Effect of chloroplast ATP synthase on reactive oxygen species metabolism in the cotton cytoplasmic male sterile line Jin A

Effect of chloroplast ATP synthase on reactive oxygen species metabolism in the cotton cytoplasmic male sterile line Jin A | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of chloroplast ATP synthase on reactive oxygen species metabolism in the cotton cytoplasmic male sterile line Jin A Li Zhang, Panpan Jing, Biao Geng, Jinjiang Shi, Jinlong Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4662060/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Abnormal programmed cell death in the tapetum is induced by reactive oxygen species (ROS), which are the main factors leading to cytoplasmic male sterility (CMS). These abnormalities are caused by genetic interactions between nuclear and cytoplasmic genes. No studies have investigated the role of chloroplast ATP synthase in ROS metabolism. Results To explore the role of chloroplast genes in ROS metabolism, sequencing of the chloroplast genome from the next generation and single-molecule real-time sequencing of chloroplast DNA from the CMS line Jin A were performed. The results showed that the length of the chloroplast genome of the CMS line Jin A was 160,042 bp, and the genome consisted of 131 genes, including 112 functional genes. Analysis of the functional annotation and sequence comparison showed that Jin A CMS plants had 29 genes annotated with single nucleotide polymorphisms compared to Gossypium hirsutum plants, including ATP synthase subunits, NAD(P) H-quinone redox reductase subunits, and photosystem complex subunit genes. Compared to those of Jin B maintainer plants, the relative expression of atpB , atpE , and atpF significantly decreased in the anthers of Jin A CMS plants at the microspore abortion stage. The relative expression of these genes in the three-line hybrids F1 significantly increased compared with that in the Jin A CMS plants. The ROS levels in the leaves increased in the atpE and atpF silenced cotton plants. Conclusions In summary, our study showed that atpE and atpF of ATP synthase subunits gene were closely related to ROS metabolism. These results provide a basic information for the analysis of ATP synthase function in cotton. cytoplasmic male sterility programmed cell death reactive oxygen species ATP synthase chloroplast genome Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Cytoplasmic male sterility (CMS) is a widespread natural phenomenon in higher plants characterized by maternal inheritance, pollen abortion, and normal pistil [ 1 , 2 ]. Anther development is a complex process that includes the proliferation and differentiation of the pollen sac multilayer membrane, specific cell apoptosis, microspore mother cell meiosis, microspore proliferation, and development. Higher plants will produce certain physiological reactions, resulting in the death of cells, tissues or organs in specific parts under changes in the external environment. This process of death caused by external signals leads to the autonomous control of cells, called programmed cell death (PCD). Abnormal PCD in the tapetum during microspore development is the main factor leading to CMS [ 3 , 4 ]. Reactive oxygen species (ROS) are the main inducers of PCD in plants [ 5 ]. CMS is a phenomenon of infertility caused by genetic interactions between nuclear and cytoplasmic genes [ 6 ]. The nuclear backgrounds of CMS and its maintainer are homologous, but there are differences in cytoplasmic genes. Previous studies have shown that the formation of CMSs is related to mitochondrial gene rearrangement [ 7 , 8 ]. Ectopic expression of Jin A- orf610A increased ROS levels in yeast (to be published). Compared with the mitochondrial and nuclear genomes, the plant chloroplast genome is more conserved in terms of structure, gene number and gene composition. Although the chloroplast genome is generally highly conserved, minor changes, such as changes in size, contraction and expansion of repeating regions, and structural rearrangement, still occur [ 9 ]. It remains unclear whether chloroplast genes are involved in the physiology and biochemistry reactions of CMS. Chloroplasts are essential organelles in higher plant cells and play important roles in the photosynthesis and metabolism of fatty acids, nitrogen and internal redox signal transfer [ 10 – 12 ]. The chloroplast genome is a typical double-linked ring structure consisting of a small single-copy area and a large single-copy area (LSC). These two regions are separated by a pair of reverse repeating regions (IRa, IRb) [ 10 ]. There are generally between 110 and 130 chloroplastic genes in plants, including genes related to photosynthesis, self-reproduction, chloroplast transcription and expression, and some unknown genes [ 13 , 14 ]. Chloroplast thylakoid membrane complexes mainly include ATP synthase complexes composed of CFo and CF1, photosystem I (PSI) complexes, photosystem II (PSII) complexes, and cytochrome 6 complexes. Chloroplast ATP synthase produces ATP via the electrochemical proton gradient generated by photosynthesis. Then, protons are conducted through the membrane-embedded Fo motor, driving ATP synthesis in the F1 head by rotary catalysis [ 15 ]. The 9 subunits of chloroplast ATP synthase are encoded by both chloroplast genome and nuclear genes, among which the α ( atpA ), β ( atpB ), and ε ( atpE ) subunits of CF1 and the 6 subunits I ( atpF ), III ( atpH ), and IV ( atpI ) subunits of CFo are encoded by chloroplast genes. The γ ( atpC ) and δ ( atpD ) subunits of CF1 and the II ( atpG ) subunit of CFo are encoded by nuclear genes [ 16 ]. The six subunits encoded by chloroplast genes are located on two gene clusters or two operons, and the subunit genes are transcribed simultaneously in each cluster. The chloroplast ATP synthase ε subunit is necessary for the recombination of the FoF1 complex, and the N-terminal structure of the ε subunit can affect the ATP synthesis ability of synthase by regulating the ability of the ε subunit to block transmembrane proton leakage [ 17 ]. The soluble and bound forms of the ε subunit are potential inhibitors of ATPase and are necessary to maintain the proton gradient [ 18 , 19 ]. ε is the smallest subunit of chloroplast ATP synthase and is critical for the binding of F1 and Fo and normal H (+) translocation [ 20 ]. The combination of the I subunit from CFo with CF1 results in proton transport. Interactions between the δ subunit and the β, γ, ε, Ⅰ, Ⅱ, Ⅲ, and Ⅳ subunits of CFo participate in preventing proton leakage [ 21 ]. The cross-linking of Ⅰ and Ⅱ-δ in chloroplast ATP synthase inhibits photophosphorylation and the loss of ATP hydrolytic activity [ 15 ]. Increasing evidence shows that chloroplast ROS is widely involved in plant responses to various biological and abiotic stresses. Chloroplasts maintain a redox state and regulate ROS metabolism through photosynthesis [ 22 , 23 ]. Chloroplasts are the main sites of ROS production in plants, and ROS are rapidly produced in chloroplasts during photosynthesis [ 24 ]. For example, the excited electrons in PSI will be transferred to oxygen molecules when the energy is excessive and the electron acceptor NADP + is insufficient, and superoxide anion (O 2 −• ) will be produced by reduction. More chemically stable hydrogen peroxide (H 2 O 2 ) is generated by O 2 −• spontaneously or with the help of superoxide dismutase on the stromal side of the thylakoid membrane [ 25 ]. In PSII, when the plastoquinone pool is in a high reduction state (e.g., high light, drought, or low CO 2 concentration), a nonradical form of highly active singlet oxygen ( 1 O 2 ) is produced [ 26 , 27 ]. In course of PSII complex repair and reassembly, 1 O 2 is generated by the combination of chlorophyll molecules and the PSII complex [ 28 ]. Chloroplast proteins participate in ROS metabolism in the CMS line. Some studies have shown that there are significant differences in the ultrastructure [ 29 ], DNA levels [ 30 ] and protein levels in the chloroplasts of cytoplasmic male sterile lines and maintainer lines. The differences in physiological and biochemical indices, chloroplast ultrastructure, relative expression and the chloroplast genome among rice heterokaryon CMS strains indicate that chloroplasts may be associated with CMS [ 31 ]. The study of the chloroplast genome contributes to elucidating the interactions between nuclear and cytoplasmic genes and photosynthesis at the molecular level. The main reason for the abortion of CMS Jin A was premature PCD of the anther tapetal layer, which was related to the excessive accumulation of ROS [ 5 ]. Moreover, the energy metabolism of CMS Jin A plants is disrupted [ 5 ]. Transcriptome and proteome studies in CMS Jin A have shown that there are differentially expressed chloroplast enzymes and genes related to ROS clearance in CMS Jin A plants compared to those in maintainer Jin B plants at the key stage of microspore abortion [ 32 , 33 ]. Chloroplast genes and proteins are related to ROS metabolism in CMS. However, whether chloroplast ATP synthase, which regulates energy metabolism, participates in ROS metabolism is unknown. To understand the relationship between ROS metabolism and chloroplast genes, we used CMS Jin A as a material for chloroplast genome sequencing. The relative expression and functions of the I subunit of chloroplast ATP synthase CFo and several small subunits (β and ε subunits) of CF1 were analyzed to determine the role of chloroplast ATP synthase subunit genes in ROS metabolism. This study provides a basis for further research on chloroplast ATP synthase function. Results Sequencing data quality control and statistics The original data volume of the next-generation sequencing (NGS) was 9037.3 Mb, and the effective data volume was 9025 Mb after mass cutting the original data. The percentage of bases with a Phred value greater than 20 was 99.26% of the total bases. The percentage of bases with a Phred value greater than 30 was 97.17%. The GC content was 36.35%. The number of subreads after filtration was 685,714 in the Jin A SMRT sequencing, the size of the subread data was 967,209,861 bp, and the largest subread