‐use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in the hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and closing across the diel cycle, which conserves water and provides a means of maintaining growth in hot, water‐limited environments. Recent genome sequencing of the constitutive model CAM species Kalanchoë fedtschenkoi provides a platform for elucidating the ensemble of proteins that link photosynthetic metabolism with stomatal movement, and that protect CAM plants from harsh environmental conditions. We describe a large‐scale proteomics analysis to characterize and compare proteins, as well as diel changes in their abundance in guard cell‐enriched epidermis and mesophyll cells from leaves of K. fedtschenkoi. Proteins implicated in processes that encompass respiration, the transport of water and CO2, stomatal regulation, and CAM biochemistry are highlighted and discussed. Diel rescheduling of guard cell starch turnover in K. fedtschenkoi compared with that observed in Arabidopsis is reported and tissue‐specific localization in the epidermis and mesophyll of isozymes implicated in starch and malate turnover are discussed in line with the contrasting roles for these metabolites within the CAM mesophyll and stomatal complex. These data reveal the proteins and the biological processes enriched in each layer and provide key information for studies aiming to adapt plants to hot and dry environments by modifying leaf physiology for improved plant sustainability.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the Phosphoglucose isomerase (PGI)-Phosphoglucose mutase (PGM)-ADP-Glucose pyrophosphorylase (AGPase) starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as Glucose-6-phosphate/Phosphate Translocators (GPTs), which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behaviour, directly affecting plant productivity.

In this protocol, we describe how to quantify starch in guard cells of using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.