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

Abstract

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.

Keywords: Cereals; drought; photosynthesis; stomata; water-use efficiency; wheat.

Fig. 1.

Overexpression of wheat EPFs in Arabidopsis leads to a decreased stomatal density. (A) Stomatal density, (B) pavement cell density, and (C) stomatal index were measured in fully expanded leaves from a series of 6-week-old transgenic Arabidopsis plants (Col-0) transformed with either TaEPF1 or TaEPF2 under control of the 35S promoter. Samples were analysed using one-way ANOVA with Fisher’s LSD test. Col-0, five plants; p35STaEPF1-1, p35STaEPF1-2, and p35STaEPF2-2, four plants; p35STaEPF2-2 and p35SAtEPF2, three plants. (A–C) show means, bars=SE.

Fig. 2.

Stomatal density is reduced in wheat (Triticum aestivum) lines overexpressing TaEPF1. (A) Stomatal densities of wild-type cv Fielder and three independently transformed TaEPF1-OE lines were measured in fully expanded leaf 1. Comparison of stomatal densities was performed using a one-way ANOVA and Fisher’s LSD test, Means that are not significantly different from each other (P<0.05) are indicated with the same letter (n=5–8 leaves). Bars=SE. (B) Image of wild-type wheat epidermis of the abaxial side of leaf 1, with stomatal files labelled (purple arrows). (C) as in (B) but a TaEPF1 transgenic plant (TaEPF-OE3) with stomatal files (purple arrows) and arrested precursor cells (green arrows). (D–G) Example images of arrested or altered cell division patterns in TaEPF-OE3 in positions predicted to form stomata normally. Scale bars (B, C)=20 µm, (D–G)=5 µm.

Fig. 3.

Wheat plants overexpressing TaEPF1 grow normally and are more water-use efficient. (A) Wheat transgenic lines TaEPF1-OE1 (moderate decrease in stomatal density) and TaEPF1-OE3 (more severe decrease in stomatal density) are not visibly distinguishable from wild-type cv Fielder plants. (B) A/C_i curves under saturating light for the wild type (white circles) and TaEPF1 overexpressors TaEPF1-OE1 and TaEPF1-OE3 (shaded circles) cannot be distinguished, indicating that biochemical limitations on photosynthesis do not differ between the lines. (C) Assimilation rates under standard growth conditions do not differ significantly between wild-type cv Fielder and TaEPF1 overexpressors TaEPF1-OE and TaEPF1-OE2 (which show a moderate and intermediate decrease in stomatal density, respectively), while the more severe line TaEPF1-OE3 shows a decrease in assimilation rate under these conditions. (D) Stomatal conductance, g_s, of the TaEPF1 overexpression lines is decreased compared with wild-type cv Fielder. (E) iWUE is higher in the TaEPF1 overexpression lines compared with wild-type cv Fielder. For (C, D, E), a one-way ANOVA was performed followed by uncorrected Fisher’s LSD test. Lines that cannot be distinguished from each other (P<0.05) are indicated with the same letter (n=5 plants per line). Bars=SE.

Fig. 4.

Seed yield is maintained in wheat plants with moderately decreased stomatal density. Plants were grown to seed in the glasshouse in two separate experiments. In (A), transgenic lines with moderate or intermediate decreases in stomatal density were analysed (TaEPF1-OE2 and TaEPF1-OE4) and in (B) transgenic lines with more extreme decreases in stomatal density (TaEPF1-OE3 and TaEPF1-OE5), with independent control lines of cv Fielder grown in both series of experiments. In (A) and (B), plants were either provided a normal watering regime (80% soil RWC) or were grown with a restricted water regime, WR (30% soil RWC). Seed yield at maturity (g of seed per plant) was measured and the yields compared (one-way ANOVA and Fisher’s LSD, n=5 plants per line). Samples designated with the same letter cannot be distinguished from each other at the 95% confidence limit. Bars are SE.

Fig. 5.

Water restriction negates any potential yield gain in elevated CO₂ conditions irrespective of stomatal density in wheat. Plants with a spectrum of decreased stomatal density (moderate, TaEPF1-OE1; intermediate, TaEPF1-OE2; extreme, TaEPF1-OE3) as well as the control, non-transgenic cv Fielder were grown to seed under (A) a control watering regime (80% FC) or (B) a restricted watering (30% FC). In addition, plants were grown under either an ambient CO₂ level (indicated by A) or elevated 1000 ppm (indicated by E). Seed yield at maturity (g of seed per plant) was measured and the yields compared (one-way ANOVA and Fisher’s LSD, n=5 plants per line). Samples designated with the same letter cannot be distinguished from each other at the 95% confidence limit. Bars are SE.