Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants

Abstract

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants. Previous studies have established that tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) play essential roles in plastid retrograde signaling during early chloroplast biogenesis; however, their functional relationship remains unknown. In this study, we generated a series of rice TPB-related gun (genome uncoupled) mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon (NF; a noncompetitive inhibitor of carotenoid biosynthesis) and/or lincomycin (Lin; a specific inhibitor of plastid translation). We show that under NF treatment, expression of plastid-encoded polymerase (PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants. We further demonstrate that the derepressed expression of PEP-transcribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome. In addition, we show that expression of photosynthesis-associated nuclear genes (PhANGs) and PEP-transcribed genes is correlated in the rice TPB-related gun mutants, with or without NF or Lin treatment. A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants. Moreover, we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants. Further, we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling. Taken together, our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

Keywords: plastid gene expression; plastid retrograde signal; tetrapyrrole biosynthesis.

Figure 1

Ultrastructure of chloroplasts and expression levels of photosynthesis-associated nuclear genes (PhANGs) in the wild type and osgun4 and osgun5 mutants under NF or Lin treatment. (A–I) Transmission electron microscopy images showing the ultrastructure of chloroplasts of the wild type (A, D, and G) and osgun4(B, E, and H) and osgun5(C, F, and I) mutants under normal conditions (A–C), NF treatment (D–F), or Lin treatment (G–I) (scale bars: 0.5 μm). The white arrow shows the vesicle-like structures in osgun5; the black arrow shows the vesicle-like structures in the wild type, osgun4, and osgun5 under NF treatment; and the red arrow shows the vesicle-like structures in the wild type, osgun4, and osgun5 under Lin treatment. (J and K) qRT–PCR assay showing the relative expression of several representative PhANGs (Lhca1, Lhcb5, Lhcb1.2, and rbcS) in the osgun4(J) and osgun5(K) mutants under NF or Lin treatment. Values are means ± SD of three replicates. Two asterisks (∗∗) mark statistically significant differences from the wild type with the same treatment (t test, p ≤ 0.01). UBIQUITIN was used as an internal control.

Figure 2

Expression levels of chloroplast-encoded genes in osgun4 and osgun5 mutants under NF or Lin treatment. (A and B) qRT–PCR assay showing the relative expression of several representative PEP-transcribed genes (psaA, psaB, psbA, and rbcL) in the osgun4(A) and osgun5(B) mutants under NF or Lin treatment. Values are means ± SD of three replicates. Two asterisks (∗∗) mark statistically significant differences from the wild type with the same treatment (t test, p ≤ 0.01). UBIQUITIN was used as an internal control. (C and D) qRT–PCR assay showing the relative expression of several representative NEP-transcribed genes (rpoA, rpoB, rpoC1, and rpoC2) in the osgun4(D) and osgun5(E) mutants under NF or Lin treatment. Values are means ± SD of three replicates. Two asterisks (∗∗) mark statistically significant differences from the wild type with the same treatment (t test, p ≤ 0.01). UBIQUITIN was used as an internal control.

Figure 3

Expression levels of chloroplast-encoded genes in the Arabidopsis gun4 and gun5 mutants under NF or Lin treatment. (A and B) qRT–PCR assay showing the relative expression of several representative PEP-transcribed genes (psbA, psbD, psbN, psaJ, and rbcL) and NEP-transcribed genes (clpP, rpoB, and rpoC1) in the Arabidopsis gun4(A) and gun5(B) mutants under NF or Lin treatment. Values are means ± SD of three replicates. Two asterisks (∗∗) mark statistically significant differences from the wild type with the same treatment (t test, p ≤ 0.01). ACTIN was used as an internal control.

Figure 4

Expression levels of chloroplast-associated genes (LHCB1.2, psbD, and rpoB) in the Arabidopsis gun1, gun4, gun5, gun1/4, and gun1/5 mutants under NF or Lin treatment, and a unifying model for the regulation of plastid retrograde signaling by TPB-related GUN genes. (A–F) qRT–PCR assay showing the relative expression of LHCB1.2(A and D), psbD(B and E), and rpoB(C and F) in the Arabidopsis gun1, gun4, gun5, gun1/4, and gun1/5 mutants under NF treatment (A–C) or Lin treatment (D–F). Values are means ± SD of three replicates. Two asterisks (∗∗) mark statistically significant differences from the wild type with the same treatment (t test, p ≤ 0.01). ACTIN was used as an internal control. (G) A model of plastid retrograde signaling in the wild type and the TPB-related gun mutants with NF or Lin treatment. Upon NF treatment, PEP activity and PGE were inhibited in the wild type, and there was thus no gun phenotype. In the TPB-related gun mutants under NF treatment, elevated expression of NEP, NEP-transcribed genes, and Sigs and increased copy number of the plastid genome haploid genome resulted in no inhibition of PGE, which led to production of an unknown signal and the gun phenotype via the PGE pathway. Upon Lin treatment, translation of PEP core subunits is inhibited, as is PGE. No signal is generated in either the wild type or the TPB-related gun mutants, and there is thus no gun phenotype. GUN1 acts downstream of the TPB-related GUN genes and participates in PGE-related retrograde signaling.