GhCTSF1, a short PPR protein with a conserved role in chloroplast development and photosynthesis, participates in intron splicing of rpoC1 and ycf3-2 transcripts in cotton

Abstract

Cotton is one of the most important textile fibers worldwide. As crucial agronomic traits, leaves play an essential role in the growth, disease resistance, fiber quality, and yield of cotton plants. Pentatricopeptide repeat (PPR) proteins are a large family of nuclear-encoded proteins involved in organellar or nuclear RNA metabolism. Using a virus-induced gene silencing assay, we found that cotton plants displayed variegated yellow leaf phenotypes with decreased chlorophyll content when expression of the PPR gene GhCTSF1 was silenced. GhCTSF1 encodes a chloroplast-localized protein that contains only two PPR motifs. Disruption of GhCTSF1 substantially reduces the splicing efficiency of rpoC1 intron 1 and ycf3 intron 2. Loss of function of the GhCTSF1 ortholog EMB1417 causes splicing defects in rpoC1 and ycf3-2, leading to impaired chloroplast structure and decreased photosynthetic rates in Arabidopsis. We also found that GhCTSF1 interacts with two splicing factors, GhCRS2 and GhWTF1. Defects in GhCRS2 and GhWTF1 severely affect intron splicing of rpoC1 and ycf3-2 in cotton, leading to defects in chloroplast development and a reduction in photosynthesis. Our results suggest that GhCTSF1 is specifically required for splicing rpoC1 and ycf3-2 in cooperation with GhCRS2 and GhWTF1.

Keywords: PPR protein; RNA splicing; chloroplast; cotton; photosynthesis.

Figure 1

Phenotypic characterization of GhCTSF1-silenced plants. (A) The leaf phenotypes of 2-month-old empty-vector control plants (CLCrV-A) and GhCTSF1-silenced plants (GhCTSF1-RNAi). Scale bars, 1 cm. (B) Quantification of the expression level of GhCTSF1 in CLCrV-A and GhCTSF1-RNAi plants. Error bars represent mean ± SD. Asterisks indicate significant differences (∗∗∗P < 0.001) by Student’s t-test. (C) Chlorophyll a and b contents of leaves from CLCrV-A and GhCTSF1-RNAi plants. Error bars indicate mean ± SD (n = 3), and asterisks indicate statistically significant differences by Student’s t-test: ∗∗P < 0.01 and ∗∗∗P < 0.001. (D) Chloroplast ultrastructure in true leaves from 2-month-old plants. Scale bars, 1 μm. The chloroplasts of GhCTSF1-RNAi plants were from the albino sector of the leaf. SG, starch grain; Thy, thylakoid; Gr, granum; PG, plastoglobule. (E–G) The quantum yield of PSI (E), electron transport rate of PSI (F), and non-photochemical quenching (G) of CLCrV-A and GhCTSF1-RNAi-2 plants. Data in (E)–(G) are shown as means ± SD of three individual replicates.

Figure 2

Domain structure and subcellular localization of GhCTSF1. (A) Schematic diagram of the functional domains of GhCTSF1. cTP, chloroplast transit peptide predicted by TargetP. (B) Quantitative RT–PCR (RT–qPCR) analysis of relative GhCTSF1 transcript levels in various tissues and fibers at different stages. The cotton UBQ7 gene was used as an internal control. Values are means ± SD (n = 3). (C) Subcellular localization of the GhCTSF1–GFP fusion protein in N. benthamiana leaf epidermal cells. GhCTSF1–GFP exhibits a chloroplast localization pattern, whereas free GFP is cytosolic and nuclear. GFP, green fluorescent protein; Chlorophyll, chlorophyll autofluorescence. (D) The GhCTSF1–mCherry construct co-localized with WTF1–GFP. Protoplasts were obtained from N. benthamiana leaves expressing GhCTSF1–mCherry and WTF1–GFP/PEND–GFP/PGL34–GFP/PIC1–GFP fusion proteins. Scale bar, 10 μm.

