Rice Phytochrome-Interacting Factor-Like1 (OsPIL1) is involved in the promotion of chlorophyll biosynthesis through feed-forward regulatory loops

Abstract

In phototrophic plants, the highly conserved and tightly regulated process of chlorophyll (Chl) biosynthesis comprises multi-step reactions involving more than 15 enzymes. Since the efficiency of Chl biosynthesis strongly affects plant productivity, understanding the underlying regulatory mechanisms in crop plants can be useful for strategies to increase grain and biomass yields. Here, we show that rice (Oryza sativa) Phytochrome-Interacting Factor-Like1 (OsPIL1), a basic helix-loop-helix transcription factor, promotes Chl biosynthesis. The T-DNA insertion knockdown ospil1 mutant showed a pale-green phenotype when grown in a natural paddy field. Transcriptome analysis revealed that several genes responsible for Chl biosynthesis and photosynthesis were significantly down-regulated in ospil1 leaves. Using promoter binding and transactivation assays, we found that OsPIL1 binds to the promoters of two Chl biosynthetic genes, OsPORB and OsCAO1, and promotes their transcription. In addition, OsPIL1 directly up-regulates the expression of two transcription factor genes, GOLDEN2-LIKE1 (OsGLK1) and OsGLK2. OsGLK1 and OsGLK2 both bind to the promoters of OsPORB and OsCAO1, as well as some of genes encoding the light-harvesting complex of photosystems, probably promoting their transcription. Thus, OsPIL1 is involved in the promotion of Chl biosynthesis by up-regulating the transcription of OsPORB and OsCAO1 via trifurcate feed-forward regulatory loops involving two OsGLKs.

Keywords: Chlorophyll biosynthesis; OsCAO1; OsGLK; OsPIL1; OsPORB; rice; transcriptional regulation.

Fig. 1.

Pale-green leaf phenotype of ospil1 mutants grown in a paddy field. (A) Gene structure and T-DNA insertion site (inverted triangle) in the 1000-bp upstream region of OsPIL1 (PFG_4A-03590.R). (B) Decrease in OsPIL1 transcript levels in the ospil1 mutant confirmed by RT-PCR. UBQ5 was used for an internal control. (C, D) Color difference in whole plants (C) and the first leaf of the main culm (D) between WT and ospil1 at 70 d after sowing (DAS). (E, F) Reduced levels of total Chl (E) and Car (F) in ospil1. The first leaves of the main culm at 50 and 90 DAS were used for analysis. Means and SD were obtained from 10 biological replicates. Significant differences between WT and ospil1 was determined by Student’s t-test (** P<0.01).

Fig. 2.

Agronomic traits of ospil1 plants. (A) Heading date, (B) plant height, (C) number of panicles per plant, (D) panicle length, (E) panicle phenotype, (F) number of grains per panicle, (G) 500-grain weight, and (H) fertility in WT and ospil1. Means and SD were obtained from at least 10 biological replicates. Significant differences were determined by Student’s t-test (* P<0.05, ** P<0.01).

Fig. 3.

OsPIL1 directly up-regulates OsPORB and OsCAO1 transcription. (A) Relative expression (ospil1/WT) of Chl biosynthetic genes. Relative expression levels of genes in ospil1 were normalized to those of the WT. Asterisks indicate significant difference between WT and ospil1 plants (* P<0.05, ** P<0.01). (B) The positions of G-boxes in the promoters of OsPORA, OsPORB, and OsCAO1 (–2000 bp to ATG) and the promoter fragments used for the yeast one-hybrid assay (Y1H), transactivation assays (blue horizontal lines), and ChIP assays (green horizontal lines). (C) The binding activity of OsPIL1 to the promoter regions of OsPORA (pOsPORA-a and pOsPORA-b), OsPORB (pOsPORB-a and pOsPORB-b), and OsCAO1 (pOsCAO1-a and pOsCAO1-b) examined by Y1H assays. Empty bait and prey plasmids (-) were used for the negative controls. The relative β-galactosidase activity was obtained by normalizing to the level of each negative control. Means and SD were obtained from more than five independent colonies. (D) OsPIL1 binding affinity to the promoter regions of OsPORA, OsPORB, and OsCAO1 in planta examined by ChIP assays. OsPIL1-GFP was transiently expressed in protoplasts isolated from 10-d-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with an anti-GFP antibody (see Methods). PP2A was used as a negative control. (E) Transactivation of OsPORB and OsCAO1 by OsPIL1. The protoplasts were co-transfected with 5 μl of effector plasmid containing 35S:OsPIL1-GFP and 3 μl of reporter plasmids containing pOsPORB-a::GUS, pOsPORB-b::GUS, and pOsCAO1-b::GUS. Empty vector was used as a vector control for the effector. Significant differences were determined by Student’s t-test (* P<0.05, ** P<0.01, *** P<0.001, NS, not significant).

Fig. 4.

OsPIL1 directly promotes the expression of OsGLK1 and OsGLK2. (A, B) Relative expression levels (ospil1/WT) of photosynthetic apparatus genes (A) and two GLK genes (B). Relative expression levels of genes in ospil1 are normalized to those of the WT. Asterisks indicate significant difference between the WT and ospil1 plants (Student’s t-test, * P<0.05, ** P<0.01). (C) Positions of the G-box (red vertical bar) and E-box (purple vertical bar) in the OsGLK1 and OsGLK2 promoters and the promoter fragments (green horizontal bars) used for ChIP and transactivation assays. (D) The binding affinity of OsPIL1 to the promoter regions of OsGLK1 and OsGLK2 in planta examined by ChIP assay. OsPIL1-GFP was transiently expressed in protoplasts isolated from 10-d-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with anti-GFP antibody (see Methods). PP2A was used as a negative control. (E) Transactivation of OsGLK1 and OsGLK2 by OsPIL1. The protoplasts were co-transfected with 5 μl of effector plasmid containing 35S:OsPIL1-GFP and 3 μl of reporter plasmids containing pOsGLK1-a::GUS, pOsGLK1-b::GUS, and pOsGLK2-a::GUS, and pOsGLK2-b::GUS. Empty vector was used as a vector control for the effector. Significant differences were determined by Student’s t-test (* P<0.05, ** P<0.01, NS, not significant).

Fig. 5.

OsGLK1 and OsGLK2 also up-regulate OsPORB and OsCAO1 transcription. (A) Binding of OsGLK1 and OsGLK2 to the promoter regions of OsPORB and OsCAO1 in planta examined by ChIP assays. OsGLK1-GFP or OsGLK2-GFP was transiently expressed in protoplasts isolated from 10-d-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with an anti-GFP antibody (see Methods). PP2A was used as a negative control. (B) Transactivation of OsPORB and OsCAO1 by OsGLK1 and OsGLK2. The protoplasts were co-transfected with 5 μl of effector plasmid containing 35S:OsGLK1-GFP or 35S:OsGLK2-GFP and 3 μl of reporter plasmids containing pOsPORB-a::GUS, pOsPORB-b::GUS, pOsCAO1-a::GUS, and pOsCAO1-b::GUS. Empty vector was used as a vector control for the effector. Significant differences were determined by Student’s t-test (** P<0.01). (C) Working model of OsPIL1-mediated up-regulation of Chl biosynthetic genes. OsPIL1 directly up-regulates the expression of OsPORB and OsCAO1 by forming trifurcate feed-forward loops involving OsGLK1 and OsGLK2. Arrows indicate direct up-regulation.