SummaryImprovement in crop water-use efficiency (WUE) is a critical priority for regions facing increased drought or diminished groundwater resources. Despite new tools for the manipulation of stomatal development, the engineering of plants with high WUE remains a challenge.We used Arabidopsis epidermal patterning factor (EPF) mutants exhibiting altered stomatal density to test whether WUE could be improved directly by manipulation of the genes controlling stomatal density. Specifically, we tested whether constitutive overexpression of EPF2 reduced stomatal density and maximum stomatal conductance (g w(max) ) sufficiently to increase WUE.We found that a reduction in g w(max) via reduced stomatal density in EPF2-overexpressing plants (EPF2OE) increased both instantaneous and long-term WUE without altering significantly the photosynthetic capacity. Conversely, plants lacking both EPF1 and EPF2 expression (epf1epf2) exhibited higher stomatal density, higher g w(max) and lower instantaneous WUE, as well as lower (but not significantly so) long-term WUE.Targeted genetic modification of stomatal conductance, such as in EPF2OE, is a viable approach for the engineering of higher WUE in crops, particularly in future high-carbon-dioxide (CO 2 ) atmospheres.

To investigate the impact of manipulating stomatal density, a collection of Arabidopsis epidermal patterning factor (EPF) mutants with an approximately 16-fold range of stomatal densities (approx. 20–325% of that of control plants) were grown at three atmospheric carbon dioxide (CO 2 ) concentrations (200, 450 and 1000 ppm), and 30 per cent or 70 per cent soil water content. A strong negative correlation between stomatal size ( S ) and stomatal density ( D ) was observed, suggesting that factors that control D also affect S . Under some but not all conditions, mutant plants exhibited abnormal stomatal density responses to CO 2 concentration, suggesting that the EPF signalling pathway may play a role in the environmental adjustment of D . In response to reduced water availability, maximal stomatal conductance was adjusted through reductions in S , rather than D . Plant size negatively correlated with D . For example, at 450 ppm CO 2 EPF2-overexpressing plants, with reduced D , had larger leaves and increased dry weight in comparison with controls. The growth of these plants was also less adversely affected by reduced water availability than plants with higher D , indicating that plants with low D may be well suited to growth under predicted future atmospheric CO 2 environments and/or water-scarce environments.

Summary Manipulation of stomatal density was investigated as a potential tool for enhancing drought tolerance or nutrient uptake.Drought tolerance and soil water retention were assessed using Arabidopsis epidermal patterning factor mutants manipulated to have increased or decreased stomatal density. Root nutrient uptake via mass flow was monitored under differing plant watering regimes using nitrogen‐15 (15N) isotope and mass spectrometry.Plants with less than half of their normal complement of stomata, and correspondingly reduced levels of transpiration, conserve soil moisture and are highly drought tolerant but show little or no reduction in shoot nitrogen concentrations especially when water availability is restricted. By contrast, plants with over twice the normal density of stomata have a greater capacity for nitrogen uptake, except when water availability is restricted.We demonstrate the possibility of producing plants with reduced transpiration which have increased drought tolerance, with little or no loss of nutrient uptake. We demonstrate that increasing transpiration can enhance nutrient uptake when water is plentiful.

BackgroundGlucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of their occurrence in the agriculturally important brassicaceae and their impact on human and animal health. There is also increasing interest in the biofumigant properties of toxic glucosinolate hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential requires rapid and accurate quantification of glucosinolates, but current commonly used methods of extraction prior to analysis involve a number of time consuming and hazardous steps; this study aimed to develop an improved method for glucosinolate extraction.ResultsThree methods previously used to extract glucosinolates from brassicaceae tissues, namely extraction in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared across tissue type (root, stem leaf) and four brassicaceae species (B. juncea, S. alba, R. sativus, and E. sativa). Cold methanol extraction was shown to perform as well or better than all other tested methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. It was also demonstrated that lyophilisation methods, routinely used during extraction to allow tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet tissue samples in cold 80% methanol is more effective.ConclusionsWe present a simplified method for extracting glucosinolates from plant tissues which does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time and cost effective. The presented method has been shown to have comparable or improved glucosinolate extraction efficiency relative to the commonly used ISO method for major glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0164-8) contains supplementary material, which is available to authorized users.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.