Reducing stomatal density by expression of a synthetic epidermal patterning factor increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop

Abstract

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (gs) has improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the relationship of photosynthesis (AN) with intracellular CO2 concentration (ci), such that photosynthesis is CO2 saturated and reductions in gs are unlikely to limit AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum led to reduced stomatal densities, reduced gs, reduced plant water use, and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to AN. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.

Keywords: Sorghum bicolor; C4 photosynthesis; stomata; water-use efficiency.

Fig. 1.

Phenotypic screen of transgenic plants. Abaxial stomatal density of independent transgenic events of sorghum (A) ubiquitously expressing the sorghum orthologue of EPIDERMAL PATTERNING FACTOR-1 (SbEPF1) and (B) ubiquitously expressing a fusion element synthesized from elements of the sorghum orthologues of EPIDERMAL PATTERNING FACTOR-2 (EPF2) and EPIDERMAL PATTERNING FACTOR-LIKE 9 (EPFL9) (SbEPF_syn). The events that displayed significantly different stomatal densities compared with the wild type (WT), according to a post-hoc Tukey test following a one-way ANOVA, are denoted by red asterisks. The independent events of SbEPF_syn that were carried forward for further investigation are highlighted in bold and underlined (ZG602-5-12b and ZG600-6-13a).

Fig. 2.

Stomatal patterning phenotype of engineered plants. Representative micrographs of the abaxial leaf surface of: (A) the wild type (WT), (B) SbEPF_syn line ZG602-5-12b (12b), and (C) SbEPF_syn line ZG600-6-13a (13a), along with the associated (D) abaxial stomatal density and (E) adaxial stomatal density at three leaf positions on the main culm (three, four, and five) for each genotype. Bars represent least square means of stomatal density for each grouping, where the errors bar represent the associated SEs. For the abaxial and the adaxial surface, the P-values from each term in a two-way ANOVA with an interaction term are inset.

Fig. 3.

Leaf gas exchange of engineered plants. Light-saturated (A) stomatal conductance (g_s), (B) net photosynthetic assimilation of CO₂ (A_N), and (C) intrinsic water-use efficiency (iWUE) of the sixth leaf on the culm when it was the youngest fully expanded leaf of the wild type (WT), SbEPF_syn line ZG602-5-12b (12b), and SbEPF_syn line ZG600-6-13a (13a). Bars represent least square means, and error bars represent associated SEs. Significant differences between genotypes are denoted as *P>0.05, ** P>0.01, or *** P>0.001.

Fig. 4.

Photosynthetic capacity of engineered plants. Fitted average photosynthetic/CO₂ response (A/c_i) curves of (A) the wild type (WT), (B) SbEPF_syn line ZG602-5-12b (12b), and (C) SbEPF_syn line ZG600-6-13a (13a), along with (D) the apparent maximum rate of carboxylation by PEPC (V_pmax), (A) the asymptote of the A/c_i curve (V_max), (F) specific leaf area (SLA), and (G) leaf nitrogen (N) content of the sixth leaf on the culm when it was the youngest fully expanded leaf. For A/c_i curves, the mean fit is represented by the solid line and the SE is denoted by the shaded area. The stomatal limitation to A_N at ambient [CO₂] (SL) is reported for each treatment. The operating point of photosynthesis at ambient [CO₂] is shown as a blue dot on the A/c_i curve. Bars represent least square means, and error bars represent associated SEs. The P-values from associated one-way ANOVA tests are inset. Where a significant effect was detected, the differences between the transgenic lines and the WT according to post-hoc testing is shown.

Fig. 5.

Water use and leaf gas exchange of engineered plants during a dry-down period. The response of (A) percentage relative soil water content, (B) stomatal conductance (g_s), and (C) net photosynthetic assimilation of CO₂ (A_N) to a 9 d water withdrawal period for wild-type (WT), SbEPF_syn line ZG602-5-12b (12b), and SbEPF_syn line ZG600-6-13a (13a) sorghum. Points and errors bars represent least square means and SEs, respectively. P-values associated with repeated measure ANOVAs are inset. Days where transgenic events ZG602-5-12b (12b) and ZG600-6-13a (13a) were significantly different from the WT for all traits are indicated by red asterisks. The statistical results of pairwise tests between each transgenic line and the WT for a specific contrast of average g_s and A_N on the first 5 d of the experiment is shown by the inset bracket.

Fig. 6.

Whole-plant phenotypic characters of engineered plants. (A) Photographs of representative plants of wild-type (WT), SbEPF_syn line ZG602-5-12b (12b), and SbEPF_syn line ZG600-6-13a (13a) plants on day 8 of the water withdrawal period. (B) Total dried above-ground biomass of all genotypes grown under water-replete conditions or subjected to the water withdrawal period. Bars represent least square means, and error bars represent associated SEs. P-values associated with each term from an associated two-way ANOVA are inset.

Fig. 7.

Photographs of representative panicles from wild-type sorghum (WT) and sorghum ubiquitously expressing a fusion element synthesized from elements of the sorghum orthologues of EPIDERMAL PATTERNING FACTOR-2 (EPF2) and EPIDERMAL PATTERNING FACTOR-LIKE 9 (EPFL9) (SbEPF_syn).