length was 38,029 bp. The subread length N50 was 1327 bp. The N90 length of the subreads was 983 bp, while the average length of the sample reads was 1411 bp. These results indicated that the constructed database and sequence were suitable for subsequent chloroplast genome assembly and bioinformatics analysis. Assembly and characteristics of the CMS Jin A chloroplast genome Chloroplast DNA in higher plants is a double-stranded covalent closed ring molecule, and its length varies with species. According to the genome assembly, the chloroplast genome length was 160,042 bp in CMS Jin A (Fig. 1 ). The genome consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA genes and 4 rRNA genes) and 19 repeat genes (Table 1). The base composition and gene distribution of each component region (LSC/SSC/IR) of the chloroplast genome were determined and summarized (Table 2), and there were four typical regions: LSC (55.37%), SSC (12.63%) and two IRs (15.99%). Functional analysis of the genome revealed that most of the genes were related to photosystem and ATP synthesis (Table 3). Five genes encoded PSI subunits, 15 genes encoded PSII subunits, 12 genes encoded NADH dehydrogenase, 6 genes encoded cytochrome b/f, 6 genes encoded ATP synthase and 1 gene encoded Rubisco large subunits. In addition, there were 9 genes encoding ribosome large subunit proteins, 12 genes encoding ribosome small subunit proteins, 4 genes encoding DNA-dependent RNA polymerase, 4 genes encoding ribosomal RNAs and 28 genes encoding transfer RNAs. Other identified genes included the mature enzyme-encoding gene matK , the protease gene clpP1 , the envelope protein-encoding gene cemA , the acetyl-cocarboxylase gene accD , and the cytochrome synthesis gene ccsA . In addition, 5 genes whose functions were unknown were identified. Five reference databases were used for gene annotation, among which the Swiss-Prot database was used to annotate the largest number of genes (Table 4), and a total of 86 protein-coding genes (including 79 genes and 7 duplicate genes, all encoding proteins) were annotated. Comparative analysis of the Gossypium hirsutum chloroplast genome The cytoplasmic background of CMS Jin A is the Gossypium hirsutum , so we chose to sequence the chloroplast genome of Gossypium hirsutum as the reference genome. A total of 29 chloroplast genes with single nucleotide (SNP) differences were obtained by sequence comparison of the chloroplast-coding protein genes between CMS Jin A and Gossypium hirsutum [ 34 ]. These DEGs mainly included ATP synthase subunit, NAD (P) H-quinone oxidoreductase subunit, and photosystem complex subunit genes. The results of the differential amino acid sequence comparison of the proteins encoded by genes are shown in Table S1 . The trnfM-CAU gene did not exist in the chloroplast genome of the sterile line Jin A. The trnfM-CAU is a differential hotspot gene in the chloroplast genome and plays an important role in the phylogenetic evolution of different species [ 35 ]. The trnfM-CAU located in the large copy region of chloroplasts in higher plants, and the deletion of trnfM-CAU indicated a change in the chloroplast genome composition in the sterile line Jin A, which may have affected normal function of the chloroplast genes. Analysis of ATP synthase subunit gene expression during the anther development in CMS Jin A plants ATP synthase plays an important role in cellular energy conversion and transfer. To explore the role of changes in chloroplast ATP synthase subunits in CMS Jin A, we first used NCBI CDD to search for and analyze the protein sequence domains encoded by three ATP synthase subunit genes, namely, atpB , atpE and atpF , and found that SNPs did not cause changes in protein domains. Next, we measured the expression levels of the atpB , atpE and atpF genes in CMS Jin A, the maintainer line Jin B and the three-line hybrid F1 (Fig. 2 ). The results showed that the expression levels of atpB , atpE and atpF in the sterile Jin A line were significantly lower than those in the maintainer line at the microspore abortion stage. Moreover, the expression levels of these three genes in the fertile restored F1 line were significantly greater than those in the Jin A sterile line. Therefore, we hypothesized that the differences in the transcription levels of these genes led to the inhibition of ATP synthesis. ROS detection in atpB , atpE , and atpF silenced cotton plants To determine the functions of atpB , atpE and atpF , gene-silenced recombinant vectors were constructed, the recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101, and cotton cotyledons were injected to obtain atpB , atpE , and atpF silenced cotton plants. The plants whose expression decreased the most were selected (Fig. 3 ). Using negative control plants with an empty pTRV2 vector, after 15 days of silencing, the cotton leaves were stained with Nitrotetrazolium blue chloride (NBT) and 3,3'-diaminobenzidine (DAB), and the ROS content of the silenced plants was determined (Fig. 3 a-c). The results showed that the accumulation of O 2 −• of the leaves of the atpE and atpF silenced plants was increased significantly compared to that of the negative control plants (Fig. 3 b). There was no significant difference in H 2 O 2 between the experimental group and the control group (Fig. 3 c). The results of the determination of 1 O 2 in leaves showed that the fluorescence color of leaves from plants silenced by atpE and atpF was deeper compared with the negative control, indicating significant accumulation of 1 O 2 (Fig. 4 ). There was no difference in the ROS content between the atpB silenced cotton plants and the control plants. Discussion At present, the whole chloroplast genome of many important crop species has been sequenced. Phylogenetic analysis of 10 chloroplast genomes of Gossypium showed that the chloroplast genome of Gossypium was relatively conserved, ranging from 159,035–160,317 bp in size, and included four typical regions: LSC, SSC and two IRs. The variation in IRs is the greatest among different cotton species and is the main determinant of chloroplast genome length differences in cotton [ 36 ] (Feng, 2014). Site-specific selection analysis revealed that some of the coding sites of 10 chloroplast genes ( atpB , atpE , rps2 , rps3 , petB , petD , ccsA , cemA , ycf1 , and rbcL ) underwent protein sequence evolution [ 37 ]. The chloroplast genome sequence of cotton was consistent with that of tobacco, and there was no rearrangement. AT-rich, with an average of 62.76%, codon preferences A and T, especially for the third codon, were as high as 69.31% [ 36 ]. In this study, the chloroplast genome of cotton CMS Jin A was 160,042 bp in size. The GC content was 37.31%. The genome consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA and 4 rRNA genes) and 19 repeat genes. These results indicate the high conservation of chloroplast genome evolution in Gossypium . Chloroplast genome of the sterile line Jin A did not contain the trnfM-CAU . The trnfM-CAU is a differential hotspot gene in the large copy region of chloroplast genome and plays a crucial role in the phylogenetic evolution of different species in higher plants [ 35 ]. Deletion of trnfM-CAU indicated a change in the chloroplast genome composition in the sterile line Jin A, which may have changed functions of the chloroplast genes. Some studies have shown that chloroplast proteins are involved in physiological and biochemical metabolism in CMS. There were significant differences in ultrastructure [ 29 ], DNA levels [ 30 ] and protein levels between the CMS and maintainer lines. Chloroplasts are associated with CMS, and there are differences in physiological and biochemical indices, chloroplast ultrastructure, relative expression and the chloroplast genome among rice heterokaryon CMS strains [ 31 ]. We sequenced the chloroplast genome of CMS Jin A and obtained 29 genes with SNPs, which are involved mainly in energy metabolism and photosystem composition, compared to those of Gossypium hirsutum. Chloroplasts, as important organelles for organic matter production in plants, play important roles in plant growth, development, and stress regulation. The levels of 1 O 2 in chloroplasts increase, triggering PCD [ 38 ]. Chloroplast genes play a crucial role in the generation of PCD in plants. In transgenic tobacco plants with plastid ndhF gene defects, reduced ROS levels are associated with delayed senescence [ 39 ]. Loss of function of the FZ1 gene, encoding a membrane GTPase, triggers ROS accumulation via chloroplast membrane damage and is sufficient to initiate the HR signaling cascade in Arabidopsis mutants [ 40 ]. The ε subunit of chloroplast ATP synthase affects the morphology and structure of the thylakoid membrane near photosystem II through its specific interaction with CF1, which makes proton loss from the thylakoid membrane difficult [ 41 ]. A comparison between the chloroplast genomes of the sterile line Jin A and the Gossypium hirsutum revealed differences in the nucleotide sequences of the atpB , atpE , and atpF genes (Table S1 ). The relative expression of genes in microspore buds during microspore abortion was measured. Quantitative qRT-PCR data showed that atpB , atpE , and atpF were significantly downregulated at the microspore abortion stage. H 2 O 2 and 1 O 2 accumulated in leaves of the atpE and atpF gene-silenced cotton. Previous studies showed that the ATP content of CMS Jin A anthers was significantly lower than that of maintainer Jin B anthers, and the energy metabolism of CMS Jin A anthers was disrupted at the microspore abortion stage [ 5 ]. On the one hand, we speculated that the ATP synthase genes atpE and atpF regulated energy metabolism through changes at the transcriptional level in CMS Jin A. On the other hand, the relative expression levels of atpE and atpF were downregulated and influenced the structure and proton transfer of photosystem II thylakoid, resulting in ROS accumulation and microspore development. This finding is consistent with the study of Shi (2001 b) [ 42 ]. Therefore, several chloroplast genes have been cloned and shown to affect ROS metabolism. More functions of chloroplast genes in plant