Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and mediate gibberellin-regulated chlorophyll biosynthesis under light conditions

Abstract

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light.

Figure 1. POR is critical for SCL-regulated chlorophyll biosynthesis in light.

(A) Phenotypes of WT (Col), MIR171c-OX, scl6 scl22 scl27, 35S::LUC-rSCL27, por-amiR, por-amiR/MIR171c-OX and por-amiR/scl6 scl22 scl27 plants grown under a 16 h/8 h light/dark cycle. Bars = 1 cm. (B) Chlorophyll content of the genotypes shown in (A). FW, fresh weight. ** indicates p values (Student's t-test) <0.01 compared with WT or between the indicated two genotypes. Error bars indicate the s.d. (n = 4). (C) PSII activity (Fv/Fm) of the leaves described in (A) treated with excess light (800 µmol m⁻² s⁻¹) for the indicated times and then incubated in the dark. Error bars indicate the s.d. (n = 18). (D) qPCR analysis of HEMAs, GUN4, GUN5, PORs and CAO transcript levels using total RNA extracted from the leaves of the genotypes shown in (A). The relative expression levels were normalized to that of ACTIN2, and the relative expression in WT plants was set as 1. Error bars represent the s.d. (n = 3). Two biological replicates were performed and provided similar results.

Figure 2. SCL-GUS accumulation and chlorophyll fluorescence intensity at the early stage of leaf growth.

(A) The GUS activity of 6-day-old transgenic plants expressing pSCL27::rSCL27-GUS. Bar = 1 mm. (B) The GUS activity of 8-day-old transgenic plants expressing pSCL27::rSCL27-GUS. Bar = 1 mm. (C) Chlorophyll autofluorescence from the leaf tip and basal cells shown in (A). Bars = 20 µm. (D) Fluorescence intensity in the tip and basal cells of 6-day-old Col and scl6 scl22 scl27 seedlings. ** represents p values (Student's t-test) <0.01 relative to wild-type. Error bars indicate the s.d. (n = 18).

Figure 3. SCL27 binds to the PORC promoter.

(A) Effect of SCL27 on the activity of three PORC promoter regions. The LUC reporter gene under the control of these promoter regions was transformed into N. benthamiana leaves, with or without 6xMYC-rSCL27 or rSCL27-VP16. The relative LUC activities were normalized to a 35S::REN internal control. Error bars indicate the s.d. (n = 4). Three biological replicates provided similar results. (B) Schematic diagram of the PORC promoter and the first exon region. Fragments Ι (−1524 bp to −1324 bp), ΙΙ (−778 bp to −598 bp), ΙΙΙ (−572 bp to −372 bp) and IV (1144 bp to 1246 bp) were used for ChIP. (C) ChIP-qPCR analysis of the relative enrichment of the DNA fragments mentioned in (B). The β-TUBULIN-2 promoter was used as a reference. Error bars indicate the s.d. (n = 3). Two biological replicates were performed and showed similar results. (D) EMSA analysis of SCL27 binding to fragments I, II and III. (E) DNA sequences. W and M contain GT and mutated-GT elements indicated by capital letters, respectively. (F) SCL27 binding to GT elements was analyzed using the indicated levels of purified SCL27 protein mixed with 1 nM of Cy5-fluorescently labeled 62-bp DNA fragments. (G) The specificity of the SCL27-DNA interaction was tested using a competition assay with 0.1, 0.2 and 0.4 µM of unlabeled W or unlabeled M fragments.

Figure 4. miR171-targeted SCLs mediate DELLA-regulated POR expression in light.

