GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Abstract

Background: The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown.

Results: In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton.

Conclusions: Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

Keywords: Chloroplast; Cotton; Leaf variegation; PPR.

Fig. 1

Phylogenetic and sequence analysis of GhYGL1d and its expression pattern. a Computational prediction for identification of leaf-related RNA editing factors in cotton. b Schematic structures of GhYGL1d proteins. Predicted targeting peptide, P, E, E+ and DYW domains are labeled on the protein sequence. The targeting peptide was predicted using the TargetP software (www.cbs.dtu.dk/services/TargetP/). The PPR motifs and domains were predicted by TPRpred software (https://toolkit.tuebingen. mpg.de/#/tools/tprpred). c Phylogenetic tree analysis of GhYGL1d proteins in plants were performed using the MEGA program (www.megasoftware.net). The phylogenetic tree was generated by MEGA5.0. d YGL1 protein in Gossypium raimondii (XP_012456442), Gossypium arboreum (XP_017631921), Theobroma cacao (XP_017979491.1), Arabidopsis thaliana (NP_173004.1), Vitis vinifera (XP_002285225.2), Glycine max (XP_003529817.2), Oryza sativa (XP_015639788.1), Zea mays (XP_008650050.1), Sorghum bicolor (XP_002440297.2), Physcomitrella patens (XP_001780298.1), Selaginella moellendorffii (XP_002964227.1), Ostreococcus tauri (XP_003078586.1), and Chlorella variabilis (XP_005844014.1) were selected to generate a bootstrap neighbor–joining phylogenetic unrooted tree. The numbers at each node represent the bootstrap values (%) calculated from 1,000 trials. The length of branches indicates the extent of divergence according to the bar scale (relative units) at the bottom.

Fig. 2

Expression and subcellular localization of GhYGL1d. a qRT-PCR analysis of GhYGL1d expression in different tissues. Total RNA was isolated from roots, stems, leaves, flowers and fibers. The GhUBQ7 gene was used as a reference gene for qRT-PCR. DPA, day post-anthesis. The values shown are means ± SE of three replicates. b GhYGL1d localizes to the chloroplast. Protoplasts from wild-type Arabidopsis were transiently transformed with a control GFP vector (designated 35S::GFP) or with a GhYGL1d-GFP vector. Fluorescence was observed by confocal microscopy of single protoplasts; green fluorescence = GFP; red = chlorophyll autofluorescence. Bars = 10 μm. c GhYGL1d localizes to the stroma and thylakoid fractions of chloroplasts. Total proteins extracted from the 35S::GhYGL1d-GFP transgenic line were used to confirm the specificity of the anti-GFP antibody. Intact chloroplasts were isolated from 35S::GhYGL1d-GFP transgenic seedlings and separated into the thylakoid and stroma fractions. Chloroplasts protein Cyt f and RbcL were used as a marker of thylakoid membrane and stroma fractions, respectively. The GFP antibody was used to detect the GhYGL1d-GFP fusion protein.

Fig. 3

GhYGL1d partially restored the function of AtECB2 in Arabidopsis. (A, B) Phenotypes of a wild-type (Col-0) plant, an atecb2 mutant and GhYGL1d complemented plants at two (a), and six (b) weeks after sowing. Scale bars are 2 cm. (C) Chlorophyll contents in a wild-type (Col-0) plant, an atecb2 mutant and GhYGL1d complemented plants. FW, fresh weight. Chl a, chlorophyll a. Chl b, chlorophyll b. Error bars indicate SD for three biological replicates. Asterisks indicate significant differences (P < 0.001) from Col-0 plants. (D) RNA editing of accD, ndhF transcripts in wild-type (Col-0) plants, atecb2 mutants and GhYGL1d complemented plants. 1# and 2# indicate GhYGL1d complemented lines.

Fig. 4

Silencing of GhYGL1d showed variegated leaves. a Cotton plants infiltrated with CLCrVA:GhYGL1d (GhYGL1d-RNAi) showed variegated leaves. The photographs were taken at approximately five weeks after infiltration. Wildtype plants transformed by an empty CLCrVA vector was used as the control. Scale bars are 2 cm. b qRT-PCR analysis of GhYGL1d transcripts in RNAi plants. The GhUBQ7 gene was used as a reference. The values shown are means ± SE of three biological replicates. Significant differences between RNAi plants and CLCrVA control plants were calculated using Student’s t-test: ***, P < 0.001; (C) Chlorophyll contents in gene-silenced plants. FW, fresh weight. Chl a, chlorophyll a. Chl b, chlorophyll b. Values are means ± SD of three replicates. Student’s t-test: ***, P < 0.001;

Fig. 5

Loss of GhYGL1d expression affects chloroplast development. a-h Chlorophyll fluorescence and morphology of the protoplasts isolated from the CLCrVA (a, e), GhYGL1d-RNAi-1(b, f), GhYGL1d-RNAi-2 (c, g) and GhYGL1d-RNAi-3 (d, h) plant leaves. (i-l) Transmission electron micrographs of plastid ultrastructures in the CLCrVA (I), GhYGL1d-RNAi-1(J), GhYGL1d-RNAi-2 (K) and GhYGL1d-RNAi-3 (L) plants. Plastids were from leaves of six-week-old plants. Three biological replicates were performed, and similar results were obtained. SG, starch grain. Thy, thylakoid. Gr, granum. Bars = 1 μm.

Fig. 6

GhYGL1d regulates plastid function-related genes. a Plastid transcriptomic comparison of CLCrVA and GhYGL1d-RNAi variegated leaves. b Expression analysis of plastid-encoded genes in CLCrVA and GhYGL1d-RNAi plants. Transcription levels were measured via quantitative real-time RT-PCR, and GhUBQ7 was used as a reference. Mean and SD values were obtained from three replicates.

Fig. 7

Analysis of photosynthetic complexes in RNAi plants and CLCrVA control plants. a Immunoblot analysis of photosynthetic proteins accumulated in CLCrVA and GhYGL1d-RNAi variegated leaves. Actin was used to check the difference in sample loading. b BN-PAGE analysis of photosynthetic complexes in CLCrVA and GhYGL1d-RNAi variegated leaves. Each lane was loaded with equal amounts of thylakoid membrane. PSI SC, PSI supercomplexes; PSI-M, PSI monomers; PSII-D, PSII dimers; PSII-M, PSII monomers; Cytb6/f, cytochrome f; and LHCII-T: PSII LHC trimers.