growth and development still need to be identified. Conclusion The chloroplast genome of CMS Jin A was 160,042 bp and consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA and 4 rRNA genes) and 19 repeat genes. Compared with the proteins encoding chloroplast genes in Gossypium hirsutum , there were 29 genes containing SNPs and lacking trnfM-CAU in CMS Jin A. The relative expression levels of atpB , atpE , and atpF were significantly downregulated in anthers at the microspore abortion stage, and atpE and atpF gene silencing led to the accumulation of ROS in cotton leaves. Materials and methods Plant materials In this study, plant materials were planted in the experimental field of Shanxi Agricultural University from 2020 to 2023. CMS Jin A, the homologous heterogenic maintainer Jin B, and the three-line hybrid F1 were managed according to the conventional field management practices for cotton. The cotton plants used for gene silencing were cultured in an artificial climate room (16 h light: 8 h darkness, 8,000 lx, 22 ~ 23°C, and 70% relative humidity). At the flowering stage, buds of CMS Jin A, maintainer line Jin B, and three-line hybrid F1 cotton plants were collected and classified according to different microspore development stages [ 41 ] (Zhao et al., 2020). Young leaves were taken for use as experimental materials. Then, these materials were quickly frozen in liquid nitrogen and stored at -80°C. DNA sequencing, data quality control and statistics An Illumina TruSeq™ Nano DNA Sample Prep Kit was used to construct the library. The experimental steps were as follows: total DNA was extracted, and the library was constructed with 1 µg of DNA as the starting point; Covaris M220 ultrasound was used to interrupt the DNA to 300–500 bp; A was added to the 3' end, and the index connector was connected (TruSeq™ Nano DNA Sample Prep Kit); library enrichment and PCR amplification were performed for 8 cycles; a 2% agarose gum recycled purpose strip (Certified Low Range Ultra Agarose) and TBS380 (Picogreen) were used for quantification, according to the proportion of data mixed on the machine; bridge PCR was performed on a cBot solid phase vector to generate clusters; and an Illumina NovaSeq sequencing platform was used for 2×150 bp sequencing. Quality control and statistics of the NGS data: Illumina NovaSeq 6000 sequencing technology was used for paired-end sequencing of the DNA samples. The original sequencing data were processed as follows: polymerase reads less than 200 bp in length and polymerase reads with masses less than 0.80 were filtered out; subreads extracted from polymerase reads and adapter sequences were filtered out; and subreads less than 200 bp in length were filtered out. Chloroplast genome assembly and annotation First, Illumina sequencing data were assembled using GetOrganelle v1.7.5 software ( https://github.com/Kinggerm/GetOrganelle ). Then, BWA v0.7.17 was used to compare the NGS assembly to the SMRT sequencing data of PacBio to extract the SMRT sequencing data of the target sample, and the extracted SMRT sequencing data were mixed with the NGS data for assembly using SPAdes v3.14.1 software. Sequences with sufficient covering depth and long assembly length were selected as candidate sequences, the chloroplast scaffold sequences were confirmed by comparison with the NT library, and the sequences were connected according to overlap. Next, the clean reads were compared to the chloroplast genome sequence, and Pilon v1.23 was used to correct the bases. Finally, the starting position and direction of the chloroplast assembly sequence were determined according to the reference genome, and the 4 chloroplast partitions were determined. The final chloroplast genome sequence was obtained by including large single-copy, small single-copy, and two inverted repeats. GeSeq ( https://chlorobox.mpimp-golm.mpg.de/geseq.html/ ) software was used to predict protein coding, tRNA and rRNA genes of the chloroplast genome (protein search identity: 60; rRNA, tRNA, DNA search identity: 35; 3rd Party tRNA annotators: tRNAscan-SE). Then, the redundancy of the predicted initial genes was removed, and the head and tail of genes and exon/intron boundaries were removed to obtain a highly accurate gene set that was corrected manually. OrganellarGenomeDRAW ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) was used to circle the sample genome. The preference value of the codon was obtained by calculating the relative synonymous codon usage (RSCU). The software used was Cusp (EMBOSS v6.6.0.0). The functional annotations of the sample amino acid sequences were analyzed via the NR ( http://www.ncbi.nlm.nih.gov/ ), GO ( http://geneontology.org/ ), eggNOG ( http://eggnogdb.embl.de/ ), KEGG ( http://www.genome.jp/kegg/ ) and Swiss-Prot ( http://www.ebi.ac.uk/UniProt ) databases. Gene expression RNA was extracted from anthers of three cotton materials using a Plant RNA Extraction Kit (Aidlab Biotechnologies Co. Ltd., China) according to the manufacturer's instructions. Anthers (0.1 g) were ground in liquid nitrogen and centrifuged at room temperature, after which absolute ethanol was added to the supernatant for RNA precipitation. After adsorption to the column, protein and pigment impurities were removed. Three distinct bands with concentrations between 600 ~ 1200 ng/µL were detected by 1% agarose gel electrophoresis, and the A260/A280 ratio was approximately 2.0; thus, the extracted RNA could be used for subsequent experiments. The PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Dalian, Inc.) was used for reverse transcription. All regions of RNA can be uniformly synthesized when an RT Primer Mix combining Random 6-mers and Oligo dT Primer is used as the primer for reverse transcription. The relative expression of genes was measured by TB Green®Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Dalian, Inc.) using the Bio-Rad CFX Connect™ fluorescence quantitative PCR assay system (Bio-Rad Laboratories, Inc.). The primer sequences were shown in Table S2 . Gene cloning and carrier construction Specific primers were designed using NCBI Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) (Table S2 ). The coding sequence (CDS) of genes was amplified, and the plasmid pMD19T was connected to the CDS of genes to transform Trans1-T1 cells (TransGen Biotech Co., Ltd.). After PCR detection, the positive bacterial solution was sent to the company (Beijing Tsingke Biotech Co., Ltd.) for sequencing. The pTRV2 plasmid was linearized using restriction enzymes (New England Biolabs Beijing) according to the manufacturer's instructions (primer and restriction enzyme sites in Table S2 ). Recombinant vectors were constructed using a ClonExpress II One Step Cloning Kit (Vazyme International LLC.). VIGS The gene-silenced recombinant vectors were subsequently transformed into Agrobacterium tumefaciens GV3101, which was subsequently injected into cotton cotyledons [ 43 ]. ROS detection NBT and DAB staining were performed according to a published method [ 44 ]. The NBT staining solution was dissolved in double-distilled water at a concentration of 0.2 mM. The samples were soaked in the solution for 2 h and then decolorized with 95% ethanol. The DAB staining solution was dissolved in redistilled water at a concentration of 1 mg/mL, and the pH was adjusted to 3.8 with concentrated hydrochloric acid. The samples were soaked in this solution for 24 h and then decolorized with acetic acid, glycerin, and 95% ethanol (1:1:3). The samples were observed using a stereomicroscope (Olympus, Germany). 1 O 2 was measured using Singlet Oxygen Sensor Green reagent (Beijing Biolab Technology Co., Ltd) according to the manufacturer’s instructions. SOSG (10 µM) was used for staining the samples. The samples were soaked in solution for 12 h and observed under a confocal microscope. The excitation and emission wavelengths were 504 nm and 525 nm, respectively. Statistical analysis All assays were independently repeated at least three times. Statistical analysis was performed using one-way ANOVA (Tukey’s multiple comparison test). Charts were drawn using GraphPad Prism 8. Abbreviations CMS Cytoplasmic male sterility PCD Programmed cell death ROS Reactive oxygen species PSI Photosystem I PSII Photosystem II O 2 −• Superoxide anion H 2 O 2 Hydrogen peroxide 1 O 2 Singlet oxygen SNP Single nucleotide polymorphisms NBT Nitrotetrazolium blue chloride DAB 3,3'-diaminobenzidine Declarations Acknowledgements We thank Dr. Jianshe Wang and Dr. Haiyan Zhao provided useful suggestion for us. Authors’ contributions LZ, PJ, BG and JS planned and designed the research. LZ, JZ, DL and YY performed experiments. LZ, JH and YQ wrote the manuscript. All authors discussed the data and approved the final version of the manuscript submitted for publication. Funding This work was supported by the National Key R&D program (No. 2018YFD0100301), Basic Research Project of Shanxi Province (No. 202203021211271), Shanxi Agricultural University Research Fund (No. 2020xshf40), and Shanxi Graduate Education Innovation Plan (No. 2021Y312). Availability of data and materials The original contributions presented in the study are publicly available. These data can be found at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1106185. Ethics approval and consent to participate No plants in this study were collected from the wild. The plant “Jin A, Jin B” used in this study were cultivated from and deposited in Cotton Breeding Laboratory of Shanxi Agricultural University. All materials were grown at the Farm Station of Shanxi Agricultural University, and all the experiments were carried out in Shanxi Agricultural University. All methods were compliance with relevant institutional, national, and international guidelines and legislation. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Chai D, Li C, Song H. Research progress of cruciferous crops CMS and related open reading frames. 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Effects of chloroplast ATP synthase ε subunit deletion mutation on the millisecond delayed luminescence fast phase intensity and ATP synthesis capacity of chloroplasts. Chinese Science Bulletin. 