Grasses exploit geometry to achieve improved guard cell dynamics.

Stomata are controllable micropores formed between two adjacent guard cells (GCs) that regulate gas flow across the plant surface.1 Grasses, among the most successful organisms on the planet and the main food crops for humanity, have GCs flanked by specialized lateral subsidiary cells (SCs).2,3,4 SCs improve performance by acting as a local pool of ions and metabolites to drive changes in turgor pressure within the GCs that open/close the stomatal pore.4,5,6,7,8 The 4-celled complex also involves distinctive changes in geometry, having dumbbell-shaped GCs compared with typical kidney-shaped stomata.2,4,9 However, the degree to which this distinctive geometry contributes to improved stomatal performance, and the underlying mechanism, remains unclear. To address this question, we created a finite element method (FEM) model of a grass stomatal complex that successfully captures experimentally observed pore opening/closure. Exploration of the model, including in silico and experimental mutant analyses, supports the importance of a reciprocal pressure system between GCs and SCs for effective stomatal function, with SCs functioning as springs to restrain lateral GC movement. Our results show that SCs are not essential but lead to a more responsive system. In addition, we show that GC wall anisotropy is not required for grass stomatal function (in contrast to kidney-shaped GCs10) but that a relatively thick GC rod region is needed to enhance pore opening. Our results demonstrate that a specific cellular geometry and associated mechanical properties are required for the effective functioning of grass stomata.

SIAMESE-RELATED1 imposes differentiation of stomatal lineage ground cells into pavement cells.

The leaf epidermis represents a multifunctional tissue consisting of trichomes, pavement cells and stomata, the specialized cellular pores of the leaf. Pavement cells and stomata both originate from regulated divisions of stomatal lineage ground cells (SLGCs), but whereas the ontogeny of the stomata is well characterized, the genetic pathways activating pavement cell differentiation remain relatively unexplored. Here, we reveal that the cell cycle inhibitor SIAMESE-RELATED1 (SMR1) is essential for timely differentiation of SLGCs into pavement cells by terminating SLGC self-renewal potency, which depends on CYCLIN A proteins and CYCLIN-DEPENDENT KINASE B1. By controlling SLGC-to-pavement cell differentiation, SMR1 determines the ratio of pavement cells to stomata and adjusts epidermal development to suit environmental conditions. We therefore propose SMR1 as an attractive target for engineering climate-resilient plants.

Candidate genes affecting stomatal density in rice (Oryza sativa L.) identified by genome-wide association.

Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.

The influences of stomatal size and density on rice abiotic stress resilience.

A warming climate coupled with reductions in water availability and rising salinity are increasingly affecting rice (Oryza sativa) yields. Elevated temperatures combined with vapour pressure deficit (VPD) rises are causing stomatal closure, further reducing plant productivity and cooling. It is unclear what stomatal size (SS) and stomatal density (SD) will best suit all these environmental extremes. To understand how stomatal differences contribute to rice abiotic stress resilience, we screened the stomatal characteristics of 72 traditionally bred varieties. We found significant variation in SS, SD and calculated anatomical maximal stomatal conductance (gsmax ) but did not identify any varieties with SD and gsmax as low as transgenic OsEPF1oe plants. Traditionally bred varieties with high SD and small SS (resulting in higher gsmax ) typically had lower biomasses, and these plants were more resilient to drought than low SD and large SS plants, which were physically larger. None of the varieties assessed were as resilient to drought or salinity as low SD OsEPF1oe transgenic plants. High SD and small SS rice displayed faster stomatal closure during increasing temperature and VPD, but photosynthesis and plant cooling were reduced. Compromises will be required when choosing rice SS and SD to tackle multiple future environmental stresses.

Stomatal Development and Gene Expression in Rice Florets.

Stomata play a fundamental role in modulating the exchange of gases between plants and the atmosphere. These microscopic structures form in high numbers on the leaf epidermis and are also present on flowers. Although leaf stomata are well studied, little attention has been paid to the development or function of floral stomata. Here, we characterize in detail the spatial distribution and development of the floral stomata of the indica rice variety IR64. We show that stomatal complexes are present at low density on specific areas of the lemma, palea and anthers and are morphologically different compared to stomata found on leaves. We reveal that in the bract-like organs, stomatal development follows the same cell lineage transitions as in rice leaves and demonstrate that the overexpression of the stomatal development regulators OsEPFL9-1 and OsEPF1 leads to dramatic changes in stomatal density in rice floral organs, producing lemma with approximately twice as many stomata (OsEPFL9-1_oe) or lemma where stomata are practically absent (OsEPF1_oe). Transcriptomic analysis of developing florets also indicates that the cellular transitions during the development of floral stomata are regulated by the same genetic network used in rice leaves. Finally, although we were unable to detect an impact on plant reproduction linked to changes in the density of floral stomata, we report alterations in global gene expression in lines overexpressing OsEPF1 and discuss how our results reflect on the possible role(s) of floral stomata.

