A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice

Abstract

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

Keywords: chlorophyll biosynthesis; geranylgeranyl diphosphate synthase; protein complex formation; rice; terpenoids.

Fig. 1.

Subcellular localization and phylogenetic analysis of rice candidate GGPPSs. (A) Transient expression of four rice PTS homologs fused to EYFP in rice leaf protoplasts showing plastidic localization. Os07g39270 (OsGGPPS1) exhibits two patterns. (Upper) Representative of 77% protoplasts. (Middle) Representative of 23% protoplasts. Chl, chlorophyll autofluorescence; cTP, chloroplast transit peptide of Os07g39270 (OsGGPPS1) fused to EYFP. (Scale bars, 5 μm.) (B) Phylogenetic analysis of 12 rice PTS homologs with AtGGPPS11 and AtSSU II. Rice plastidic PTSs are shown in bold. (C) Immunoblot analysis of Os07g39270 (OsGGPPS1) and Os02g44780 (OsGRP) subchloroplast localization. Env, envelope membranes; Str, stroma; TM, thylakoid membrane. Tic110 and RbcL were used as markers for the envelope and stroma fractions, respectively. OsGGR, rice geranylgeranyl reductase; OsLIL3, rice light-harvesting-like protein 3.

Fig. 2.

Functional characterization of four rice plastidic PTS homologs. (A) Diagram showing the carotenoid biosynthesis pathway in Erwinia herbicola. Plasmid pAC-94N contains the E. herbicola carotenoid biosynthetic genes, crtB, crtI, and crtY, with the exception of crtE. When coexpressed with a functional GGPPS, the pathway is complemented and β-carotene is produced. (B) Complementation assay for detecting GGPPS activity of the rice PTS homologs that have chloroplast localization. Functionally characterized GGPPS from Antirrhinum majus (AmLSU) and pET32b empty vector were used as positive and negative controls, respectively. Data are means ± SEM (n = 3 biological replicates). **P < 0.01; Student’s t test. (C) LC-MS/MS chromatograms showing the products generated by Os01g14630 (OsGPPS), Os07g39270 (OsGGPPS1), Os02g44780 (OsGRP), and a mixture of Os07g39270 and Os02g44780 (1:1) from IPP and DMAPP in vitro, as well as GPP, FPP, and GGPP standards (1 μM each).

Fig. 3.

Analysis of OsGGPPS1 and Os02g44780 interactions. (A) Y2H detection of protein–protein interactions. Yeast cells harboring both constructs (Left) were spotted on nonselective (−LW) and selective medium supplied with X-α-Gal (−LWAH+X-α-Gal). Interaction strength was measured by β-galactosidase activity. AD, activation domain of pGAD-T7; and BD, DNA binding domain of pGBK-T7, to which OsGGPPS1 and Os02g44780 were fused; EV, empty vector. Known interaction between Arabidopsis AtGGPPS11 and AtSSU II was used as a positive control. Data are means ± SEM (n = 3). (B) Pull-down analysis of OsGGPPS1 binding to His-tagged Os02g44780. (C) BiFC detection of protein–protein interactions in rice leaf protoplasts. Coexpressed fusion constructs are indicated. Fluorescence of reconstructed EYFP is shown in the “EYFP” panel. Chl, chlorophyll autofluorescence. (Scale bars, 5 μm.)

Fig. 4.

Interactions among OsGGPPS1/OsGRP heterodimer, OsGGR, and OsLIL3. (A) Y2H assay of OsGGPPS1 and OsGGR interaction. OsGGPPS1 and OsGGR were fused with the DNA binding domain (BD) of pGBK-T7 and the activation domain (AD) of pGAD-T7, respectively. Tenfold serial dilutions of yeast cells expressing both fusion proteins were spotted on nonselective (−LW) medium and selective medium supplied with X-α-Gal (−LWAH+X-α-Gal). (B and C) BiFC detection of the interactions of OsGGPPS1 (B) or OsGRP (C) with OsGGR and OsLIL3. (D) BiFC detection of the interaction between OsLIL3 and OsGGR. (E and F) BiFC assays of the colocalization of OsGGPPS1 homodimer with OsLIL3 (E) and OsGGR (F). (G and H) BiFC assays of the colocalization of OsGGPPS1/OsGRP heterodimer with OsLIL3 (G) and OsGGR (H). (Scale bars, 5 μm.)

Fig. 5.

Effect of down- and up-regulation of OsGGPPS1 and OsGRP expression on GGPP-derived terpenoids in rice. (A–D) Relative OsGGPPS1 (A and B) and OsGRP (C and D) transcript levels. (E–H) Relative plant height. (I–L) Relative chlorophyll levels. (M–P) Relative carotenoid levels. Analyzed lines are shown at the bottom. WT (Dj), osggpps-1 and osgrp-1: wild-type, OsGGPPS1 and OsGRP T-DNA insertion lines in Oryza sativa L. cv. Donjing (Dj) background. All overexpression and RNAi lines are in Oryza sativa L. cv. Nipponbare (Np) background. OE#GG2, 5, and 6, transgenic lines overexpressing OsGGPPS1; OE#GRP1-3, transgenic lines overexpressing OsGRP; RNAi#GRP1-2, transgenic OsGRP-RNAi lines. (Q–T) Representative plants of each of the lines. In each analyzed parameter, corresponding WT level was set as 1. Data are means ± SEM (n = 3). *P < 0.05; **P < 0.01; Student’s t test. (U) OsGGPPS1 protein levels in total chloroplast preparation and thylakoid membrane (TM) and stroma (Str) fractions of the WT and OsGRP overexpressing plants.