2003; 24: 2539-2543. Senior AE. The proton-translocating ATPase of Escherichia coli. Annu. Rev. Biophy. Biophy. Chem. 1990; 19: 7-41. McCarty RE, Evron Y, Johnson EA. The chloroplast ATP synthase: A rotary enzyme?. Annu. Rev. Plant Physiol. Plant Mol. Bio. 2000; 51: 83-109. Zhang L, Peng L. Research progress on regulation mechanisms of biogenesis of chloroplast ATP synthase. Journal of Plant physiology. 2019; 55: 703-710. Shi X, Wei J, Shen Y. Interaction between chloroplast ATP synthase CF 1 and CFo subunits. Chinese Science Bulletin. 2001 a; 18: 1550-1554. Cejudo FJ, Ojeda V, Delgado-Requerey V, González M, Pérez-Ruiz JM. Chloroplast redox regulatory mechanisms in plant adaptation to light and darkness. Front. Plant Sci. 2019; 10: 380. Hoh D, Froehlich JE, Kramer DM. Redox regulation in chloroplast thylakoid lumen: the pmf changes everything, again. Plant Cell & Environment. 2023. Wu L, Wu J, Liu Y, Gong X, Xu J, Lin D, Dong Y. The rice pentatricopeptide repeat gene TCD10 is needed for chloroplast development under cold stress. Rice (New York). 2016; 9: 67. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 2006; 141: 391-396. Krieger-Liszkay A. Singlet oxygen production in photosynthesis. J. Exp. Bot. 2005; 56: 337-346. Mark AG, Gibbs JG, Lee TC, Fischer P. Hybrid nanocolloids with programmed thre e-dimensional shape and material composition. Nat. Mater. 2013; 12: 802-807. Dogra V, Kim C. Singlet oxygen metabolism: from genesis to signaling. Front. Plant Sci. 2020; 10: 1640. Li L, Li J. Chloroplast thylakoid membrane polypeptides and cytoplasmic male sterility. Cur. Genet. 1986; 10: 947-949. Galau GA, Wilkins TA. Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii . Theor. Appl. Genet. 1989; 78: 23-30. Tang D, Wei F, Kashif MH, Khan A, Li Z, Shi Q, Jia R, Xie H, Zhang L, Li B, Chen P, Zhou R. Analysis of chloroplast differences in leaves of rice isonuclear alloplasmic lines. Protoplasma. 2018; 255: 863-871. Yang P, Han J, Huang J. Proteome analysis of cytoplasmic male sterility and its maintaince in JA-CMS Cotton. Scientia Agricultura Sinica. 2014; 47: 3929-3940. Yang P. Study on the differential transcriptomics and proteomics analysis between JA-CMS and its maintaince of cotton. Henan Agricultural University. 2016 (in Chinese). Lee SB, Kaittanis C, Jansen RK, Hostetler JB, Tallon LJ, Town CD, Daniell H. The complete chloroplast genome sequence of Gossypium hirsutum : organization and phylogenetic relationships to other angiosperms. BMC Genomics. 2006; 7: 61. NAWAL AFZAL. Comparative chloroplast genomics of Aceraceae species: Insights into sequence variations and phylogenetic evolution. Xi'an, Shanxi Province: Northwest University. 2020. Feng K. Chloroplast genome sequences of ten species of Gossypium : structural organization and phylogenetic analyses. Beijing: Chinese Academy of Agricultural Sciences. 2014 (in Chinese). Wu Y, Liu F, Yang DG, Li W, Zhou XJ, Pei XY, Liu YG, He KL, Zhang WS, Ren ZY, Zhou KH, Ma XF, Li ZH. Comparative chloroplast genomics of Gossypium species: insights into repeat sequence variations and phylogeny. Front. Plant Sci. 2018; 9: 376. Kim C, Meskauskiene R, Zhang S, Lee KP, Lakshmanan Ashok M, Blajecka K, Herrfurth C, Feussner I, Apel K. Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway. Plant Cell. 2012; 24: 3026-3039. Zapata JM, Guéra A, Esteban-Carrasco A, Martín M, Sabater B. Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF -defective tobacco. Cell Death Differ. 2005; 12: 1277-1284. Ambastha V, Tripathy BC, Tiwari BS. Programmed cell death in plants: A chloroplastic connection. Plant Signal. Behav. 2015; 10: e989752. Shi X. Study on the structure and function of chloroplast ATP synthase subunits in higher plants. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 2001 b (in Chinese). Zhao H, Wang J, Qu Y, Peng R, Magwanga RO, Liu F, Huang J. Transcriptomic and proteomic analyses of a new cytoplasmic male sterile line with a wild Gossypium bickii genetic background. BMC genomics. 2020; 21: 859. Pang J, Zhu Y, Li Q, Liu J, Tian Y, Liu Y, Wu J. Development of Agrobacterium -mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense . PloS One. 2013; 8: e73211. Wu J, Sun Y, Zhao Y, Zhang J, Luo L, Li M, Wang J, Yu H, Liu G, Yang L, Xiong G, Zhou JM, Zuo J, Wang Y, Li J. Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species. Cell Res. 2015; 25: 621-633. Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx Table1.xlsx Table2.xlsx Table3.xlsx Table4.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4662060","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322680075,"identity":"2a55b9f5-fae1-448f-acbe-e62726859055","order_by":0,"name":"Li Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""},{"id":322680076,"identity":"17063ff4-f8e8-4856-ab5a-85efac3f92f0","order_by":1,"name":"Panpan Jing","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Jing","suffix":""},{"id":322680077,"identity":"970fead4-0119-4327-85fc-90bcf47eed36","order_by":2,"name":"Biao Geng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Geng","suffix":""},{"id":322680078,"identity":"ab58807d-b4b6-4990-8865-87884432cf1a","order_by":3,"name":"Jinjiang Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinjiang","middleName":"","lastName":"Shi","suffix":""},{"id":322680079,"identity":"5894ecde-9b04-47e6-998e-c605f2e9716c","order_by":4,"name":"Jinlong Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinlong","middleName":"","lastName":"Zhang","suffix":""},{"id":322680080,"identity":"6031a312-3ad0-4a14-8fb2-c2f725e147c0","order_by":5,"name":"Dong Liang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Liang","suffix":""},{"id":322680081,"identity":"5d60052e-deac-4f66-8f68-03fc1bfc642e","order_by":6,"name":"Yujie Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Yang","suffix":""},{"id":322680082,"identity":"827e2ffd-2486-4b70-98df-4435dbec0d6d","order_by":7,"name":"Yunfang Qu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yunfang","middleName":"","lastName":"Qu","suffix":""},{"id":322680083,"identity":"721eef7e-0d7e-491b-9bb8-d4fc33055426","order_by":8,"name":"Jinling Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACAzDJYyEH5TMTrUXCmFQtDBKJDURrMZdIfvbwi4xE+vwZyc8kGCqsExvYzx7Aq8VyRpq5sQyPRO6GM8fMJBjOpCc28OQl4HfYjQQzaQmQFvYGMwnGtsOJDRI8BgS0pH8DaUmXb2b/JsH4jygtOWaSH3gkEhiO9wBtaSBGy5k3ZdLAQDbccOZMsUXCsXTjNp4cAlqOp2+T/NljIy8/I33jjQ811rL97GfwawEBZt4eKCsBiNkIqgcCxh8/iFE2CkbBKBgFIxYAAHZ9PxT5lCyiAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Jinling","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-06-30 09:23:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4662060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4662060/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61287731,"identity":"b3196695-86f6-4b56-b3d7-78780dc1c5cc","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1470537,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome assembly, size and features of CMS Jin A. Genes lying outside of the outer layer circle are transcribed in the counter clockwise direction, whereas genes inside are transcribed in the clockwise direction. The colored bars indicate different functional groups. The darker gray area in the inner circle denotes GC content while the lighter gray corresponds to AT content of the genome.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/3e278586488dde997120b591.png"},{"id":61287729,"identity":"58545a9e-65d2-43c3-a688-12ddaf7c8f13","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67881,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression analysis of ATP synthase subunit genes during the anther development in CMS Jin A. (a) The relative expression of\u003cem\u003e atpB\u003c/em\u003e; (b) The relative expression of\u003cem\u003e atpE\u003c/em\u003e; (c) The relative expression of \u003cem\u003eatpF\u003c/em\u003e. S (before the microspore abortion stage), M1 (the microspore abortion stage) and M2 (after the microspore abortion stage). Values are means ± SD of three replicates. “*” represents significant differences between sterile line and its maintainer (* P \u0026lt; 0.05; ** P \u0026lt; 0.01, Tukey’s multiple comparison tests).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/53f84f3a443364b197c0bf08.png"},{"id":61287738,"identity":"8ab3bf91-2a01-48ea-a11c-609ad1e87266","added_by":"auto","created_at":"2024-07-29 06:45:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223449,"visible":true,"origin":"","legend":"\u003cp\u003eThe detection of ROS in gene-silenced cotton leaves. (a) Determination of gene expression in leaves of gene-silenced plants; Values are means ± SD of three replicates. “*” represents significant differences between sterile line and maintainer (* P \u0026lt; 0.05; ** P \u0026lt; 0.01, Tukey’s multiple comparison tests). (b) The detection of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−•\u003c/sup\u003e in gene-silenced plant leaves; (c) The detection of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in gene-silenced plant leaves. Bar = 1 mm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/cefef035fdb07ee8ccdfc2ec.png"},{"id":61287734,"identity":"09abce18-1099-4d17-aa85-cc111ba496d1","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1144534,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e in gene-silenced cotton leaves. GFP, green fluorescence; Bright, bright field; Merged, merged field, bar = 1 mm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/2339f9c864ae22d15d6ace98.png"},{"id":66655846,"identity":"63cc038a-7993-4c2d-b1fe-75219ea1ddb5","added_by":"auto","created_at":"2024-10-15 08:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3470546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/203e31ce-176f-4a51-9585-603485760d01.pdf"},{"id":61288644,"identity":"641f0895-41e6-4b03-a01f-ca44faf45908","added_by":"auto","created_at":"2024-07-29 06:53:56","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12017,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/d9b38876db9bd77ac3a4a50e.xlsx"},{"id":61287733,"identity":"9b05932e-a64d-450e-9e1c-88cbad2936b4","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10520,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/e82247c10854e14b6cfb09a8.xlsx"},{"id":61287736,"identity":"53197491-3e83-4001-886d-f00c974f1091","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9324,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/fb48664f74c7f486e73b4a6a.xlsx"},{"id":61287732,"identity":"8f3cc3d1-e134-432d-88ff-56207d259cb5","added_by":"auto","created_at":"2024-07-29 06:45:56","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9665,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/9ed6b1746bb73e2cd8e1a3ca.xlsx"},{"id":61287737,"identity":"35c632a4-9916-46b7-aecd-1264a7f4dfd3","added_by":"auto","created_at":"2024-07-29 06:45:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10625,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/724d6afc2ae37751c13f46d0.xlsx"},{"id":61287739,"identity":"d52641ff-0119-48b5-a08d-9eee1d03c6dd","added_by":"auto","created_at":"2024-07-29 06:45:57","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":9050,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4662060/v1/07aa0991bc5e11e1cce49933.