Serine carboxypeptidase 46 Regulates Grain Filling and Seed Germination in Rice (Oryza sativa L.)

Abstract

Serine carboxypeptidase (SCP) is one of the largest groups of enzymes catalyzing proteolysis for functional protein maturation. To date, little is known about the function of SCPs in rice. In this study, we present a comprehensive analysis of the gene structure and expression profile of 59 rice SCPs. SCP46 is dominantly expressed in developing seeds, particularly in embryo, endosperm and aleurone layers, and could be induced by ABA. Functional characterization revealed that knock-down of SCP46 resulted in smaller grain size and enhanced seed germination. Furthermore, scp46 seed germination became less sensitive to the ABA inhibition than the Wild-type did; suggesting SCP46 is involved in ABA signaling. As indicated by RNA-seq and qRT-PCR analysis, numerous grain filling and seed dormancy related genes, such as SP, VP1 and AGPs were down-regulated in scp46. Yeast-two-hybrid assay also showed that SCP46 interacts with another ABA-inducible protein DI19-1. Taken together, we suggested that SCP46 is a master regulator of grain filling and seed germination, possibly via participating in the ABA signaling. The results of this study shed novel light into the roles of SCPs in rice.

Fig 1. Phylogenetic analysis of rice and maize SCPs.

The branch length scale bar indicates the evolutionary distance. Red line corresponds to SCP46.

Fig 2. Expression analysis of SCPs in tissues and response to phytohormone treatments.

(A) Hierarchical cluster display of the expression profile of rice SCPs in callus, leaf, shealth, root, panicle, 7, 14 and 21 DAP seeds. Color bar on the top represents the log2 expression values. Blue, white and Purple indicate the low, medium and high expression values respectively. (B) Schematical representation of the gene structure of SCP46. (C) qRT-PCR analysis of SCP46 in various rice tissues. Different characters indicate a statistically significant difference at P<0.05. (D-F) qRT-PCR analysis of SCP46 in response to ABA (D), BR (E) and GA (F) treatment at 5 different time points. ** indicates P<0.01 by t-test in comparison with 0h. (G-J) mRNA in-situ hybridization assay of SCP46 in seeds at early (G), medium (H) and late stages (I); (J) shows the negative control by using sense probe for hybridization. Em: embryo; En: endosperm; PL: pericap layer; AL: aleuron layer; Bar = 1.5 mm.

Fig 3. Phenotypical characterization of scp46 grain size and seed germination.

(A-B) Comparison of the width (A) and length (B) of scp46 and WT grains; (C) qRT-PCR analysis of SCP46 expression level in the seeds of RNAi and WT lines. 1000-grain-weight (D), grain length (E) and width (F) of RNAi and WT lines. (G) Seed germination of scp46 and WT seeds under 0, 2 and 5 μM ABA. Pictures were taken at 120 hours after the germination assay. (H) Germination time courses of scp46 and WT seeds. Asterisks indicate the significance of differences between Wild-type and scp46 lines as determined by Student’s t test analysis: ** P<0.01.

Fig 4. Expression analysis of SCP46 regulated genes by qRT-PCR.

(A) qRT-PCR validation of the DEGs revealed by RNA-seq experiments; (B) the expression change of rice grain filling genes in scp46 and WT. 7 DAP seed cDNA was used as templates for this analysis. All values are based on three technical repeats and presented as means±SE.* indicates P<0.05, ** indicates P<0.01 by t-test.

Fig 5. Expressoin profile of DI19-1 and yeast-two-hybrid assay of SCP46-DI19-1 interaction.

(A) Expression profile of DI19-1 in different rice tissues; Different characters indicate a statistically significant difference at P<0.05. (B) SCP46 expression in response to ABA treatment; * indicates P<0.05, ** indicates P<0.01 by t-test in comparison with 0h. (C) yeast-two-hybrid assay of SCP46-DI19-1 interaction. pGBKT7-Lam+pGADT7-T and pGBKT7-53+pGADT7-T were used as negative and positive controls as instructed by the Kit. DDO: double drop out medium (SD/-Trp-Leu); QDO: quarter drop out medium (SD/-Trp-Leu-Ade-His/+X-α-Gal).