Starch Metabolism in Guard Cells and its Impact on Stomatal Function

Abstract

The appearance of stomata over 400 million years ago represents a major evolutionary invention, which enabled plants to conquer land. Stomata are turgordriven valves enclosed by a pair of highly specialized guard cells allowing the exchange of carbon dioxide, oxygen and water between the leaf interior and the environment. The reversible changes in guard cell turgor result from the uptake and release of a variety of osmotically active solutes. Much of the guard cell research conducted over the past 80 years focused on transmembrane ion transport. Although its importance for stomatal movement regulation is inarguably, adjustment of the stomatal pore size requires a complex network of interactions between ion transport, metabolism and solute partitioning. Carbon metabolism has been implicated with stomatal aperture control ever since von Mohl (1856) and Lloyd (1908) observed the presence of starch granules in guard cells. However, the lack of suitable experimental techniques imposed by the microscopic size of guard cells has long hindered the investigation of metabolic rearrangements during stomatal movements. We only recently resolved the diurnal temporal dynamics of starch turnover in guard cells. Substantial amounts of stomatal starch are present throughout the majority of day and night. After dawn, this starch gets transiently fully hydrolyzed by the concerted action of the glucan hydrolases a-amylase 3 (AMY3) and b-amylase 1 (BAM1) accelerating stomatal opening. This process is directly triggered by the bluelight- dependent activation of the plasma membrane H+-ATPase, which activity links membrane ion transport to guard cell carbon metabolism. However, one big unresolved question was how guard cell starch degradation integrates with light-induced ion transport processes in the control of stomatal opening kinetics. In Chapter I of this thesis, we demonstrate that guard cell starch degradation does not directly affect the capacity for the transport of H+, K+ and Cl- ions across the plasma membrane, suggesting that starch degradation does not directly affect the ability for ion transport. Moreover, we examined a long-lasting hypothesis that malate accumulates upon guard cell starch mobilization. Using newly developed enzymatic quantification assays, we revealed that the major end product of blue light-induced starch breakdown in the guard cells of Arabidopsis thaliana is glucose and not malate. The rapid generation of glucose from starch is thus essential for fast stomatal opening and contributes to the coordination between photosynthesis and transpiration. Although guard cell starch plays this essential role during stomatal opening, starch synthesis in guard cells is poorly understood. In Chapter I, we further demonstrate that the capacity of guard cells for autonomous CO2 fixation is limited and starch synthesis largely depends on the supply of mesophyll-derived substrates. Along this line of evidence, the two proton-coupled hexose carriers Sugar Transport Proteins 1 and 4 (STPs) were identified as the major plasma membranelocalized monosaccharide transporters in guard cells in Chapter II. Using a large set of physiological and biochemical techniques, along with phenotyping technology, allowed us to show that their combined activity is required for glucose uptake to guard cells at dawn, delivering carbon substrates for starch synthesis and light-induced stomatal opening. In Chapter III, in a review under revision for the “New Phytologist”, we provide a critical summary of the latest research about guard cell carbon metabolism and identify remaining knowledge gaps. Chapter IV reveals that in addition to the STPs, three members (1, 4 and 5) of the hexose facilitators from the Sugars Will Eventually be Exported (SWEET) family are highly expressed in guard cells and supply them with sugar precursors for starch synthesis. We further provide evidence that guard cell starch acts as a sink for osmolytes during high CO2-induced stomatal closure. We identified the three responsible vacuolar exporters involved in this process: the Aluminum-activated Malate Transporter 4 (ALMT4), the Early Response to Dehydration Like 6 (ERDL6) and the Sucrose-H+ symporter SUC4. Chapter V deals with the enzymatic pathway(s) of guard cell starch synthesis as guard cells show traits of both auto- and heterotrophic tissues. We demonstrate that the enzymes of the classical leaf pathway of starch synthesis play also a significant role in guard cells. Concomitant with hexose uptake to guard cells for starch synthesis, we identified the Glucose-6-phosphate/Phosphate Translocator 1 (GPT1), which catalyzes glucose uptake into chloroplasts, as an essential player of starch accumulation in guard cells. Given the largely heterotrophic nature of guard cells, in Chapter VI, we conducted research in collaboration with the lab of Dr. Boon-Leong Lim (University of Hong Kong) to uncover how guard cell chloroplasts obtain energy for metabolic processes. We used genetically encoded ATP and NADPH biosensors and demonstrated that guard cell chloroplasts surprisingly import cytosolic ATP via the Nucleotide Transporter 1 (NTT1), which is used among other processes for the formation of starch.