Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood. We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP-l-fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-l-Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l-fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant-water relations.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

Conserved cellular patterning in the mesophyll of rice leaves.

The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed.

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.

Elevated CO2 Priming as a Sustainable Approach to Increasing Rice Tiller Number and Yield Potential.

Tillering and yield are linked in rice, with significant efforts being invested to understand the genetic basis of this phenomenon. However, in addition to genetic factors, tillering is also influenced by the environment. Exploiting experiments in which seedlings were first grown in elevated CO2 (eCO2) before transfer and further growth under ambient CO2 (aCO2) levels, we found that even moderate exposure times to eCO2 were sufficient to induce tillering in seedlings, which was maintained in plants grown to maturity plants in controlled environment chambers. We then explored whether brief exposure to eCO2 (eCO2 priming) could be implemented to regulate tiller number and yield in the field. We designed a cost-effective growth system, using yeast to increase the CO2 level for the first 24 days of growth, and grew these seedlings to maturity in semi-field conditions in Malaysia. The increased growth caused by eCO2 priming translated into larger mature plants with increased tillering, panicle number, and improved grain filling and 1000 grain weight. In order to make the process more appealing to conventional rice farmers, we then developed a system in which fungal mycelium was used to generate the eCO2 via respiration of sugars derived by growing the fungus on lignocellulosic waste. Not only does this provide a sustainable source of CO2, it also has the added financial benefit to farmers of generating economically valuable oyster mushrooms as an end-product of mycelium growth. Our experiments show that the system is capable of generating sufficient CO2 to induce increased tillering in rice seedlings, leading eventually to 18% more tillers and panicles in mature paddy-grown crop. We discuss the potential of eCO2 priming as a rapidly implementable, broadly applicable and sustainable system to increase tillering, and thus yield potential in rice.

Defining the scope for altering rice leaf anatomy to improve photosynthesis: a modelling approach.

Leaf structure plays an important role in photosynthesis. However, the causal relationship and the quantitative importance of any single structural parameter to the overall photosynthetic performance of a leaf remains open to debate. In this paper, we report on a mechanistic model, eLeaf, which successfully captures rice leaf photosynthetic performance under varying environmental conditions of light and CO2 . We developed a 3D reaction-diffusion model for leaf photosynthesis parameterised using a range of imaging data and biochemical measurements from plants grown under ambient and elevated CO2 and then interrogated the model to quantify the importance of these elements. The model successfully captured leaf-level photosynthetic performance in rice. Photosynthetic metabolism underpinned the majority of the increased carbon assimilation rate observed under elevated CO2 levels, with a range of structural elements making positive and negative contributions. Mesophyll porosity could be varied without any major outcome on photosynthetic performance, providing a theoretical underpinning for experimental data. eLeaf allows quantitative analysis of the influence of morphological and biochemical properties on leaf photosynthesis. The analysis highlights a degree of leaf structural plasticity with respect to photosynthesis of significance in the context of attempts to improve crop photosynthesis.

Memory efficient constrained optimization of scanning-beam lithography.

This article describes a memory efficient method for solving large-scale optimization problems that arise when planning scanning-beam lithography processes. These processes require the identification of an exposure pattern that minimizes the difference between a desired and predicted output image, subject to constraints. The number of free variables is equal to the number of pixels, which can be on the order of millions or billions in practical applications. The proposed method splits the problem domain into a number of smaller overlapping subdomains with constrained boundary conditions, which are then solved sequentially using a constrained gradient search method (L-BFGS-B). Computational time is reduced by exploiting natural sparsity in the problem and employing the fast Fourier transform for efficient gradient calculation. When it comes to the trade-off between memory usage and computational time we can make a different trade-off compared to previous methods, where the required memory is reduced by approximately the number of subdomains at the cost of more computations. In an example problem with 30 million variables, the proposed method reduces memory requirements by 67% but increases computation time by 27%. Variations of the proposed method are expected to find applications in the planning of processes such as scanning laser lithography, scanning electron beam lithography, and focused ion beam deposition, for example.