(A and D) Phenotypes of the indicated plants grown under long-day conditions. Bars = 1 cm. (B, C, E and F) Chlorophyll content (B and E) and relative PORC mRNA levels (C and F) of the plants shown in (A and D). ** indicates p values (Student's t-test) <0.01 between the indicated two genotypes; error bars indicate the s.d. (n = 4). PORC expression levels were normalized to that of ACTIN2, and the level of PORC expression in Col or Ler was set as 1. Error bars indicate the s.d. (n = 3). Two biological replicates were performed and showed similar results. FW, fresh weight. (G) Domain mapping of the interaction between SCL27 and RGA in yeast. (H) BiFC analysis of the interaction between SCL27 and RGA. Bars = 50 µm. (I) Co-IP assay of the interaction between SCL27 and RGA using a transient expression assay in N. benthamiana leaves. Fusion proteins were detected by immunoblotting with anti-MYC or anti-HA antibodies. (J) Transgenic plants over-expressing 6xMYC-rSCL27 were used for co-IP. Arabidopsis proteins were detected by anti-MYC or anti-RGA antibodies. (K) Effect of DELLA binding to SCL27 on PORC promoter activity. The pPORC-1685::LUC reporter gene was transformed with 6xMYC-rSCL27 and/or RGAd17-3xHA in N. benthamiana. Relative LUC activities were normalized to the 35S::REN internal control. The LUC/REN ratio in the leaves transformed with the vector was set as 1. Error bars indicate the s.d. (n = 4). Three biological replicates showed similar results. (L) The binding of SCL27 to DNA was analyzed using EMSA in the presence of RGA. GST was used as a control. (M) in vivo analysis of the binding of SCLs to PORC promoter regions in the presence of GA₃ or PAC. Three-week-old 6xMYC-rSCL27-OX plants treated with GA or PAC for 2 days were used for ChIP and qPCR experiments. The β-TUBULIN-2 promoter was used as a reference. Error bars indicate the s.d. (n = 3). Two biological replicates showed similar results.

Figure 5. SCL27 activates MIR171 gene expression in a feedback manner.

(A) qPCR analysis of MIR171a, MIR171b and MIR171c expression in Col, scl6 scl22 scl27 and LUC-rSCL27-OX plants. Relative expression levels of MIR171 genes were normalized to that of ACTIN2, and the relative expression in WT plants was set as 1. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (B) 35S::6xMYC-rSCL27-GR/scl6 scl22 scl27 transgenic plants were treated with DEX (10 µM) or untreated (Mock) for 20 days. Bars = 1 cm. (C) Chlorophyll content of the plants shown in (B). ** indicates p value (Student's t-test) <0.01; Error bars indicate the s.d. (n = 4). (D) Relative expression of PORC, MIR171a, MIR171b and MIR171c in the plants shown in (B). Expression levels were normalized to that of ACTIN2. Expression levels in plants without DEX were set as 1 for each gene. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (E) Relative activity of the MIR171a promoter. pMIR171a::LUC was transformed into N. benthamiana leaves with or without co-transformation of 6xMYC-rSCL27. Relative LUC activities were normalized to the 35S::REN internal control. Error bars indicate the s.d. (n = 4). Three biological replicates showed similar results. (F) Schematic diagram of MIR171a promoter regions V (−726 bp to −495 bp), VΙ (−260 bp to −71 bp) and VΙΙ (the precursor of MIR171a), which were used for ChIP experiments. (G) Relative enrichment of MIR171a promoter fragments in the immuno-precipitates. Leaves of 3-week-old Col and 6xMYC-rSCL27-OX plants were used for ChIP experiments. The enriched DNA fragments were quantified using qPCR. The β-TUBULIN-2 promoter was used as a reference. Error bars indicate the s.d. (n = 3). Similar results were obtained from three independent immuno-precipitation experiments.

Figure 6. A working model of GA-regulated chlorophyll biosynthesis under the light condition.

In light, GA biosynthesis is inhibited, resulting in an increase in DELLAs. Accumulated DELLAs bind to SCLs and subsequently terminate the SCL suppression of PORC expression. On the other hand, light promotes miR171 expression, leading to a decrease in SCL expression. Thus, the inhibition of SCLs on PORC expression is maximally relieved in light. miR171 and its target SCLs form a feedback regulatory loop that maintains the light-dependent diurnal oscillation of miR171 and SCL expression. Arrows indicate verified positive regulation and the chlorophyll biosynthesis pathway; bars indicate verified negative regulation.