Stomatal clustering in Begonia associates with the kinetics of leaf gaseous exchange and influences water use efficiency

Stomata are microscopic pores formed by specialized cells in the leaf epidermis and permit gaseous exchange between the interior of the leaf and the atmosphere. Stomata in most plants are separated by at least one epidermal pavement cell and, individually, overlay a single substomatal cavity within the leaf. This spacing is thought to enhance stomatal function. Yet, there are several genera naturally exhibiting stomata in clusters and therefore deviating from the one-cell spacing rule with multiple stomata overlaying a single substomatal cavity. We made use of two Begonia species to investigate whether clustering of stomata alters guard cell dynamics and gas exchange under different light and dark treatments. Begonia plebeja, which forms stomatal clusters, exhibited enhanced kinetics of stomatal conductance and CO2 assimilation upon light stimuli that in turn were translated into greater water use efficiency. Our findings emphasize the importance of spacing in stomatal clusters for gaseous exchange and plant performance under environmentally limited conditions

Speedy grass stomata: emerging molecular and evolutionary features

No abstract available

Hydrogen sulphide regulates inward-rectifying K+ channels in conjunction with stomatal closure

Hydrogen sulphide (H2S) is the third biological gasotransmitter and, in animals, affects many physiological processes by modulating ion channels. H2S has been reported to protect plants from oxidative stress in diverse physiological responses. H2S closes stomata, but the underlying mechanism remains elusive. Here we report the selective inactivation of current carried by inward-rectifying K+ channels (IKIN) of tobacco guard cells and demonstrate its close parallel with stomatal closure evoked by submicromolar concentrations of H2S. Experiments to scavenge H2S suggested an effect that is separable from that of abscisic acid, which is associated with water stress. Thus, H2S appears to associate with a new and as yet unresolved signalling pathway that selectively targets IKIN

Stomatal clustering and hydrogen sulfide: unraveling novel aspects of stomatal behavior

No abstract available

Physiological and cell biological characterisation of two novel membrane proteins in Arabidopsis thaliana

This thesis describes the characterisation of AtPQL4 and AtPQL6, two members of a hitherto uncharacterized gene family of A. thaliana with six members, AtPQL1-6. As their counterparts in other species and kingdoms AtPQL proteins contain seven transmembrane domains and two copies of the so-called ‘PQ-loop’ domain. AtPQL4 and AtPQL6 show high amino acid sequence identity between each other and they are the closest A. thaliana homologues of the mammalian LEC35/MPDU1 protein, which has been shown to be required for all types of Man-P-Dol dependent glycosylation in the ER. To address the functional homology between the two AtPQL proteins and MPDU1, the sub-cellular localisation of AtPQL4-GFP and AtPQL6-GFP fusion proteins was investigated. Confocal laser scanning microscopy analysis of Nicotiana tabacum leaf cells expressing AtPQL4-GFP or AtPQL6-GFP showed fluorescence patterns typical for ER. ER localisation of AtPQL4 and AtPQL6 was further confirmed by co-expression with the ER marker, YFP-HDEL. A second set of experiments employed YFP-fusion proteins of AtSYP121, a plasma membrane SNARE protein, and AtTIP2, a tonoplast aquaporin. Confocal microscopy confirmed plasma membrane/tonoplast localisation of the YFP proteins when expressed on their own in tobacco leaf cells. However, both proteins were found to be retained in the ER when co-expressed with AtPQL4-GFP or AtPQL6-GFP fusion proteins. These new findings point to a role of AtPQL4 and AtPQL6 in protein processing in the ER thereby enforcing previous results from microarray experiments indicating ER-stress in AtPQL4 and AtPQL6 mutants. Finally, a number of AtPQL4 and AtPQL6 knockout and AtPQL6 overexpressor lines were tested under a variety of environmental stresses to investigate the function of the two AtPQLs at whole-plant level. Low sucrose conditions resulted in growth inhibition of mutants compared to wild type plants. Considering previous findings that (a) AtPQL4 and AtPQL6 are localised in the ER (b) ATPQL mutants show differential expression of genes involved in the unfolded protein response (UPR) and (c) over-expression of AtPQL4 and AtPQL6 impacts on the targeting of other proteins, the observed phenotype could be linked to the unfolded protein response and autophagy that occurs during sugar starvation.. In conclusion, it is proposed that AtPQL4 and AtPQL6 proteins function in retaining membrane proteins for sufficient time in the ER to allow ER-quality control and related processes to take place. Further experiments to investigate such function are discussed

Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation

Stomatal spacing safeguards stomatal dynamics by facilitating guard cell ion transport independent of the epidermal solute reservoir

Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO(2) assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K(+) channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K(+) ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K(+) and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K(+) accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells

Systems Dynamic Modeling of a Guard Cell Cl −

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K+ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H+ load on the cytosol and promoted Ca2+ influx to elevate cytosolic pH (pHi) and free cytosolic Ca2+ concentration ([Ca2+]i), in turn regulating the K+ channels. We have confirmed these predictions, measuring pHi and [Ca2+]i in vivo, and report that experimental manipulation of pHi and [Ca2+]i is sufficient to recover K+ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K+ channels. Additionally, these findings underscore the importance of H+-coupled anion transport for pHi homeostasis

A guide to photosynthetic gas exchange measurements:Fundamental principles, best practice and potential pitfalls

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step‐by‐step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data‐modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas‐exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance inArabidopsis thalianaand its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+channel activities and changes in stomatal conductance of theslac1Cl-channel andost2H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grants BB/L019205/1 and BB/M001601/1 to M.R.B., BB/L001276/1 to M.R.B. and S.R., and BB/I001187/1 to H.G. and T.L