Induced Genetic Variations in Stomatal Density and Size of Rice Strongly Affects Water Use Efficiency and Responses to Drought Stresses.

Rice (Oryza sativa L.) is an important food crop relied upon by billions of people worldwide. However, with increasing pressure from climate change and rapid population growth, cultivation is very water-intensive. Therefore, it is critical to produce rice that is high-yielding and genetically more water-use efficient. Here, using the stabilized fast-neutron mutagenized population of Jao Hom Nin (JHN) - a popular purple rice cultivar - we microscopically examined hundreds of flag leaves to identify four stomatal model mutants with either high density (HD) or low density (LD) stomata, and small-sized (SS) or large-sized (LS) stomata. With similar genetic background and uniformity, the stomatal model mutants were used to understand the role of stomatal variants on physiological responses to abiotic stress. Our results show that SS and HD respond better to increasing CO2 concentration and HD has higher stomatal conductance (gs) compared to the other stomatal model mutants, although the effects on gas exchange or overall plant performance were small under greenhouse conditions. In addition, the results of our drought experiments suggest that LD and SS can better adapt to restricted water conditions, and LD showed higher water use efficiency (WUE) and biomass/plant than other stomatal model mutants under long-term restricted water treatment. Finally, our study suggests that reducing stomata density and size may play a promising role for further work on developing a climate-ready rice variety to adapt to drought and heat stress. We propose that low stomata density and small size have high potential as genetic donors for improving WUE in climate-ready rice.

Small EPIDERMAL PATTERNING FACTOR-LIKE2 peptides regulate awn development in rice.

The EPIDERMAL PATTERNING FACTOR (EPF) and EPF-LIKE (EPFL) family of small secreted peptides act to regulate many aspects of plant growth and development; however, their functions are not widely characterized in rice (Oryza sativa). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology to individually knockout each of 11 EPF/EPFL genes in the rice cultivar Kasalath. Loss of function of most OsEPF/EPFL genes generated no obvious phenotype alteration, while disruption of OsEPFL2 in Kasalath caused a short or no awn phenotype and reduced grain size. OsEPFL2 is strongly expressed in the young panicle, consistent with a role in regulating awn and grain development. Haplotype analysis indicated that OsEPFL2 can be classified into six major haplotypes. Nucleotide diversity and genetic differentiation analyses suggested that OsEPFL2 was positively selected during the domestication of rice. Our work to systematically investigate the function of EPF/EPFL peptides demonstrates that different members of the same gene family have been independently selected for their ability to regulate a similar biological function and provides perspective on rice domestication.

The origin and evolution of stomata.

The acquisition of stomata is one of the key innovations that led to the colonisation of the terrestrial environment by the earliest land plants. However, our understanding of the origin, evolution and the ancestral function of stomata is incomplete. Phylogenomic analyses indicate that, firstly, stomata are ancient structures, present in the common ancestor of land plants, prior to the divergence of bryophytes and tracheophytes and, secondly, there has been reductive stomatal evolution, especially in the bryophytes (with complete loss in the liverworts). From a review of the evidence, we conclude that the capacity of stomata to open and close in response to signals such as ABA, CO2 and light (hydroactive movement) is an ancestral state, is present in all lineages and likely predates the divergence of the bryophytes and tracheophytes. We reject the hypothesis that hydroactive movement was acquired with the emergence of the gymnosperms. We also conclude that the role of stomata in the earliest land plants was to optimise carbon gain per unit water loss. There remain many other unanswered questions concerning the evolution and especially the origin of stomata. To address these questions, it will be necessary to: find more fossils representing the earliest land plants, revisit the existing early land plant fossil record in the light of novel phylogenomic hypotheses and carry out more functional studies that include both tracheophytes and bryophytes.

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening.