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of chloroplast ATP synthase on reactive oxygen species metabolism in the cotton cytoplasmic male sterile line Jin A","fulltext":[{"header":"Background","content":"\u003cp\u003eCytoplasmic male sterility (CMS) is a widespread natural phenomenon in higher plants characterized by maternal inheritance, pollen abortion, and normal pistil [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Anther development is a complex process that includes the proliferation and differentiation of the pollen sac multilayer membrane, specific cell apoptosis, microspore mother cell meiosis, microspore proliferation, and development. Higher plants will produce certain physiological reactions, resulting in the death of cells, tissues or organs in specific parts under changes in the external environment. This process of death caused by external signals leads to the autonomous control of cells, called programmed cell death (PCD). Abnormal PCD in the tapetum during microspore development is the main factor leading to CMS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Reactive oxygen species (ROS) are the main inducers of PCD in plants [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCMS is a phenomenon of infertility caused by genetic interactions between nuclear and cytoplasmic genes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The nuclear backgrounds of CMS and its maintainer are homologous, but there are differences in cytoplasmic genes. Previous studies have shown that the formation of CMSs is related to mitochondrial gene rearrangement [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ectopic expression of Jin A-\u003cem\u003eorf610A\u003c/em\u003e increased ROS levels in yeast (to be published). Compared with the mitochondrial and nuclear genomes, the plant chloroplast genome is more conserved in terms of structure, gene number and gene composition. Although the chloroplast genome is generally highly conserved, minor changes, such as changes in size, contraction and expansion of repeating regions, and structural rearrangement, still occur [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It remains unclear whether chloroplast genes are involved in the physiology and biochemistry reactions of CMS.\u003c/p\u003e \u003cp\u003eChloroplasts are essential organelles in higher plant cells and play important roles in the photosynthesis and metabolism of fatty acids, nitrogen and internal redox signal transfer [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The chloroplast genome is a typical double-linked ring structure consisting of a small single-copy area and a large single-copy area (LSC). These two regions are separated by a pair of reverse repeating regions (IRa, IRb) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. There are generally between 110 and 130 chloroplastic genes in plants, including genes related to photosynthesis, self-reproduction, chloroplast transcription and expression, and some unknown genes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChloroplast thylakoid membrane complexes mainly include ATP synthase complexes composed of CFo and CF1, photosystem I (PSI) complexes, photosystem II (PSII) complexes, and cytochrome 6 complexes. Chloroplast ATP synthase produces ATP via the electrochemical proton gradient generated by photosynthesis. Then, protons are conducted through the membrane-embedded Fo motor, driving ATP synthesis in the F1 head by rotary catalysis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The 9 subunits of chloroplast ATP synthase are encoded by both chloroplast genome and nuclear genes, among which the α (\u003cem\u003eatpA\u003c/em\u003e), β (\u003cem\u003eatpB\u003c/em\u003e), and ε (\u003cem\u003eatpE\u003c/em\u003e) subunits of CF1 and the 6 subunits I (\u003cem\u003eatpF\u003c/em\u003e), III (\u003cem\u003eatpH\u003c/em\u003e), and IV (\u003cem\u003eatpI\u003c/em\u003e) subunits of CFo are encoded by chloroplast genes. The γ (\u003cem\u003eatpC\u003c/em\u003e) and δ (\u003cem\u003eatpD\u003c/em\u003e) subunits of CF1 and the II (\u003cem\u003eatpG\u003c/em\u003e) subunit of CFo are encoded by nuclear genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The six subunits encoded by chloroplast genes are located on two gene clusters or two operons, and the subunit genes are transcribed simultaneously in each cluster. The chloroplast ATP synthase ε subunit is necessary for the recombination of the FoF1 complex, and the N-terminal structure of the ε subunit can affect the ATP synthesis ability of synthase by regulating the ability of the ε subunit to block transmembrane proton leakage [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The soluble and bound forms of the ε subunit are potential inhibitors of ATPase and are necessary to maintain the proton gradient [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ε is the smallest subunit of chloroplast ATP synthase and is critical for the binding of F1 and Fo and normal H (+) translocation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The combination of the I subunit from CFo with CF1 results in proton transport. Interactions between the δ subunit and the β, γ, ε, Ⅰ, Ⅱ, Ⅲ, and Ⅳ subunits of CFo participate in preventing proton leakage [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The cross-linking of Ⅰ and Ⅱ-δ in chloroplast ATP synthase inhibits photophosphorylation and the loss of ATP hydrolytic activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreasing evidence shows that chloroplast ROS is widely involved in plant responses to various biological and abiotic stresses. Chloroplasts maintain a redox state and regulate ROS metabolism through photosynthesis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Chloroplasts are the main sites of ROS production in plants, and ROS are rapidly produced in chloroplasts during photosynthesis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For example, the excited electrons in PSI will be transferred to oxygen molecules when the energy is excessive and the electron acceptor NADP\u003csup\u003e+\u003c/sup\u003e is insufficient, and superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u0026bull;\u003c/sup\u003e) will be produced by reduction. More chemically stable hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) is generated by O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u0026bull;\u003c/sup\u003e spontaneously or with the help of superoxide dismutase on the stromal side of the thylakoid membrane [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In PSII, when the plastoquinone pool is in a high reduction state (e.g., high light, drought, or low CO\u003csub\u003e2\u003c/sub\u003e concentration), a nonradical form of highly active singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) is produced [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In course of PSII complex repair and reassembly, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e is generated by the combination of chlorophyll molecules and the PSII complex [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Chloroplast proteins participate in ROS metabolism in the CMS line. Some studies have shown that there are significant differences in the ultrastructure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], DNA levels [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and protein levels in the chloroplasts of cytoplasmic male sterile lines and maintainer lines. The differences in physiological and biochemical indices, chloroplast ultrastructure, relative expression and the chloroplast genome among rice heterokaryon CMS strains indicate that chloroplasts may be associated with CMS [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The study of the chloroplast genome contributes to elucidating the interactions between nuclear and cytoplasmic genes and photosynthesis at the molecular level.\u003c/p\u003e \u003cp\u003eThe main reason for the abortion of CMS Jin A was premature PCD of the anther tapetal layer, which was related to the excessive accumulation of ROS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, the energy metabolism of CMS Jin A plants is disrupted [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Transcriptome and proteome studies in CMS Jin A have shown that there are differentially expressed chloroplast enzymes and genes related to ROS clearance in CMS Jin A plants compared to those in maintainer Jin B plants at the key stage of microspore abortion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Chloroplast genes and proteins are related to ROS metabolism in CMS. However, whether chloroplast ATP synthase, which regulates energy metabolism, participates in ROS metabolism is unknown. To understand the relationship between ROS metabolism and chloroplast genes, we used CMS Jin A as a material for chloroplast genome sequencing. The relative expression and functions of the I subunit of chloroplast ATP synthase CFo and several small subunits (β and ε subunits) of CF1 were analyzed to determine the role of chloroplast ATP synthase subunit genes in ROS metabolism. This study provides a basis for further research on chloroplast ATP synthase function.