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.

Model-Based Nonlinear Feedback Controllers for Pressure Control of Soft Pneumatic Actuators Using On/Off Valves.

This article describes the application and comparison of three nonlinear feedback controllers for low-level control of soft actuators driven by a pressure source and single high-speed on/off solenoid valve. First, a mathematical model of the pneumatic system is established and the limitations of the open-loop system are evaluated. Next, a model of the pneumatic system is developed using Simscape Fluids to evaluate the performance of various control strategies. In this article, State-Dependent Riccati Equation control, sliding mode control, and feedback linearization are considered. To improve robustness to model uncertainties, the sliding mode and feedback linearization control strategies are augmented with integral action. The model of the pneumatic system is also used to develop a feedforward component, which is added to a PI controller with anti-windup. The simulation and experimental results demonstrate the effectiveness of the proposed controllers for pressure tracking.

Experimental analysis of tip vibrations at higher eigenmodes of QPlus sensors for atomic force microscopy.

QPlus sensors are non-contact atomic force microscope probes constructed from a quartz tuning fork and a tungsten wire with an electrochemically etched tip. These probes are self-sensing and offer an atomic-scale spatial resolution. Therefore, qPlus sensors are routinely used to visualize the chemical structure of adsorbed organic molecules via the so-called bond imaging technique. This is achieved by functionalizing the AFM tip with a single CO molecule and exciting the sensor at the first vertical cantilever resonance mode. Recent work using higher-order resonance modes has also resolved the chemical structure of single organic molecules. However, in these experiments, the image contrast can differ significantly from the conventional bond imaging contrast, which was suspected to be caused by unknown vibrations of the tip. This work investigates the source of these artefacts by using a combination of mechanical simulation and laser vibrometry to characterize a range of sensors with different tip wire geometries. The results show that increased tip mass and length cause increased torsional rotation of the tuning fork beam due to the off-center mounting of the tip wire, and increased flexural vibration of the tip. These undesirable motions cause lateral deflection of the probe tip as it approaches the sample, which is rationalized to be the cause of the different image contrast. The results also provide a guide for future probe development to reduce these issues.

Ploidy influences wheat mesophyll cell geometry, packing and leaf function.

Leaf function is influenced by leaf structure, which is itself related not only to the spatial arrangement of constituent mesophyll cells, but also their size and shape. In this study, we used confocal microscopy to image leaves of Triticum genotypes varying in ploidy level to extract 3D information on individual mesophyll cell size and geometry. Combined with X-ray Computed Tomography and gas exchange analysis, the effect of changes in wheat mesophyll cell geometry upon leaf structure and function were investigated. Mesophyll cell size and shape were found to have changed during the course of wheat evolution. An unexpected linear relationship between mesophyll cell surface area and volume was discovered, suggesting anisotropic scaling of mesophyll cell geometry with increasing ploidy. Altered mesophyll cell size and shape were demonstrated to be associated with changes in mesophyll tissue architecture. Under experimental growth conditions, CO2 assimilation did not vary with ploidy, but stomatal conductance was lower in hexaploid plants, conferring a greater instantaneous water-use efficiency. We propose that as wheat mesophyll cells have become larger with increased ploidy, this has been accompanied by changes in cell geometry and packing which limit water loss while maintaining carbon assimilation.

Estimating uncertainty: A Bayesian approach to modelling photosynthesis in C3 leaves.

The Farquhar-von Caemmerer-Berry (FvCB) model is extensively used to model photosynthesis from gas exchange measurements. Since its publication, many methods have been developed to measure, or more accurately estimate, parameters of this model. Here, we have created a tool that uses Bayesian statistics to fit photosynthetic parameters using concurrent gas exchange and chlorophyll fluorescence measurements whilst evaluating the reliability of the parameter estimation. We have tested this tool on synthetic data and experimental data from rice leaves. Our results indicate that reliable parameter estimation can be achieved whilst only keeping one parameter, Km , that is, Michaelis constant for CO2 by Rubisco, prefixed. Additionally, we show that including detailed low CO2 measurements at low light levels increases reliability and suggests this as a new standard measurement protocol. By providing an estimated distribution of parameter values, the tool can be used to evaluate the quality of data from gas exchange and chlorophyll fluorescence measurement protocols. Compared to earlier model fitting methods, the use of a Bayesian statistics-based tool minimizes human interaction during fitting, reducing the subjectivity which is essential to most existing tools. A user friendly, interactive Bayesian tool script is provided.