Stomata regulate plant water use and photosynthesis by controlling leaf gas exchange. They do this by reversibly opening the pore formed by two adjacent guard cells, with the limits of this movement ultimately set by the mechanical properties of the guard cell walls and surrounding epidermis.1,2 A body of evidence demonstrates that the methylation status and cellular patterning of pectin wall polymers play a core role in setting the guard cell mechanical properties, with disruption of the system leading to poorer stomatal performance.3-6 Here we present genetic and biochemical data showing that wall arabinans modulate guard cell flexibility and can be used to engineer stomata with improved performance. Specifically, we show that a short-chain linear arabinan epitope associated with the presence of rhamnogalacturonan I in the guard cell wall is required for full opening of the stomatal pore. Manipulations leading to the novel accumulation of longer-chain arabinan epitopes in guard cell walls led to an increase in the maximal pore aperture. Using computational modeling combined with atomic force microscopy, we show that this phenotype reflected a decrease in wall matrix stiffness and, consequently, increased flexing of the guard cells under turgor pressure, generating larger, rounder stomatal pores. Our results provide theoretical and experimental support for the conclusion that arabinan side chains of pectin modulate guard cell wall stiffness, setting the limits for cell flexing and, consequently, pore aperture, gas exchange, and photosynthetic assimilation.

Rice Stomatal Mega-Papillae Restrict Water Loss and Pathogen Entry.

Rice (Oryza sativa) is a water-intensive crop, and like other plants uses stomata to balance CO2 uptake with water-loss. To identify agronomic traits related to rice stomatal complexes, an anatomical screen of 64 Thai and 100 global rice cultivars was undertaken. Epidermal outgrowths called papillae were identified on the stomatal subsidiary cells of all cultivars. These were also detected on eight other species of the Oryza genus but not on the stomata of any other plant species we surveyed. Our rice screen identified two cultivars that had "mega-papillae" that were so large or abundant that their stomatal pores were partially occluded; Kalubala Vee had extra-large papillae, and Dharia had approximately twice the normal number of papillae. These were most accentuated on the flag leaves, but mega-papillae were also detectable on earlier forming leaves. Energy dispersive X-Ray spectrometry revealed that silicon is the major component of stomatal papillae. We studied the potential function(s) of mega-papillae by assessing gas exchange and pathogen infection rates. Under saturating light conditions, mega-papillae bearing cultivars had reduced stomatal conductance and their stomata were slower to close and re-open, but photosynthetic assimilation was not significantly affected. Assessment of an F3 hybrid population treated with Xanthomonas oryzae pv. oryzicola indicated that subsidiary cell mega-papillae may aid in preventing bacterial leaf streak infection. Our results highlight stomatal mega-papillae as a novel rice trait that influences gas exchange, stomatal dynamics, and defense against stomatal pathogens which we propose could benefit the performance of future rice crops.

Leaf temperature responses to ABA and dead bacteria in wheat and Arabidopsis.

Stomatal densities, aperture openness and their responsiveness to environmental change determine plant water loss and regulate entry of pathogens. Stomatal responsiveness is usually assessed on restricted areas of leaves or isolated epidermal peels floated in solution. Analyzing these responses in the whole plant context could give valuable additional information, for example on the role of mesophyll in stomatal responses. We analyzed stomatal responses to the phytohormone abscisic acid (ABA) and pathogenic elicitors in intact plants by dynamic measurement of leaf temperature. We tested whether ABA-induced stomatal closure in wheat requires external nitrate and whether bacterial elicitor-induced stomatal closure can be detected by dynamic thermal imaging in intact Arabidopsis. We found that wheat was hypersensitive to all applied treatments, as even mock-treated leaves showed a strong increase in leaf temperature. Nevertheless, ABA activated stomatal closure in wheat independent of exogenous nitrate. Pathogenic elicitors triggered a fast and transient increase in leaf temperature in intact Arabidopsis, indicating short-term stomatal closure. The data suggest that the dynamics of pathogen-induced stomatal closure is different in whole plants compared to epidermal peels, where elicitor-induced stomatal closure persists longer. We propose that dynamic thermal imaging could be applied to address the effect of pathogenic elicitors on stomatal behavior in whole plants to complement detached sample assays and gain a better understanding of stomatal immunity.

Stomatal responses to carbon dioxide and light require abscisic acid catabolism in Arabidopsis.