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequencing data quality control and statistics\u003c/h2\u003e \u003cp\u003eThe original data volume of the next-generation sequencing (NGS) was 9037.3 Mb, and the effective data volume was 9025 Mb after mass cutting the original data. The percentage of bases with a Phred value greater than 20 was 99.26% of the total bases. The percentage of bases with a Phred value greater than 30 was 97.17%. The GC content was 36.35%.\u003c/p\u003e \u003cp\u003eThe number of subreads after filtration was 685,714 in the Jin A SMRT sequencing, the size of the subread data was 967,209,861 bp, and the largest subread length was 38,029 bp. The subread length N50 was 1327 bp. The N90 length of the subreads was 983 bp, while the average length of the sample reads was 1411 bp. These results indicated that the constructed database and sequence were suitable for subsequent chloroplast genome assembly and bioinformatics analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAssembly and characteristics of the CMS Jin A chloroplast genome\u003c/h2\u003e \u003cp\u003eChloroplast DNA in higher plants is a double-stranded covalent closed ring molecule, and its length varies with species. According to the genome assembly, the chloroplast genome length was 160,042 bp in CMS Jin A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genome consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA genes and 4 rRNA genes) and 19 repeat genes (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe base composition and gene distribution of each component region (LSC/SSC/IR) of the chloroplast genome were determined and summarized (Table\u0026nbsp;2), and there were four typical regions: LSC (55.37%), SSC (12.63%) and two IRs (15.99%).\u003c/p\u003e \u003cp\u003eFunctional analysis of the genome revealed that most of the genes were related to photosystem and ATP synthesis (Table\u0026nbsp;3). Five genes encoded PSI subunits, 15 genes encoded PSII subunits, 12 genes encoded NADH dehydrogenase, 6 genes encoded cytochrome b/f, 6 genes encoded ATP synthase and 1 gene encoded Rubisco large subunits. In addition, there were 9 genes encoding ribosome large subunit proteins, 12 genes encoding ribosome small subunit proteins, 4 genes encoding DNA-dependent RNA polymerase, 4 genes encoding ribosomal RNAs and 28 genes encoding transfer RNAs. Other identified genes included the mature enzyme-encoding gene \u003cem\u003ematK\u003c/em\u003e, the protease gene \u003cem\u003eclpP1\u003c/em\u003e, the envelope protein-encoding gene \u003cem\u003ecemA\u003c/em\u003e, the acetyl-cocarboxylase gene \u003cem\u003eaccD\u003c/em\u003e, and the cytochrome synthesis gene \u003cem\u003eccsA\u003c/em\u003e. In addition, 5 genes whose functions were unknown were identified. Five reference databases were used for gene annotation, among which the Swiss-Prot database was used to annotate the largest number of genes (Table\u0026nbsp;4), and a total of 86 protein-coding genes (including 79 genes and 7 duplicate genes, all encoding proteins) were annotated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative analysis of the\u003c/b\u003e \u003cb\u003eGossypium hirsutum\u003c/b\u003e \u003cb\u003echloroplast genome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cytoplasmic background of CMS Jin A is the \u003cem\u003eGossypium hirsutum\u003c/em\u003e, so we chose to sequence the chloroplast genome of \u003cem\u003eGossypium hirsutum\u003c/em\u003e as the reference genome. A total of 29 chloroplast genes with single nucleotide (SNP) differences were obtained by sequence comparison of the chloroplast-coding protein genes between CMS Jin A and \u003cem\u003eGossypium hirsutum\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These DEGs mainly included ATP synthase subunit, NAD (P) H-quinone oxidoreductase subunit, and photosystem complex subunit genes. The results of the differential amino acid sequence comparison of the proteins encoded by genes are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The \u003cem\u003etrnfM-CAU\u003c/em\u003e gene did not exist in the chloroplast genome of the sterile line Jin A. The \u003cem\u003etrnfM-CAU\u003c/em\u003e is a differential hotspot gene in the chloroplast genome and plays an important role in the phylogenetic evolution of different species [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The \u003cem\u003etrnfM-CAU\u003c/em\u003e located in the large copy region of chloroplasts in higher plants, and the deletion of \u003cem\u003etrnfM-CAU\u003c/em\u003e indicated a change in the chloroplast genome composition in the sterile line Jin A, which may have affected normal function of the chloroplast genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of ATP synthase subunit gene expression during the anther development in CMS Jin A plants\u003c/b\u003e \u003c/p\u003e \u003cp\u003eATP synthase plays an important role in cellular energy conversion and transfer. To explore the role of changes in chloroplast ATP synthase subunits in CMS Jin A, we first used NCBI CDD to search for and analyze the protein sequence domains encoded by three ATP synthase subunit genes, namely, \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e, and found that SNPs did not cause changes in protein domains. Next, we measured the expression levels of the \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e genes in CMS Jin A, the maintainer line Jin B and the three-line hybrid F1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results showed that the expression levels of \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e in the sterile Jin A line were significantly lower than those in the maintainer line at the microspore abortion stage. Moreover, the expression levels of these three genes in the fertile restored F1 line were significantly greater than those in the Jin A sterile line. Therefore, we hypothesized that the differences in the transcription levels of these genes led to the inhibition of ATP synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eROS detection in\u003c/b\u003e \u003cb\u003eatpB\u003c/b\u003e, \u003cb\u003eatpE\u003c/b\u003e, \u003cb\u003eand\u003c/b\u003e \u003cb\u003eatpF\u003c/b\u003e \u003cb\u003esilenced cotton plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the functions of \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e, gene-silenced recombinant vectors were constructed, the recombinant plasmids were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, and cotton cotyledons were injected to obtain \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, and \u003cem\u003eatpF\u003c/em\u003e silenced cotton plants. The plants whose expression decreased the most were selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing negative control plants with an empty pTRV2 vector, after 15 days of silencing, the cotton leaves were stained with Nitrotetrazolium blue chloride (NBT) and 3,3'-diaminobenzidine (DAB), and the ROS content of the silenced plants was determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). The results showed that the accumulation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u0026bull;\u003c/sup\u003e of the leaves of the \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e silenced plants was increased significantly compared to that of the negative control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). There was no significant difference in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e between the experimental group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe results of the determination of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e in leaves showed that the fluorescence color of leaves from plants silenced by \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e was deeper compared with the negative control, indicating significant accumulation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There was no difference in the ROS content between the \u003cem\u003eatpB\u003c/em\u003e silenced cotton plants and the control plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAt present, the whole chloroplast genome of many important crop species has been sequenced. Phylogenetic analysis of 10 chloroplast genomes of \u003cem\u003eGossypium\u003c/em\u003e showed that the chloroplast genome of \u003cem\u003eGossypium\u003c/em\u003e was relatively conserved, ranging from 159,035\u0026ndash;160,317 bp in size, and included four typical regions: LSC, SSC and two IRs. The variation in IRs is the greatest among different cotton species and is the main determinant of chloroplast genome length differences in cotton [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (Feng, 2014). Site-specific selection analysis revealed that some of the coding sites of 10 chloroplast genes (\u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, \u003cem\u003erps2\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003eccsA\u003c/em\u003e, \u003cem\u003ecemA\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, and \u003cem\u003erbcL\u003c/em\u003e) underwent protein sequence evolution [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The chloroplast genome sequence of cotton was consistent with that of tobacco, and there was no rearrangement. AT-rich, with an average of 62.76%, codon preferences A and T, especially for the third codon, were as high as 69.31% [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, the chloroplast genome of cotton CMS Jin A was 160,042 bp in size. The GC content was 37.31%. The genome consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA and 4 rRNA genes) and 19 repeat genes. These results indicate the high conservation of chloroplast genome evolution in \u003cem\u003eGossypium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eChloroplast genome of the sterile line Jin A did not contain the \u003cem\u003etrnfM-CAU\u003c/em\u003e. The \u003cem\u003etrnfM-CAU\u003c/em\u003e is a differential hotspot gene in the large copy region of chloroplast genome and plays a crucial role in the phylogenetic evolution of different species in higher plants [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Deletion of \u003cem\u003etrnfM-CAU\u003c/em\u003e indicated a change in the chloroplast genome composition in the sterile line Jin A, which may have changed functions of the chloroplast genes.