Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields. We engineered the high-yielding rice cultivar 'IR64' to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance. Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO , rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions. Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

The development and patterning of stomata in the plant epidermis has emerged as an ideal system for studying fundamental plant developmental processes. Over the past twenty years most studies of stomata have used the model dicotyledonous plant Arabidopsis thaliana. However, cultivated monocotyledonous grass (or Gramineae) varieties provide the majority of human nutrition, and future research into grass stomata could be of critical importance for improving food security. Recent studies using Brachypodium distachyon, Hordeum vulgare (barley) and Oryza sativa (rice) have led to the identification of the core transcriptional regulators essential for stomatal initiation and progression in grasses, and begun to unravel the role of secretory signaling peptides in controlling stomatal developmental. This review revisits how stomatal developmental unfolds in grasses, and identifies key ontogenetic steps for which knowledge of the underpinning molecular mechanisms remains outstanding.

SummaryThe pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.

BackgroundLeaf cellular architecture plays an important role in setting limits for carbon assimilation and, thus, photosynthetic performance. However, the low density, fine structure, and sensitivity to desiccation of plant tissue has presented challenges to its quantification. Classical methods of tissue fixation and embedding prior to 2D microscopy of sections is both laborious and susceptible to artefacts that can skew the values obtained. Here we report an image analysis pipeline that provides quantitative descriptors of plant leaf intercellular airspace using lab-based X-ray computed tomography (microCT). We demonstrate successful visualisation and quantification of differences in leaf intercellular airspace in 3D for a range of species (including both dicots and monocots) and provide a comparison with a standard 2D analysis of leaf sections.ResultsWe used the microCT image pipeline to obtain estimates of leaf porosity and mesophyll exposed surface area (Smes) for three dicot species (Arabidopsis, tomato and pea) and three monocot grasses (barley, oat and rice). The imaging pipeline consisted of (1) a masking operation to remove the background airspace surrounding the leaf, (2) segmentation by an automated threshold in ImageJ and then (3) quantification of the extracted pores using the ImageJ ‘Analyze Particles’ tool. Arabidopsis had the highest porosity and lowest Smes for the dicot species whereas barley had the highest porosity and the highest Smes for the grass species. Comparison of porosity and Smes estimates from 3D microCT analysis and 2D analysis of sections indicates that both methods provide a comparable estimate of porosity but the 2D method may underestimate Smes by almost 50%. A deeper study of porosity revealed similarities and differences in the asymmetric distribution of airspace between the species analysed.ConclusionsOur results demonstrate the utility of high resolution imaging of leaf intercellular airspace networks by lab-based microCT and provide quantitative data on descriptors of leaf cellular architecture. They indicate there is a range of porosity and Smes values in different species and that there is not a simple relationship between these parameters, suggesting the importance of cell size, shape and packing in the determination of cellular parameters proposed to influence leaf photosynthetic performance.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0367-7) contains supplementary material, which is available to authorized users.

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.

Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure.