Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene

Abstract

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.

Fig. 1.

The hrd-D mutant phenotype in Arabidopsis. (A) Rosette leaf phenotype of WT and hrd-D mutant with smaller, slightly curled, thicker deep-green leaves. (B) Cryo-fracture scanning electron microscopy section of leaves of WT and hrd-D mutant, showing more mesophyll cell layers. (C) Root structure of WT and hrd-D mutant, showing more profuse secondary and tertiary roots at the root base. (D) Cross-section of WT and hrd-D roots, showing increased cortical cell layers (lighter stained) and compact stele in the mutant.

Fig. 2.

The hrd-D mutant and expression analysis. (A) The hrd-D mutant genomic region, showing adjacent genes, annotated as AP2-like and aldolase, with their promoters located, respectively, 3.1 kb and 2.4 kb from the CaMV35S enhancer tetramer of the I-ATag transposon insert (10). (B) RT-PCR expression analysis of the AP2-like and aldolase genes, using RNA from rosette leaves of two WT (Wt1, Wt2) samples and the hrd-D mutant. The aldolase gene is highly expressed and unchanged, whereas the AP2-like gene is overexpressed in the hrd-D mutant, with the weak lower bands being primer–dimers.

Fig. 3.

Stress tolerance/resistance by overexpression of HRD in Arabidopsis. (A) Drought-resistance tests of Arabidopsis WT and the hrd-D mutant line, treated for 9–12 days without water. The first row is at 9 days of dehydration (DOD), followed by plants treated for 11 and 12 DOD that were subsequently watered to reveal surviving plants. (B) Mutant hrd-D and WT Arabidopsis treated at 300 mM NaCl concentrations, showing bleached/dead plants and surviving hrd-D plants.

Fig. 4.

Phenotype of HRD overexpression in rice. (A) Rice HRD overexpression line compared with WT Nipponbare under well watered (control) and water-stress (70% field capacity) conditions. (B) Leaf cross-section of WT and HRD overexpression lines, observed under fluorescence microscope, revealing red chlorophyll fluorescence and blue vascular bundles surrounded by the bundle sheath cells marked with an arrow. (C) Number of bundle sheath cells in WT compared with HRD overexpressors, which show significant increase (n > 5, P = 7.5 × 10⁻¹⁰).

Fig. 5.

Physiological analyses of rice HRD overexpression lines showing improved WUE. (A and B) The HRD lines and WT Nipponbare tested under well watered (white) and drought stress (shaded) conditions. Bars indicate SE (n > 3). All parameters are significant at 1% with calculated P values shown for HRD vs. WT. (A) WUE by gravimetric determination (P = 1.6 × 10⁻⁰⁴). (B) MTR (P = 2 × 10⁻²). (C) NAR (P = 2.27 × 10⁻⁵). (D) Total biomass (P = 9.9 × 10⁻¹⁰). (E) Shoot biomass (P = 7.4 × 10⁻⁶). (F) Root biomass (P = 1 × 10⁻⁷). (G) Instantaneous WUE (P = 1.4 × 10⁻⁴). (H) Carbon assimilation (P = 2.9 × 10⁻⁴). (I) Relative quantum yield of PSII at steady-state photosynthesis (P = 2 × 10⁻³).