Figure 3

Silencing of GhCTSF1 expression results in impaired intron splicing of ycf3-2 and rpoC1. (A) Log2 average fold change of read counts across the complete plastid genome. (B) Chloroplast transcript levels in GhCTSF1-silenced plants assessed by organelle RNA-seq. The values are presented as log2 ratios of chloroplast gene expression in GhCTSF1-silenced plants (GhCTSF1-RNAi) relative to that in empty-vector control plants (CLCrV-A). Three independent biological replicates were analyzed. Error bars represent means ± SD. (C) RT–qPCR was performed to analyze transcript levels of ycf3 and rpoC1. Ubiquitin was used as an internal control. Representative results from three biological replicates are shown. Error bars indicate mean ± SD. Asterisks indicate statistically significant differences by Student’s t-test: ∗∗∗P < 0.001. (D) RT–PCR analysis of the intron splicing of ycf3-2 and rpoC1 in GhCTSF1-RNAi and CLCrV-A plants. U, unspliced transcripts; S, spliced transcripts. All PCR products were confirmed by sequencing. (E) RT–qPCR analysis of splicing efficiency of ycf3-2 and rpoC1. Primers spanned adjacent exons and introns to measure differences in splicing efficiency. U, unspliced transcripts; S, spliced transcripts. Error bars indicate mean ± SD. (F) Expression levels of PEP-dependent chloroplast genes. The values are presented as log2 ratios of gene expression in GhCTSF1-silenced plants (GhCTSF1-RNAi) relative to that in empty-vector control plants (CLCrV-A). Data are shown as means ± SD.

Figure 4

Mutation of EMB1417 results in disrupted chloroplast development and impaired RNA splicing of ycf3-2 and rpoC1. (A) A. thaliana EMB1417 is a homolog of GhCTSF1 in G. hirsutum. The phylogenetic tree was constructed with MEGA v.5.2 (https://www.megasoftware.net). Numbers are percentage bootstrap values for 1000 replicates. (B) The cotyledon phenotypes of three EMB1417 mutant lines. Pictures were scored using 5-day-old seedlings grown on half-strength MS plates at 22°C. Scale bar, 1 mm. (C) Sequencing results for the three EMB1417 mutant lines. (D) Phenotype of the EMB1417-KO3 mutant. Pictures were scored at 10, 20, and 40 days. Scale bar, 2.5 mm. CD, cotyledon; FL, first leaf; TL, fourth leaf. (E) Chlorophyll a and b contents of EMB1417-KO3 plants. Error bars indicate mean ± SD (n = 3), and asterisks indicate statistically significant differences by Student’s t-test: ∗∗∗P < 0.001. (F) RT–PCR analysis of the intron splicing of ycf3-2 and rpoC1 in EMB1417-KO3 plants. U, unspliced transcripts; S, spliced transcripts. clpP-1 was used as a control. All PCR products were confirmed by sequencing. (G) Analysis of the splicing efficiency of ycf3-2 and rpoC1 in the EMB1417-KO3 mutant assessed by RT–qPCR. Error bars represent ± SD of three biological replicates. (H) Expression levels of PEP-dependent chloroplast genes. The values are presented as log2 ratios of gene expression in EMB1417-KO3 plants relative to that in wild-type (WT) plants. Data are shown as means ± SD. (I–K) The quantum yield of PSI (I), electron transport rate of PSI (J), and non-photochemical quenching (K) of EMB1417-KO3 plants. Data in (F)–(H) are shown as means ± SD of three individual replicates.