\u003c/p\u003e \u003cp\u003eSome studies have shown that chloroplast proteins are involved in physiological and biochemical metabolism in CMS. There were significant differences in ultrastructure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], DNA levels [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and protein levels between the CMS and maintainer lines. Chloroplasts are associated with CMS, and there are differences in physiological and biochemical indices, chloroplast ultrastructure, relative expression and the chloroplast genome among rice heterokaryon CMS strains [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We sequenced the chloroplast genome of CMS Jin A and obtained 29 genes with SNPs, which are involved mainly in energy metabolism and photosystem composition, compared to those of \u003cem\u003eGossypium hirsutum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eChloroplasts, as important organelles for organic matter production in plants, play important roles in plant growth, development, and stress regulation. The levels of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e in chloroplasts increase, triggering PCD [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Chloroplast genes play a crucial role in the generation of PCD in plants. In transgenic tobacco plants with plastid \u003cem\u003endhF\u003c/em\u003e gene defects, reduced ROS levels are associated with delayed senescence [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Loss of function of the \u003cem\u003eFZ1\u003c/em\u003e gene, encoding a membrane GTPase, triggers ROS accumulation via chloroplast membrane damage and is sufficient to initiate the HR signaling cascade in \u003cem\u003eArabidopsis\u003c/em\u003e mutants [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ε subunit of chloroplast ATP synthase affects the morphology and structure of the thylakoid membrane near photosystem II through its specific interaction with CF1, which makes proton loss from the thylakoid membrane difficult [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A comparison between the chloroplast genomes of the sterile line Jin A and the \u003cem\u003eGossypium hirsutum\u003c/em\u003e revealed differences in the nucleotide sequences of the \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, and \u003cem\u003eatpF\u003c/em\u003e genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The relative expression of genes in microspore buds during microspore abortion was measured. Quantitative qRT-PCR data showed that \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, and \u003cem\u003eatpF\u003c/em\u003e were significantly downregulated at the microspore abortion stage. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e accumulated in leaves of the \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e gene-silenced cotton. Previous studies showed that the ATP content of CMS Jin A anthers was significantly lower than that of maintainer Jin B anthers, and the energy metabolism of CMS Jin A anthers was disrupted at the microspore abortion stage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. On the one hand, we speculated that the ATP synthase genes \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e regulated energy metabolism through changes at the transcriptional level in CMS Jin A. On the other hand, the relative expression levels of \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e were downregulated and influenced the structure and proton transfer of photosystem II thylakoid, resulting in ROS accumulation and microspore development. This finding is consistent with the study of Shi (2001 b) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, several chloroplast genes have been cloned and shown to affect ROS metabolism. More functions of chloroplast genes in plant growth and development still need to be identified.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe chloroplast genome of CMS Jin A was 160,042 bp and consisted of 131 genes, including 112 functional genes (79 protein-coding genes, 29 tRNA and 4 rRNA genes) and 19 repeat genes. Compared with the proteins encoding chloroplast genes in \u003cem\u003eGossypium hirsutum\u003c/em\u003e, there were 29 genes containing SNPs and lacking \u003cem\u003etrnfM-CAU\u003c/em\u003e in CMS Jin A. The relative expression levels of \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, and \u003cem\u003eatpF\u003c/em\u003e were significantly downregulated in anthers at the microspore abortion stage, and \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e gene silencing led to the accumulation of ROS in cotton leaves.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eIn this study, plant materials were planted in the experimental field of Shanxi Agricultural University from 2020 to 2023. CMS Jin A, the homologous heterogenic maintainer Jin B, and the three-line hybrid F1 were managed according to the conventional field management practices for cotton. The cotton plants used for gene silencing were cultured in an artificial climate room (16 h light: 8 h darkness, 8,000 lx, 22\u0026thinsp;~\u0026thinsp;23\u0026deg;C, and 70% relative humidity).\u003c/p\u003e \u003cp\u003eAt the flowering stage, buds of CMS Jin A, maintainer line Jin B, and three-line hybrid F1 cotton plants were collected and classified according to different microspore development stages [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Zhao et al., 2020). Young leaves were taken for use as experimental materials. Then, these materials were quickly frozen in liquid nitrogen and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDNA sequencing, data quality control and statistics\u003c/h2\u003e \u003cp\u003eAn Illumina TruSeq\u0026trade; Nano DNA Sample Prep Kit was used to construct the library. The experimental steps were as follows: total DNA was extracted, and the library was constructed with 1 \u0026micro;g of DNA as the starting point; Covaris M220 ultrasound was used to interrupt the DNA to 300\u0026ndash;500 bp; A was added to the 3' end, and the index connector was connected (TruSeq\u0026trade; Nano DNA Sample Prep Kit); library enrichment and PCR amplification were performed for 8 cycles; a 2% agarose gum recycled purpose strip (Certified Low Range Ultra Agarose) and TBS380 (Picogreen) were used for quantification, according to the proportion of data mixed on the machine; bridge PCR was performed on a cBot solid phase vector to generate clusters; and an Illumina NovaSeq sequencing platform was used for 2\u0026times;150 bp sequencing.\u003c/p\u003e \u003cp\u003eQuality control and statistics of the NGS data: Illumina NovaSeq 6000 sequencing technology was used for paired-end sequencing of the DNA samples. The original sequencing data were processed as follows: polymerase reads less than 200 bp in length and polymerase reads with masses less than 0.80 were filtered out; subreads extracted from polymerase reads and adapter sequences were filtered out; and subreads less than 200 bp in length were filtered out.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eChloroplast genome assembly and annotation\u003c/h2\u003e \u003cp\u003eFirst, Illumina sequencing data were assembled using GetOrganelle v1.7.5 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Kinggerm/GetOrganelle\u003c/span\u003e\u003cspan address=\"https://github.com/Kinggerm/GetOrganelle\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, BWA v0.7.17 was used to compare the NGS assembly to the SMRT sequencing data of PacBio to extract the SMRT sequencing data of the target sample, and the extracted SMRT sequencing data were mixed with the NGS data for assembly using SPAdes v3.14.1 software. Sequences with sufficient covering depth and long assembly length were selected as candidate sequences, the chloroplast scaffold sequences were confirmed by comparison with the NT library, and the sequences were connected according to overlap.\u003c/p\u003e \u003cp\u003eNext, the clean reads were compared to the chloroplast genome sequence, and Pilon v1.23 was used to correct the bases. Finally, the starting position and direction of the chloroplast assembly sequence were determined according to the reference genome, and the 4 chloroplast partitions were determined. The final chloroplast genome sequence was obtained by including large single-copy, small single-copy, and two inverted repeats.\u003c/p\u003e \u003cp\u003eGeSeq (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/geseq.html/\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/geseq.html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software was used to predict protein coding, tRNA and rRNA genes of the chloroplast genome (protein search identity: 60; rRNA, tRNA, DNA search identity: 35; 3rd Party tRNA annotators: tRNAscan-SE). Then, the redundancy of the predicted initial genes was removed, and the head and tail of genes and exon/intron boundaries were removed to obtain a highly accurate gene set that was corrected manually. OrganellarGenomeDRAW (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to circle the sample genome. The preference value of the codon was obtained by calculating the relative synonymous codon usage (RSCU). The software used was Cusp (EMBOSS v6.6.0.0).\u003c/p\u003e \u003cp\u003eThe functional annotations of the sample amino acid sequences were analyzed via the NR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://geneontology.org/\u003c/span\u003e\u003cspan address=\"http://geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), eggNOG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://eggnogdb.embl.de/\u003c/span\u003e\u003cspan address=\"http://eggnogdb.