A rice small GTPase, Rab6a, is involved in the regulation of grain yield and iron nutrition in response to CO2 enrichment

Abstract

Despite extensive studies on the effects of elevated atmospheric CO2 concentrations ([CO2]) on rice, the molecular mechanisms and signaling events underlying the adaptation of plants remain largely elusive. Here, we report that OsRab6a, which encodes a small GTPase, is involved in the regulation of rice growth, grain yield, and accumulation of iron (Fe) in response to elevated [CO2] (e[CO2]). We generated transgenic plants with OsRab6a-overexpression (-OE) together with OsRab6a-RNAi lines, and found no differences in growth and grain yield among them and wild-type (WT) plants under ambient [CO2] conditions. Under e[CO2] conditions, growth and grain yield of the WT and OsRab6a-OE plants were enhanced, with a greater effect being observed in the latter. In contrast, there were no effects of e[CO2] on growth and grain yield of the OsRab6a-RNAi plants. Photosynthetic rates in both the WT and OsRab6a-OE plants were stimulated by e[CO2], with the magnitude of the increase being higher in OsRab6a-OE plants. Fe concentrations in vegetative tissues and the grain of the WT and transgenic plants were reduced by e[CO2], and the magnitude of the decrease was lower in the OE plants than in the WT and RNAi plants. Genes associated with Fe acquisition in the OsRab6a-OE lines exhibited higher levels of expression than those in the WT and the RNAi lines under e[CO2]. Analysis of our data using Dunnett's multiple comparison test suggested that OsRab6a is an important molecular regulator that underlies the adaptation of rice to e[CO2] by controlling photosynthesis and Fe accumulation.

Keywords: Oryza sativa; Rab6a; Elevated CO2 concentrations; Fe acquisition; grain Fe content; photosynthesis; small GTPase.

Fig. 1.

Time course of OsRab6a expression in rice seedlings under elevated [CO₂] in (A) shoots and (B) roots. Wild-type seedlings of 30-d-old were placed in an open-top chamber with [CO₂] of ~770 μmol mol⁻¹ for 5 d. Expression is relative to the value before treatment (0 d), which was set as 1, and actin was used as the reference gene. Data are means (±SE), n=3. Different letters indicate significant differences at P<0.05 between the expression level of OsRab6a under ambient and elevated [CO₂] treatments, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05)

Fig. 2.

Effects of elevated [CO₂] (e[CO₂]) on the growth of rice wild-type (WT) seedlings and transgenic lines with OsRab6a-overexpression (OE) or OsRab6a-RNAi (Ri). Seedlings at 30 d old were placed in open-top chambers receiving either ambient [CO₂] (~380 μmol mol⁻¹) or e[CO₂] (~770 μmol mol⁻¹) and measurements were taken after 20 d (seedling stage) and after 60 d (tillering stage). (A–C) Seedling stage: (A) plant height, (B) shoot dry weight, and (C) root dry weight. (D–F) Tillering stage: (D) plant height, (E) shoot dry weight, and (F) root dry weight. Data are means (±SE), n=8, with two plants being sampled in each of four replicate chambers. Asterisks indicate a significant difference between ambient [CO₂] and e[CO₂] for a given genotype, as determined using Student’s t-test (P<0.05). Different letters indicate significant differences between the genotypes within the same [CO₂] treatment, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05).

Fig. 3.

Effects of elevated [CO₂] (e[CO₂]) on the net rate of photosynthesis (P_n) in rice wild-type (WT) seedlings and transgenic lines with OsRab6a-overexpression (OE) or OsRab6a-RNAi (Ri). Seedlings of 30-d-old were placed in open-top chambers receiving either ambient [CO₂] (~380 μmol mol⁻¹) or e[CO₂] (~770 μmol mol⁻¹) and measurements were taken (A) after 20 d (seedling stage) and (B) after 60 d (tillering stage). Data are means (±SE) n=8, with two plants being sampled in each of four replicate chambers. Asterisks indicate a significant difference between ambient [CO₂] and e[CO₂] for a given genotype, as determined using Student’s t-test (P<0.05). Different letters indicate significant differences between the genotypes within the same [CO₂] treatment, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05).

Fig. 4.

Effects of elevated [CO₂] (e[CO₂]) on final grain yield in rice wild-type (WT) seedlings and transgenic lines with OsRab6a-overexpression (OE) or OsRab6a-RNAi (Ri). Seedlings of 30-d-old were placed in open-top chambers receiving either ambient [CO₂] (~380 μmol mol⁻¹) or e[CO₂] (~770 μmol mol⁻¹) and grown to maturity. Data are means (±SE), n=8, with two plants being sampled in each of four replicate chambers. Asterisks indicate a significant difference between ambient [CO₂] and e[CO₂] for a given genotype, as determined using Student’s t-test (P<0.05). Different letters indicate significant differences between the genotypes within the same [CO₂] treatment, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05).

Fig. 5.

Effects of elevated [CO₂] (e[CO₂]) on Fe concentrations in rice wild-type (WT) seedlings and transgenic lines with OsRab6a-overexpression (OE) or OsRab6a-RNAi (Ri). Seedlings at 30-d-old were placed in open-top chambers receiving either ambient [CO₂] (~380 μmol mol⁻¹) or e[CO₂] (~770 μmol mol⁻¹) and measurements were taken (A) after 20 d (seedling stage) and (B) after 60 d (tillering stage). (A, B) Seedling stage: Fe concentrations in (A) shoots and (B) roots. (C, D) Tillering stage: Fe concentrations in (C) shoots and (D) roots. (E) Fe concentrations in grains. Data are means (±SE), with one plant in each of four replicate chambers being measured. Asterisks indicate a significant difference between ambient [CO₂] and e[CO₂] for a given genotype, as determined using Student’s t-test (P<0.05). Different letters indicate significant differences between the genotypes within the same [CO₂] treatment, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05).

Fig. 6.

Effects of elevated [CO₂] (e[CO₂]) on expression of (A) OsNAS1, (B) OsNAS2, and (C) OsIRT1 in rice wild-type (WT) seedlings and transgenic lines with OsRab6a-overexpression (OE) or OsRab6a-RNAi (Ri). Seedlings of 30-d-old were placed in an open-top chamber receiving either ambient [CO₂] (~380 μmol mol⁻¹) or e[CO₂] (~770 μmol mol⁻¹) and root samples were taken after 10 d. Expression is relative to the value in the WT under ambient [CO₂], which was set as 1, and actin was used as the reference gene. Data are means (±SE), n=3. Asterisks indicate a significant difference between ambient [CO₂] and e[CO₂] for a given genotype, as determined using Student’s t-test (P<0.05). Different letters indicate significant differences between the genotypes within the same [CO₂] treatment, as determined using ANOVA followed by Dunnett’s multiple comparison test (P<0.05).