Stomata are pores in the plant epidermis that control carbon dioxide uptake and water loss. They are major regulators of global carbon and water cycles [1]. Several signaling components that regulate stomatal development have been characterized. These include a putative secretory peptide EPF1, LRR receptor components TMM and ER, and a peptidase SDD1 [2-4]. We have identified EPF2, a peptide related to EPF1 that is expressed in proliferating cells of the stomatal lineage, known as meristemoids, and in guard mother cells, the progenitors of stomata. EPF2 expression during leaf development affects stomatal density on the mature leaf. In the absence of EPF2, excessive numbers of cells enter the stomatal lineage and produce numerous small epidermal cells that express stomatal lineage reporter genes, whereas plants overexpressing EPF2 produce virtually no stomata. Results from genetic experiments indicate that EPF2 regulates a different aspect of stomatal development to EPF1 and are consistent with EPF2 acting in a pathway to regulate stomatal density that involves ER and TMM, but not SDD1. We propose that EPF2 is expressed earlier in leaf development than EPF1 and is involved in determining the number of cells that enter, and remain in, the stomatal lineage.

The epidermal patterning factor (EPF) family of secreted signaling peptides regulate the frequency of stomatal development in model dicot and basal land plant species. Here, we identify and manipulate the expression of a barley (Hordeum vulgare) ortholog and demonstrate that when overexpressed HvEPF1 limits entry to, and progression through, the stomatal development pathway. Despite substantial reductions in leaf gas exchange, barley plants with significantly reduced stomatal density show no reductions in grain yield. In addition, HvEPF1OE barley lines exhibit significantly enhanced water use efficiency, drought tolerance, and soil water conservation properties. Our results demonstrate the potential of manipulating stomatal frequency for the protection and optimization of cereal crop yields under future drier environments.

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.

Summary• The putative secretory peptides epidermal patterning factor 1 (EPF1) and EPF2 act as negative regulators of stomatal clustering and density early in Arabidopsis leaf development.• Here, we investigated whether the related peptide gene epidermal patterning factor-like 9 (EPFL9), which is coexpressed with EPF1 and stomatal density and distribution 1 (SDD1), also plays a role in controlling stomatal development.• Plants manipulated to constitutively overexpress EPFL9 showed increased stomatal density and clustering, and those manipulated to have reduced EPFL9 expression showed reduced stomatal density with no clustering, confirming that EPFL9 is a regulator of stomatal development. Genetic analysis was consistent with EPFL9 acting independently of EPF1 to control stomatal clustering, independently of EPF2 to regulate stomatal density, and independently of SDD1 to control both stomatal clustering and density.• These findings demonstrate that at least three secretory peptides independently regulate stomatal development. Surprisingly, EPFL9 acts to increase, rather than decrease, stomatal density and clustering. However, in common with EPF1 and EPF2, EPFL9 is unlikely to be a substrate for proteolysis by SDD1.