In plants, stomata control water loss and CO2 uptake. The aperture and density of stomatal pores, and hence the exchange of gases between the plant and the atmosphere, are controlled by internal factors such as the plant hormone abscisic acid (ABA) and external signals including light and CO2. In this study, we examine the importance of ABA catabolism in the stomatal responses to CO2 and light. By using the ABA 8'-hydroxylase-deficient Arabidopsis thaliana double mutant cyp707a1 cyp707a3, which is unable to break down and instead accumulates high levels of ABA, we reveal the importance of the control of ABA concentration in mediating stomatal responses to CO2 and light. Intriguingly, our experiments suggest that endogenously produced ABA is unable to close stomata in the absence of CO2. Furthermore, we show that when plants are grown in short day conditions ABA breakdown is required for the modulation of both elevated [CO2]-induced stomatal closure and elevated [CO2]-induced reductions in leaf stomatal density. ABA catabolism is also required for the stomatal density response to light intensity, and for the full range of light-induced stomatal opening, suggesting that ABA catabolism is critical for the integration of stomatal responses to a range of environmental stimuli.

Dynamic thermal imaging confirms local but not fast systemic ABA responses.

Abscisic acid (ABA) signals regulating stomatal aperture and water loss are usually studied in detached leaves or isolated epidermal peels and at infrequent timepoints. Measuring stomatal ABA responses in attached leaves across a time course enables the study of stomatal behaviour in the physiological context of the plant. Infrared thermal imaging is often used to characterize steady-state stomatal conductance via comparisons of leaf surface temperature but is rarely used to capture stomatal responses over time or across different leaf surfaces. We used dynamic thermal imaging as a robust, but sensitive, tool to observe stomatal ABA responses in a whole plant context. We detected stomatal responses to low levels of ABA in both monocots and dicots and identified differences between the responses of different leaves. Using whole plant thermal imaging, stomata did not always behave as described previously for detached samples: in Arabidopsis, we found no evidence for fast systemic ABA-induced stomatal closure, and in barley, we observed no requirement for exogenous nitrate during ABA-induced stomatal closure. Thus, we recommend dynamic thermal imaging as a useful approach to complement detached sample assays for the study of local and systemic stomatal responses and molecular mechanisms underlying stomatal responses to ABA in the whole plant context.

Stomata and Sporophytes of the Model Moss Physcomitrium patens.

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.

The influence of stomatal morphology and distribution on photosynthetic gas exchange.

The intricate and interconnecting reactions of C3 photosynthesis are often limited by one of two fundamental processes: the conversion of solar energy into chemical energy, or the diffusion of CO2 from the atmosphere through the stomata, and ultimately into the chloroplast. In this review, we explore how the contributions of stomatal morphology and distribution can affect photosynthesis, through changes in gaseous exchange. The factors driving this relationship are considered, and recent results from studies investigating the effects of stomatal shape, size, density and patterning on photosynthesis are discussed. We suggest that the interplay between stomatal gaseous exchange and photosynthesis is complex, and that a disconnect often exists between the rates of CO2 diffusion and photosynthetic carbon fixation. The mechanisms that allow for substantial reductions in maximum stomatal conductance without affecting photosynthesis are highly dependent on environmental factors, such as light intensity, and could be exploited to improve crop performance.

CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.

Mesophyll porosity is modulated by the presence of functional stomata.

The formation of stomata and leaf mesophyll airspace must be coordinated to establish an efficient and robust network that facilitates gas exchange for photosynthesis, however the mechanism by which this coordinated development occurs remains unclear. Here, we combine microCT and gas exchange analyses with measures of stomatal size and patterning in a range of wild, domesticated and transgenic lines of wheat and Arabidopsis to show that mesophyll airspace formation is linked to stomatal function in both monocots and eudicots. Our results support the hypothesis that gas flux via stomatal pores influences the degree and spatial patterning of mesophyll airspace formation, and indicate that this relationship has been selected for during the evolution of modern wheat. We propose that the coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops, providing a target for future improvement.

Reduced stomatal density in bread wheat leads to increased water-use efficiency.

Wheat is a staple crop, frequently cultivated in water-restricted environments. Improving crop water-use efficiency would be desirable if grain yield can be maintained. We investigated whether a decrease in wheat stomatal density via the manipulation of epidermal patterning factor (EPF) gene expression could improve water-use efficiency. Our results show that severe reductions in stomatal density in EPF-overexpressing wheat plants have a detrimental outcome on yields. However, wheat plants with a more moderate reduction in stomatal density (i.e. <50% reduction in stomatal density on leaves prior to tillering) had yields indistinguishable from controls, coupled with an increase in intrinsic water-use efficiency. Yields of these moderately reduced stomatal density plants were also comparable with those of control plants under conditions of drought and elevated CO2. Our data demonstrate that EPF-mediated control of wheat stomatal development follows that observed in other grasses, and we identify the potential of stomatal density as a tool for breeding wheat plants that are better able to withstand water-restricted environments without yield loss.