Genetic manipulation of abscisic acid receptors enables modulation of water use efficiency

ABSTRACT

The phytohormone abscisic acid (ABA) is a key signal molecule for controlling stomatal movement and stress responses such as drought. Genetic manipulation of ABA receptors has recently been reported to improve plant water use efficiency (WUE). We therefore compared details of WUE traits between the ABA receptor quintuple mutant and wheat ABA receptor-overexpressing Arabidopsis lines. Biomass and seed productivities per liter of water were both reduced in the receptor mutant but improved in the transgenic Arabidopsis lines. This result suggests that appropriate modulation of ABA receptors can extend crop potential productivity.

The phytohormone abscisic acid (ABA) has multiple roles in plant physiological processes, including stomatal closure, stress response, seed dormancy, and growth regulation. These ABA actions are elicited through a signal transduction pathway that is initiated by ABA binding to soluble PYRABACTIN RESISTANCE 1 (PYR1)/PYR1- LIKE (PYL) (PYR/PYL) receptors.¹ Multiple PYR/PYL homologs are conserved across plant species.² Recently, genetically modified plants overexpressing ABA receptors have been shown to improve water use efficiency (WUE) without any trade-offs in growth or yield.^3–6 For instance, Yang et al. generated Arabidopsis lines overexpressing each PYR/PYL and found that PYL4 and PYL12 improve WUE.³ These transgenic lines were able to generate more biomass from smaller amounts of water. In addition, we have observed that grain yield from 1 L of water is enhanced in wheat by overexpression of a wheat PYR/PYL homolog (TaPYL4).⁴ Furthermore, exogenous ABA application can improve WUE in Arabidopsis and wheat.⁵ Appropriate activation of ABA receptors can therefore be used as a new agricultural method to improve crop productivity in water- limited areas. Despite these findings, details of WUE traits in ABA receptor mutants have not been reported. We therefore investigated WUE and ABA response traits using the ABA receptor pyr1pyl1pyl2pyl4pyl5 quintuple mutant and TaPYL4-overexpressing Arabidopsis.

In a previous study, we characterized nine PYR/PYL homologs in the wheat genome and verified their functions in the ABA signaling pathway by an in vitro assay.⁴ Among these homologs, TaPYL4 effectively improved the water-saving function of wheat. We accordingly generated Arabidopsis transgenic lines overexpressing TaPYL4, which resulted in two transgenic lines (#14 and #16) in which the TaPYL4 transgene was highly expressed (Figure 1a). Two ABA-responsive genes (AtRD29B and AtMAPKKK18) were induced more strongly in the two transgenic lines compared with the wild type at the same ABA concentrations (Figure 1b). The ABA receptor gene can thus also function in Arabidopsis plants. In the quintuple mutant, in contrast, the expressions of the two ABA- responsive genes were negligible under exogenous ABA treatment (Figure 1b). Thermal imaging analysis revealed that the two transgenic lines had decreased transpiration due to increased ABA sensitivity, whereas transpiration of the quintuple mutant was increased as a result of its reduced ABA sensitivity (Figure 1c). Consequently, the transgenic lines exhibited more drought tolerance compared with the wild type, whereas the quintuple mutant was sensitive to drought (Figure 1d).

Figure 1.

Molecular and physiological traits of an ABA receptor mutant and wheat TaPYL4-overexpressing Arabidopsis lines. (a) Expression levels of TaPYL4 among wild type (WT), ABA receptor quintuple mutant (quin), and #14 and # 16 lines. (b) Expression levels of ABA-responsive genes in each genotype. Error bars represent SD (n = 3). A Tukey–Kramer test was performed to assess the statistical significance (P < .05) of differences among genotypes. (c) Thermal imaging of intact leaves of each genotype. (d) Representative photographs of each genotype before and after drought stress. The number of surviving plants (out of the total number tested) per treatment is shown at the bottom right of each image.

To investigate long-term transpiration activity, we measured ¹³C composition.⁷ The degree of ¹³C composition among genotypes was correlated with their transpirational levels (Figures 1c and 2a). Indeed, the transgenic lines consumed a smaller amount of water than the wild type, whereas the quintuple mutant used more (Figure 2b). Although the transgenic lines consumed less water than the wild type, they had comparable biomasses and seed yield, consistent with a previous study (Figure 2c).³ In contrast, the biomass and seed yield of the quintuple mutant was significantly lower than that of the wild type even though the mutant consumed more water (Figure 2c). Finally, we calculated plant biomass and seed yield per liter of input water. Compared with the wild type, transgenic line #14 had a 40% and 25% higher biomass and seed yield per liter of input water, respectively, while the quintuple mutant had corresponding decreases of 33% and 37% (Figure 2d).

Figure 2.

Comparison of biomass production, seed yield, and water consumption among wild type, ABA receptor mutant (quin), and wheat TaPYL4-overexpressing Arabidopsis (#14 and #16) lines. (a) Carbon isotope composition (δ¹³C) of each genotype. Error bars represent SD (n = 6). (b) Water consumption volume over the lifetime of each genotype. (c) Total above-ground biomass (dry weight: DW) and seed yield per plant. (d) Total above-ground biomass (DW) and seed yield produced per plant from 1 L of water. Error bars represent SD (B–D: n = 5). A Tukey–Kramer test was performed to assess the statistical significance (P < .05) of differences among genotypes.

This study has revealed that wheat ABA-receptor overexpression in Arabidopsis improves biomass and seed production per input water without growth inhibition, similar to the results of our previous investigation of wheat.⁴ Conversely, disruption of multiple ABA receptors, including dimeric types (PYR1, PYL1, and PYL2), leads to water- wasteful traits and reduces biomass and seed productivities in Arabidopsis. In contrast to our present findings, the pyl1pyl4pyl6 triple mutant improves rice grain productivity in paddy fields, where water is not limited, but the rice has reduced drought tolerance.⁸ Unlike the triple mutant, transgenic rice plants overexpressing an ABA receptor acquire drought tolerance but exhibit growth inhibition.⁹ The optimal method for genetic manipulation of ABA receptors may thus differ among plant species and may depend on land water-availability. In any case, ABA receptor genes are excellent targets for improving crop yield productivity.

Funding Statement

This work was supported by grants from KAKENHI (17H05009 to M.O.), the Joint Research Program of Arid Land Research Center, Tottori University (30C2007 to M.O.), Project Marginal Region Agriculture of Tottori University (to H.T.), and JST SATREPS (JPMJSA1805 to H.T.)the Joint Research Program of Arid Land Research Center, Tottori University [30C2007].