An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice

Abstract

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Fig. 1.

The drought sensitivity of the eibi1.b wild barley mutant and its cuticle structure. (A) Wild-type (Left) and eibi1.b (Right) seedlings. The leaf comprises the three regions: the EmBL, the NEZ below the point of emergence (POE), and the EZ above the point of leaf insertion (POLI). (B) Water loss from detached leaves of eibi1 and wild-type plants. (C) Electron micrographs of the cuticle in EmBL, NEZ, and EZ of wild-type plants and the eibi1.b mutant. Arrows indicate the cuticle thickness. (D) Major cutin monomers and wax components in the EmBL of the eibi1.b mutant and wild type. All bars represent mean ± SD (n = 5). Asterisks denote significant differences (P < 0.0001) between wild type and mutant as determined by student's t tests. (E) Epidermis cells with (eibi1.b) or without (wild type) cytoplasmic protrusions into the vacuole. c, cytoplasm; p, protrusions; v, vacuole.

Fig. 2.

Positional cloning of eibi1 and its functional assignment. (A) The Eibi1 locus maps to barley chromosome 3H and its ortholog to rice chromosome 1. Numbers to the left of the barley map indicate the number of observed recombination events in a population of 9,070 gametes. Rice and barley orthologs are connected by dashed lines. (B) Exon/intron structure of barley Eibi1. The single-nucleotide difference between the wild-type and the eibi1.b mutant sequence is indicated. The red dashed line indicates the 9-bp deletion in eibi1.c exon 10. Untranslated regions are indicated by an empty box at each end. (C) The γ irradiation-induced eibi1.c mutant and its wild type at the flowering stage. (D) Water-loss test of detached leaves from eibi1.c and OUH602. (E) Exon/intron structure of rice OsABCG31 indicating Tos17 insertions (arrows). (F) Wild-type (one tall seedling) and osabcg31.b mutants (four dwarf seedlings) at the three-leaf stage. (G) Enlarged view of dwarf seedlings in F. (H) Water loss in the detached leaf of the wild type and the mutant. (I) Mutant and wild-type leaves of barley air dried for 1 h and rice air dried for 0.5 h. (J) Toluidine blue staining of leaf segments of mutants and wild types. (Scale bar: 20 mm for wild barley; 5 mm for rice.)

Fig. 3.

Eibi1 gene expression and EIBI1 immunolocalization. The Eibi1 expression pattern as revealed by (A) RT-PCR and by (B) quantitative RT-PCR; data shown are means ± SD of three biological replicates. (C) Longitudinal section and (D) cross-section of the young shoot demonstrating gene expression of Eibi1 in the developing leaves of wild barley 23-19, as detected by in situ hybridization with an antisense (Upper image) and sense (Lower image) probe. c, coleoptile; l, leaf. (E–H) Immunofluorescent localization of EIBI1 in cross-sections taken 5–15 mm above the root–shoot junction in wild barley 23-19 (E and F) and rice 'Nipponbare' (G and H) seedlings at the three-leaf stage. The fluorescence signal is shown in red. F and H are enlargements of E and G, respectively. e, epidermis cell; m, mature leaf; y, young leaf.