ZmFdC2 Encoding a Ferredoxin Protein With C-Terminus Extension Is Indispensable for Maize Growth

Abstract

As important electron carriers, ferredoxin (Fd) proteins play important roles in photosynthesis, and the assimilation of CO₂, nitrate, sulfate, and other metabolites. In addition to the well-studied Fds, plant genome encodes two Fd-like protein members named FdC1 and FdC2, which have extension regions at the C-terminus of the 2Fe-2S cluster. Mutation or overexpression of FdC genes caused alterations in photosynthetic electron transfer rate in rice and Arabidopsis. Maize genome contains one copy of each FdC gene. However, the functions of these genes have not been reported. In this study, we identified the ZmFdC2 gene by forward genetics approach. Mutation of this gene causes impaired photosynthetic electron transport and collapsed chloroplasts. The mutant plant is seedling-lethal, indicating the indispensable function of ZmFdC2 gene in maize development. The ZmFdC2 gene is specifically expressed in photosynthetic tissues and induced by light treatment, and the encoded protein is localized on chloroplast, implying its specialized function in photosynthesis. Furthermore, ZmFdC2 expression was detected in both mesophyll cells and bundle sheath cells, the two cell types specialized for C4 and C3 photosynthesis pathways in maize. Epigenomic analyses showed that ZmFdC2 locus was enriched for active histone modifications. Our results demonstrate that ZmFdC2 is a key component of the photosynthesis pathway and is crucial for the development of maize.

Keywords: ZmFdC2; electron transfer; growth; maize; photosynthesis.

Figure 1

pas1 is defective in plant growth and chloroplast development. (A), Phenotype of wild-type and pas1 mutant at the 3-leaf stage. Bar = 3 cm. (B), Phenotype of the wild-type and pas1 mutant at the 4-leaf stage. Bar = 3 cm. (C), The chlorophyll contents of the wild-type and pas1 mutant. (D–I), Transmission electron microscopy for chloroplast structure of the wild-type and pas1. (D–F), HZM; (G–I), pas1.

Figure 2

Map based cloning of ZmFdC2 gene and the alignment of FdC2 proteins from various species. (A), ZmFdC2 gene was roughly mapped between marker M1 and M2. (B), The ZmFdC2 gene was further localized to a 104 kb region between M4 and M5. (C), Three candidate genes were annotated in the mapped region. (D), The diagram representation of the ZmFdC2 gene structure. (E), The C to T mutation in ZmFdC2 gene led to an extra stop codon. (F), Sequence alignment of FdC2 proteins from various species. The solid and dashed lines under the letters indicate the 2Fer-2S cluster and the C-terminus extensions, respectively. The amino acids in yellow rectangles denote the conserved –CxxxxCxxCxn–C– motif, and the asterisk indicates the mutated position in ZmFdC2. (G), Phylogenetic tree of all FdC2 homologs from selected species.

Figure 3

Chloroplast localization of ZmFdC2 and photosynthetic electron transport parameters of HZM and pas1 mutant. (A). Subcellular localization of ZmFdC2-GFP. ZmFdC2-GFP plasmid was transformed into maize protoplast, and fluorescence was detected with confocal microscopy. Left, ZmFdC2-GFP; middle, chloroplast autofluorescence; right, overlay of GFP and chloroplast signals. (B), Photosynthetic electron transport parameters of HZM and pas1 mutant. Chlorophyll fluorescence parameters for PSII capacity (ϕII), the photosynthetic electron transfer rate (ETR), photochemical quenching (qP), and non-photochemical quenching (NPQ) were determined. Values are mean ± S.D. of at least six independent measurements. Significances were determined with Student's t-test. “^*” indicates p < 0.05 and “^**” indicates p < 0.01.

Figure 4

ZmFdC2 interacted with PET proteins of maize. ZmFdI, ZmFdII, ZmFdC2 and ZmFdC2m were constructed into yeast two-hybrid bait plasmid (BD), and other maize PET genes were constructed into prey plasmid (AD). Yeast transformants were selected on DDO (SD/-Leu/-Trp) agar medium and the protein interactions were tested on TDO (SD/-Leu/-Trp/-His) and QDO (SD/-Leu/-Trp/-His/-Ade) agar medium.

Figure 5

Tissue specificity and light induction of ZmFdC2 expression. (A), Real-time PCR of ZmFdC2 in different maize tissues. All expression values in different tissues were normalized to that of root. (B), Expression of different genes in mesophyll cells and bundle sheath cells of maize leaves. The expression of each gene in two cell types was normalized to its expression in the total RNA. PEPC and ZmFdI served as positive control for mesophylls; PCK and ZmFdII served as positive control for bundle sheath cells. (C), Real-time PCR of ZmFdC2 before and after light treatment. One-week-old seedlings grown in the dark were transferred to light and the youngest leaves were collected at different time points for RNA analyses. All expression values were normalized to that of 0 h of light treatment. (D), Real-time PCR of ZmFdC2 before and after nitrate addition. Two-week-old seedlings grown under low nitrate concentration (1.6 mM KNO₃) were treated with 16 mM KNO₃, and the youngest leaves were harvested at different time points for RNA analyses. All expression values were normalized to 0 h of nitrate treatment. All experiments were conducted for three biological replicates and results were presented as mean ± SD. Maize Actin gene served as internal control in all experiments except for (B).

Figure 6

Differentially expressed genes in pas1 mutant. The youngest leaves of seedlings at three-leaf stage were collected for the analyses. (A), GO analysis of the DEGs from RNA-seq data between the wild-type and pas1 mutant. RNA-seqs were performed for three biological replicates, and DEGs were defined as fold change > 2 and p < 0.05. (B,C), expression validation of some DEGs from RNA-seq data. The selected DEGs included chloroplast-coding (B) and nuclear-coding (C) genes. The expression of each gene was normalized to HZM and maize Actin gene served as internal control. “^**” indicates p < 0.01.

Figure 7

Epigenetic modifications associated with ZmFdC2 locus. (A), Reads of DNA methylation, mRNA and small RNA on ZmFdC2 locus. (B), Enrichments of four histone modifications on ZmFdC2 locus. RNA-seq and all epigenetic data were generated from the same samples and downloaded from the publication (Wang et al., 2009).