Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production

Abstract

The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha(-1).

Figure 1.

Phenotypic analysis of the ysa mutant plants. A to C, Phenotypes of Pei'ai64S (left) and ysa mutant (right) seedlings at 1 (A), 2 (B), and 3 (C) weeks after sowing. D, The pigment contents in leaves of 1-week-old ysa mutants are much lower than that in Pei'ai64S. E, The pigment contents in leaves of 6-week-old ysa mutants are similar to that of Pei'ai64S plants. Chla, Chlorophyll a; Chlb, chlorophyll b; Chl, total chlorophyll; Car, carotenoid. Bars represent sds of three measurements. Student’s t test was performed on the raw data; asterisk indicates statistical significance at P < 0.01.

Figure 2.

Transmission electron microscopic images of chloroplasts of wild-type Pei'ai64S and ysa mutant plants at 10 d (A and D), 18 d (B and E), and 30 d (C and F) after sowing. Chloroplasts of Pei'ai64S have abundant, well-ordered stacks membranes after sowing for 10, 18, or 30 d, but ysa have normal stacked membranes only after 30 d.

Figure 3.

Cloning of the YSA gene. A, The YSA locus was initially mapped to the centromeric region between markers RM411 and RM8208 on chromosome 3 (Chr. 3). B, Mapping of the YSA locus with markers P1 to P3 developed based on bacterial artificial chromosome clone sequence (BAC1, OSJNBa0010D22; BAC2, OSJNBb0085A04; BAC3, OSJNBa0027H16; BAC4, OSJNBb0056O10; BAC5, OSJNBb0042N11; BAC6, OSJNBa0087M10). C, Fine mapping of the YSA locus with markers P4 to P9. The YSA locus was narrowed down to a genomic DNA region of 45 kb between dCAPS markers P6 and P7. The number of recombinants identified from 2,083 recessive individuals was shown for each marker. D, Diagram of the predicted ORFs (highlighted with arrows) and the mutation site. A 5-bp deletion (underlined) in ORF4 results in a premature stop codon. E, The difference between ysa mutant and Pei'ai64S is shown by the size of amplified genomic DNA by a newly developed InDel marker P9. F, Complementation of the ysa mutant. The wild-type Pei'ai64S plants (left) and the ysa mutants transformed with pYSA vector (right) shows normal green leaves, whereas the mutant transformed with pYSAT vector has albino leaves. G, Chloroplasts of plants transformed with pYSAT have few membrane stacks. H, Chloroplasts of plants transformed with pYSA have abundant and well-ordered stacks.

Figure 4.

Sequence analysis of YSA. A, The YSA protein has 15 PPR motifs, whereas the ysa mutant protein has only 10 PPR motifs (P). B, Comparison of the PPR motifs of YSA. Amino acids fully or semiconserved are shaded black and gray, respectively. C, Amino acid sequence alignment of YSA and MEE40. Amino acids fully or semiconserved are shaded black and gray, respectively.

Figure 5.

Expression analysis of YSA. A to D, GUS staining shows that YSA is highly expressed in young leaves and stem, but not in the roots and mature stems. E, Transcript levels of YSA in leaves of different developmental stages. The YSA RNA level in the Pei'ai64S plants of two-leaf stage was set to 1.0, and the relative YSA RNA levels in other developmental stages were calculated accordingly. F, YSA expression in 10-d-old etiolated Pei'ai64S plants after different times of illumination. After growing in darkness for 10 d, the etiolated rice seedlings were illuminated for 4, 8, 12, 16, or 24 h. The YSA RNA level in seedlings growing under illumination was set to 1.0, and the relative YSA RNA level in seedlings growing under dark or dark-to-light condition were calculated accordingly. Seedlings growing under continuous light or dark were chosen as the control. Error bars (sds) are based on three independent experiments. Bars with different letters indicate significant differences at P < 0.05 level based on the one-way ANOVA assay.

Figure 6.

Expression analysis of genes associated with chlorophyll biosynthesis, photosynthesis, or chloroplast development by real-time PCR. The relative expression level of each gene was normalized using Actin1 as an internal control. The expression level of each gene at the two-leaf stage in Pei'ai64S was set as 1.0 and other samples were calculated accordingly. Bars represent sds of three independent experiments. Asterisks indicate statistically significant differences compared with Pei'ai64S (Student’s t test: *P < 0.05; **P < 0.01).

Figure 7.

Subcellular localization of YSA protein. Fluorescence signals were visualized using confocal laser-scanning microscopy. Green fluorescence shows GFP, red fluorescence indicates chloroplast autofluorescence, and yellow fluorescence indicates images with the two types of fluorescence merged. A, GFP signals of the YSA-GFP fusion protein. B, GFP signals from the transit peptide of ribulose bisphosphate carboxylase small subunit (control). C, GFP signals from the nuclear localization signal of fibrillarin (control). D, Empty GFP vector without a specific targeting sequence. E, Untransformed chloroplasts. F, Subcellular localization of YSA protein in rice protoplasts. Bars = 5 μm. A to E are Arabidopsis protoplasts and F is rice protoplasts.

Figure 8.

Application of the ysa marker in T/PGMS propagation and two-line hybrid seed production. A, The ysa marker identifies the green off-type seedlings during propagation of T/PGMS seeds. B, ysa identifies albino off-type seedlings during two-line hybrid seed production.