On the roles of organic acids transport and sucrose metabolism controlling stomatal movements

Stomata control the concomitant exchange of CO2 and transpiration in land plants. While a constant supply of CO2 is need to maintain the rate of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. The factors affecting stomatal movement are directly coupled with the cellular networks of guard cells. Although the guard cell has been used as a model for characterization of signaling pathways, several important questions about its functioning remain elusive. Current modeling approaches describe the stomatal conductance in terms of relatively few easy-tomeasure variables being unsuitable for in silico design of genetic manipulation strategies. Here, we argue that a system biology approach, combining modeling and high-throughput experiments, may be used to elucidate the mechanisms underlying stomata control and to determine targets for modulation of stomatal responses to environment. In support of our opinion, we review studies demonstrating how highthroughput approaches have provided a systems-view of guard cells. Finally, we emphasize the opportunities and challenges of genome-scale modeling and large-scale data integration for in silico manipulation of guard cell functions to improve crop yields, particularly under stress conditions which are of pertinence both to climate change and water use efficiency. INTRODUCTION Stomata are microscopic pores formed by pairs of specialized epidermal guard cells, which control the essential exchange of CO2 with the environment concomitantly with transpiration in land plants. Although plants are in constant need of sufficient quantities of CO2 to maintain their photosynthetic rates, the water losses associated with the CO2 entry must also be strictly regulated to prevent dehydration and the subsequent unfavourable changes of metabolic state that this invokes. Furthermore, the influx of CO2 and the absorption of minerals depend highly on the availability of water, which is usually a major limiting factor for the development and growth of terrestrial plants. For these reasons, most terrestrial plants are coated with a water-impermeable cuticle layer that prevents evaporation and water loss (Bessire et al. 2007; Schreiber 2010; Buschhaus & Jetter 2011). Accordingly, the majority of water lost to transpiration, and CO2 absorption for that matter, occurs through stomatal pores rendering the stomata as the main control point, which regulates the flow of gases between plants and atmosphere (Schroeder et al. 2001). Changes in guard cell metabolism can regulate the magnitude of the pore size in response to a range of environmental and endogenous plant stimulus (Kim et al. 2010). This regulation allows plants to occupy habitats with fluctuating environmental conditions. Therefore, it has been postulated that stomata must be important contributors to speciation and evolutionary adaptation (Hetherington & Woodward 2003). Moreover, stomatal behaviour and stomatal density are of fundamental importance not only for individual plant performance, but also for crop productivity and the terrestrial CO2 and water cycles (Buckley & Mott 2013; Schroeder et al. 2013). Stomatal pores open in response to an increase in guard cell volume driven by influx of water, which in turn is the result of a decrease in the water potential of the guard cell. Conversely, during stomatal closure, an efflux of osmolytes from guard cells is necessary with an associated increase in guard cell water potential, leading to efflux of water (Schroeder et al. 2001; Kim et al. 2010; Araújo et al. 2011a,c). Therefore, water potential is one of the key determinants of stomatal movements that is opening and closing. In addition to the control of water loss, stomatal movements directly affect CO2 assimilation in response to environmental cues by integrating a variety of stimuli, such as: light, vapour pressure deficit (VPD), abscisic acid (ABA) and CO2 concentration (Blatt 2000; Hetherington & Woodward 2003; Shimazaki et al. 2007; Kim et al. 2010; Hubbard et al. 2012). Although guard cells have often been used as a model system in which to discover ABA receptors (Liu et al. 2007; Pandey et al. 2009; Park et al. 2009) and for the in vitro characterization of the ABA signalling pathway (Fujii et al. 2009), several important questions about guard cell functioning, particularly under suboptimal conditions, remain elusive. For over a century, guard cells have been extensively studied, and considerable efforts have been made to better Correspondence: W. L. Araújo. Fax: +55 31 3899.2580; e-mail: wlaraujo@ufv.br Plant, Cell and Environment (2015) 38, 1457–1470 doi: 10.1111/pce.12517 bs_bs_banner © 2015 John Wiley & Sons Ltd 1457 understand their structure, development and physiology (Bessey 1898; Bergmann & Sack 2007; Mott 2009; Berry et al. 2010; Casson & Hetherington 2010; Kim et al. 2010; Dow & Bergmann 2014). In addition, the relative ease of the isolation of the guard cells, which gate the stomatal pore, has facilitated experimental interrogation contributing to the increase in the understanding of these cells relative to other plant cell types (Hetherington & Brownlee 2004; Israelsson et al. 2006; Kim et al. 2010). These advances have rendered the guard cell as one of the best studied plant cell models for membrane transport, signalling and homeostasis (Blatt 2000; Roelfsema & Hedrich 2010; Hills et al. 2012). Thorough understanding of stomatal behaviour and its regulation represents an important step towards obtaining plants with better water-use efficiency, which can be defined as the carbon fixed per unit of water (Lawson & Blatt 2014; Lawson et al. 2014; Way et al. 2014). Here, our first aim was to focus on high-throughput studies that have recently made considerable progress in elucidating the molecular and genetic bases underlying stomatal function and movement. However, the complexity of interactions involved in regulatory processes ranging from metabolite transport to the cellautonomous metabolic and buffering activities of guard cells remains the main barrier to understand the regulatory networks governing stomatal movement (Blatt et al. 2013). In this context, we suggest that a systems biology approach offers itself as a comprehensive way to aid in elucidation of the mechanisms governing stomatal function and the molecular hierarchies underpinning the stomata control. Furthermore and given that systems biology approaches necessitate data-driven modelling to integrate datasets capturing various read-outs of the cell as well as to generate and validate (or refute) hypotheses, they can also ultimately be used to predict biotechnological targets for the manipulation of stomatal behaviour. To this end, our second aim was to outline the steps that are prerequisites for coupling reverse engineering of stomatal responses with in silico design and modelling in order to direct genetic manipulation for improved crop yield and water-use efficiency (Fig. 1). We additionally suggest an alternative approach to those commonly used to illuminate stomatal behaviour and in doing so hope to bridge the gap between ecophysiological and molecular views of this important cell-to-environment interaction. Why apply the systems biology approach to study stomatal function? Over the last 20 years we have witnessed an increasing interest in the understanding of the molecular mechanisms by which guard cells sense and respond to stimuli such as ABA, plant water status, light, CO2, temperature and humidity.As a consequence, guard cells have become an important model for studies of membrane transport and cell signalling (Assmann 2010). Advances in our understanding over this time period are mostly due to the identification and characterization of mutants deficient in stomatal closing and opening coupled with the currently challenging, but all the same feasible, isolation and screening of large populations of guard cells, employed in various high-throughput technologies (Table 1). Several seminal studies and numerous reviews (Tallman 2004; Shimazaki et al. 2007; Kim et al. 2010; Meyer et al. 2010; Araújo et al. 2011a; Song et al. 2014), point out that stomatal Known function of genes regulating stomatal control v Transcriptomics