Figure 5

GhCTSF1 interacts with GhCRS2 and GhWTF1. (A) Yeast two-hybrid assay showing that GhCTSF1 interacts with GhCAF2, GhCRS1, GhCRS2, GhCFM2, GhWTF1, GhOTP51, and GhCTSF1 itself. Full-length GhCTSF1 was fused with the DNA-binding domain in pGBKT7, and the potential interactors were separately fused with the activation domain (AD) in pGADT7. Transformed yeast was grown on SD/-Trp-Leu (SD-TL) and SD/-Trp-Leu-His-Ade (SD-TLHA) dropout plates to test protein interactions. The empty pGADT7 vector was co-transformed with GhCTSF1 as a negative control. (B) Bimolecular fluorescence complementation assay showing that GhCTSF1 interacts with GhWTF1, GhCRS2, and GhOTP51 in N. benthamiana leaf epidermal cells. GhCTSF1 was fused to the C-terminal fragment of YFP (cYFP), and GhWTF1, GhCRS2, or GhOTP51 was fused to the N-terminal fragment of YFP (nYFP). Chlorophyll autofluorescence (chlorophyll) was used to reveal the chloroplasts. Bright, bright-field image under transmitted light; Merge, merged image of YFP, chlorophyll, and Bright. Excitation/emission wavelengths were 510/530 nm for YFP and 488/670 nm for chlorophyll autofluorescence. Scale bar, 50 μm. (C) Co-IP analysis shows the interaction between GhCTSF1 and GhWTF1/GhCRS2 using a transient expression system in tobacco leaves. IP was performed using an anti-Myc matrix, and the co-immunoprecipitated proteins were detected with anti-HA antibodies. This assay was repeated three times with similar results. (D) MBP-pull-down assay demonstrating the interaction between GhCTSF1 proteins and GhWTF1/GhCRS2.

Figure 6

Loss of GhWTF1 and GhCRS2 will greatly delay chloroplast development and impair photosynthesis in cotton. (A and B) KO of GhWTF1 and GhCRS2 was verified by gold-standard Sanger sequencing. (C and D) Cotyledon and leaf phenotypes of GhWTF1 and GhCRS2 KO plants. Pictures of cotyledons and leaves were scored at 10 and 60 days, respectively. Scale bars, 2.5 cm in (C) and 5 cm in (D). (E) Chlorophyll a and b contents of leaves from GhWTF1 and GhCRS2 KO plants. Error bars indicate mean ± SD (n = 3), and asterisks indicate statistically significant differences by Student’s t-test: ∗∗∗P < 0.001. (F) Chloroplast ultrastructure in true leaves from 60-day-old plants. Chloroplasts of GhWTF1 and GhCRS2 KO plants were obtained from the albino sector of the leaf. SG, starch grain; Thy, thylakoid; Gr, granum; PG, plastoglobule. (G–I) The quantum yield of PSI (G), electron transport rate of PSI (H), and non-photochemical quenching (I) of GhWTF1 and GhCRS2 KO plants. Data in (G)–(I) are shown as means ± SD of three individual replicates. (J) RT–qPCR analysis of splicing efficiency of ycf3-2 and rpoC1 in GhWTF1 and GhCRS2 KO plants. The values are presented as log2 ratios of gene expression in GhWTF1 and GhCRS2 KO plants relative to that in WT plants. Data are shown as means ± SD. (K) Expression levels of PEP-dependent chloroplast genes.

Figure 7

GhWTF1 associates with ycf3 and rpoC1 transcripts. (A) Immunoblots showing the WTF1–HA protein in cotton cotyledons. (B–D) RIP–qPCR assay to analyze the region of WTF1 binding to clpP, rpoC1, and ycf3. Top, diagram depicting the structures of clpP, ycf3, and rpoC1 transcripts. The locations of regions used for RIP–qPCR analysis are indicated by black lines. Bottom, relative enrichment of each region in clpP, rpoC1, and ycf3. GFP-HA was used as the control. Data are means ± SD, n = 3; ∗∗∗P < 0.001. (E) Gel-mobility shift assays (EMSAs) demonstrating the binding activity of recombinant GhWTF1 to rpoC1 and ycf3-2. (F) A proposed working model for the involvement of CTSF1-mediated RNA splicing in chloroplast development and photosynthesis. The nuclear-encoded PPR protein CTSF1 is imported into plastids and interacts with CRS2 and WTF1 to form part of the plastid RNA splicing complex. The splicing complex then associates with the pre-mRNA and promotes the intron splicing of rpoC1 and ycf3-2. As a result, the spliced plastid rpoC1 transcripts maintain high activity of PEP, which plays an essential role in chloroplast development by activating expression of photosynthesis-associated plastid-encoded genes (PhAPGs). Meanwhile, the spliced ycf3 transcript promotes assembly of the PSI complex, which in turn functions in photosynthesis.