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.

A review of demodulation techniques for multifrequency atomic force microscopy.

This article compares the performance of traditional and recently proposed demodulators for multifrequency atomic force microscopy. The compared methods include the lock-in amplifier, coherent demodulator, Kalman filter, Lyapunov filter, and direct-design demodulator. Each method is implemented on a field-programmable gate array (FPGA) with a sampling rate of 1.5 MHz. The metrics for comparison include the sensitivity to other frequency components and the magnitude of demodulation artifacts for a range of demodulator bandwidths. Performance differences are demonstrated through higher harmonic atomic force microscopy imaging.

Cellular perspectives for improving mesophyll conductance.

After entering the leaf, CO2 faces an intricate pathway to the site of photosynthetic fixation embedded within the chloroplasts. The efficiency of CO2 flux is hindered by a number of structural and biochemical barriers which, together, define the ease of flow of the gas within the leaf, termed mesophyll conductance. Previous authors have identified the key elements of this pathway, raising the prospect of engineering the system to improve CO2 flux and, thus, to increase leaf photosynthetic efficiency. In this review, we provide a perspective on the potential for improving the individual elements that contribute to this complex parameter. We lay particular emphasis on generation of the cellular architecture of the leaf which sets the initial boundaries of a number of mesophyll conductance parameters, incorporating an overview of the molecular transport processes which have been proposed as major facilitators of CO2 flux across structural boundaries along the pathway. The review highlights the research areas where future effort might be invested to increase our fundamental understanding of mesophyll conductance and leaf function and, consequently, to enable translation of these findings to improve the efficiency of crop photosynthesis.

The developmental relationship between stomata and mesophyll airspace.

The quantitative and spatial coordination of stomatal pores in the epidermis and airspaces in the underlying mesophyll tissue is vital for efficient gas exchange in the leaf. The mechanisms that determine the distribution of stomata in the epidermis have been studied extensively, but how this relates to the regulation of mesophyll airspace configuration is poorly understood. Recent studies have investigated how development is coordinated between these tissue layers. The evidence suggests that multiple mechanisms are likely to work concurrently to coordinate stomatal and mesophyll development for optimal leaf gas exchange, and that both genetic and physiological factors contribute to this regulation. Such advances in our understanding of leaf development have important implications for potential improvement of crop water use efficiency.

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.

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.

Multimodal atomic force microscopy with optimized higher eigenmode sensitivity using on-chip piezoelectric actuation and sensing.

Atomic force microscope (AFM) cantilevers with integrated actuation and sensing provide several distinct advantages over conventional cantilever instrumentation. These include clean frequency responses, the possibility of down-scaling and parallelization to cantilever arrays as well as the absence of optical interference. While cantilever microfabrication technology has continuously advanced over the years, the overall design has remained largely unchanged; a passive rectangular shaped cantilever design has been adopted as the industry wide standard. In this article, we demonstrate multimode AFM imaging on higher eigenmodes as well as bimodal AFM imaging with cantilevers using fully integrated piezoelectric actuation and sensing. The cantilever design maximizes the higher eigenmode deflection sensitivity by optimizing the transducer layout according to the strain mode shape. Without the need for feedthrough cancellation, the read-out method achieves close to zero actuator/sensor feedthrough and the sensitivity is sufficient to resolve the cantilever Brownian motion.

Investigating the microstructure of plant leaves in 3D with lab-based X-ray computed tomography.

BACKGROUND: Leaf 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. RESULTS: We 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. CONCLUSIONS: Our 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.