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Swiss-Prot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/UniProt\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/UniProt\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene expression\u003c/h2\u003e \u003cp\u003e RNA was extracted from anthers of three cotton materials using a Plant RNA Extraction Kit (Aidlab Biotechnologies Co. Ltd., China) according to the manufacturer's instructions. Anthers (0.1 g) were ground in liquid nitrogen and centrifuged at room temperature, after which absolute ethanol was added to the supernatant for RNA precipitation. After adsorption to the column, protein and pigment impurities were removed. Three distinct bands with concentrations between 600\u0026thinsp;~\u0026thinsp;1200 ng/\u0026micro;L were detected by 1% agarose gel electrophoresis, and the A260/A280 ratio was approximately 2.0; thus, the extracted RNA could be used for subsequent experiments. The PrimeScript\u0026trade; RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Dalian, Inc.) was used for reverse transcription. All regions of RNA can be uniformly synthesized when an RT Primer Mix combining Random 6-mers and Oligo dT Primer is used as the primer for reverse transcription.\u003c/p\u003e \u003cp\u003eThe relative expression of genes was measured by TB Green\u0026reg;Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) (Takara Bio Dalian, Inc.) using the Bio-Rad CFX Connect\u0026trade; fluorescence quantitative PCR assay system (Bio-Rad Laboratories, Inc.). The primer sequences were shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene cloning and carrier construction\u003c/h2\u003e \u003cp\u003eSpecific primers were designed using NCBI Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The coding sequence (CDS) of genes was amplified, and the plasmid pMD19T was connected to the CDS of genes to transform Trans1-T1 cells (TransGen Biotech Co., Ltd.). After PCR detection, the positive bacterial solution was sent to the company (Beijing Tsingke Biotech Co., Ltd.) for sequencing.\u003c/p\u003e \u003cp\u003eThe pTRV2 plasmid was linearized using restriction enzymes (New England Biolabs Beijing) according to the manufacturer's instructions (primer and restriction enzyme sites in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Recombinant vectors were constructed using a ClonExpress II One Step Cloning Kit (Vazyme International LLC.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVIGS\u003c/h2\u003e \u003cp\u003eThe gene-silenced recombinant vectors were subsequently transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, which was subsequently injected into cotton cotyledons [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eROS detection\u003c/h2\u003e \u003cp\u003eNBT and DAB staining were performed according to a published method [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The NBT staining solution was dissolved in double-distilled water at a concentration of 0.2 mM. The samples were soaked in the solution for 2 h and then decolorized with 95% ethanol. The DAB staining solution was dissolved in redistilled water at a concentration of 1 mg/mL, and the pH was adjusted to 3.8 with concentrated hydrochloric acid. The samples were soaked in this solution for 24 h and then decolorized with acetic acid, glycerin, and 95% ethanol (1:1:3). The samples were observed using a stereomicroscope (Olympus, Germany). \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was measured using Singlet Oxygen Sensor Green reagent (Beijing Biolab Technology Co., Ltd) according to the manufacturer\u0026rsquo;s instructions. SOSG (10 \u0026micro;M) was used for staining the samples. The samples were soaked in solution for 12 h and observed under a confocal microscope. The excitation and emission wavelengths were 504 nm and 525 nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll assays were independently repeated at least three times. Statistical analysis was performed using one-way ANOVA (Tukey\u0026rsquo;s multiple comparison test). Charts were drawn using GraphPad Prism 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCMS Cytoplasmic male sterility \u003c/p\u003e\n\u003cp\u003ePCD Programmed cell death \u003c/p\u003e\n\u003cp\u003eROS Reactive oxygen species \u003c/p\u003e\n\u003cp\u003ePSI Photosystem I\u003c/p\u003e\n\u003cp\u003ePSII Photosystem II\u003c/p\u003e\n\u003cp\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u0026bull;\u003c/sup\u003e Superoxide anion\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Hydrogen peroxide\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e Singlet oxygen\u003c/p\u003e\n\u003cp\u003eSNP Single nucleotide polymorphisms\u003c/p\u003e\n\u003cp\u003eNBT Nitrotetrazolium blue chloride \u003c/p\u003e\n\u003cp\u003eDAB 3,3\u0026apos;-diaminobenzidine\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Jianshe Wang and Dr. Haiyan Zhao provided useful suggestion for us.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLZ, PJ, BG and JS planned and designed the research. LZ, JZ, DL and YY performed experiments. LZ, JH and YQ wrote the manuscript. All authors discussed the data and approved the final version of the manuscript submitted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D program (No. 2018YFD0100301), Basic Research Project of Shanxi Province (No. 202203021211271), Shanxi Agricultural University Research Fund (No. 2020xshf40), and Shanxi Graduate Education Innovation Plan (No. 2021Y312).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are publicly available. These data can be found at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1106185.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo plants in this study were collected from the wild. The plant \u0026ldquo;Jin A, Jin B\u0026rdquo; used in this study were cultivated from and deposited in Cotton Breeding Laboratory of Shanxi Agricultural University. All materials were grown at the Farm Station of Shanxi Agricultural University, and all the experiments were carried out in Shanxi Agricultural University. All methods were compliance with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChai D, Li C, Song H. Research progress of cruciferous crops CMS and related open reading frames. The 11th Symposium of Cruciferous Vegetable Branch of Chinese Horticultural Society. 2013 (in Chinese).\u003c/li\u003e\n\u003cli\u003eZhao X, Zhou S, Gu H. Progress, problems and countermeasures of three-line hybrid cotton breeding in Xinjiang. 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Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 2006; 141: 391-396. \u003c/li\u003e\n\u003cli\u003eKrieger-Liszkay A. Singlet oxygen production in photosynthesis. J. Exp. Bot. 2005; 56: 337-346. \u003c/li\u003e\n\u003cli\u003eMark AG, Gibbs JG, Lee TC, Fischer P. Hybrid nanocolloids with programmed thre e-dimensional shape and material composition. Nat. Mater. 2013; 12: 802-807. \u003c/li\u003e\n\u003cli\u003eDogra V, Kim C. Singlet oxygen metabolism: from genesis to signaling. Front. Plant Sci. 2020; 10: 1640.\u003c/li\u003e\n\u003cli\u003eLi L, Li J. Chloroplast thylakoid membrane polypeptides and cytoplasmic male sterility. Cur. Genet. 1986; 10: 947-949. \u003c/li\u003e\n\u003cli\u003eGalau GA, Wilkins TA. 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Comparative chloroplast genomics of \u003cem\u003eGossypium\u003c/em\u003e species: insights into repeat sequence variations and phylogeny. Front. Plant Sci. 2018; 9: 376. \u003c/li\u003e\n\u003cli\u003eKim C, Meskauskiene R, Zhang S, Lee KP, Lakshmanan Ashok M, Blajecka K, Herrfurth C, Feussner I, Apel K. Chloroplasts of \u003cem\u003eArabidopsis\u003c/em\u003e are the source and a primary target of a plant-specific programmed cell death signaling pathway. Plant Cell. 2012; 24: 3026-3039. \u003c/li\u003e\n\u003cli\u003eZapata JM, Gu\u0026eacute;ra A, Esteban-Carrasco A, Mart\u0026iacute;n M, Sabater B. Chloroplasts regulate leaf senescence: delayed senescence in transgenic \u003cem\u003endhF\u003c/em\u003e-defective tobacco. Cell Death Differ. 2005; 12: 1277-1284. \u003c/li\u003e\n\u003cli\u003eAmbastha V, Tripathy BC, Tiwari BS. Programmed cell death in plants: A chloroplastic connection. Plant Signal. Behav. 2015; 10: e989752. \u003c/li\u003e\n\u003cli\u003eShi X. Study on the structure and function of chloroplast ATP synthase subunits in higher plants. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 2001 b (in Chinese).\u003c/li\u003e\n\u003cli\u003eZhao H, Wang J, Qu Y, Peng R, Magwanga RO, Liu F, Huang J. Transcriptomic and proteomic analyses of a new cytoplasmic male sterile line with a wild \u003cem\u003eGossypium bickii\u003c/em\u003e genetic background. BMC genomics. 2020; 21: 859. \u003c/li\u003e\n\u003cli\u003ePang J, Zhu Y, Li Q, Liu J, Tian Y, Liu Y, Wu J. Development of \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated virus-induced gene silencing and performance evaluation of four marker genes in \u003cem\u003eGossypium barbadense\u003c/em\u003e. PloS One. 2013; 8: e73211. \u003c/li\u003e\n\u003cli\u003eWu J, Sun Y, Zhao Y, Zhang J, Luo L, Li M, Wang J, Yu H, Liu G, Yang L, Xiong G, Zhou JM, Zuo J, Wang Y, Li J. Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species. Cell Res. 2015; 25: 621-633.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cytoplasmic male sterility, programmed cell death, reactive oxygen species, ATP synthase, chloroplast genome","lastPublishedDoi":"10.21203/rs.3.rs-4662060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4662060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAbnormal programmed cell death in the tapetum is induced by reactive oxygen species (ROS), which are the main factors leading to cytoplasmic male sterility (CMS). These abnormalities are caused by genetic interactions between nuclear and cytoplasmic genes. No studies have investigated the role of chloroplast ATP synthase in ROS metabolism.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTo explore the role of chloroplast genes in ROS metabolism, sequencing of the chloroplast genome from the next generation and single-molecule real-time sequencing of chloroplast DNA from the CMS line Jin A were performed. The results showed that the length of the chloroplast genome of the CMS line Jin A was 160,042 bp, and the genome consisted of 131 genes, including 112 functional genes. Analysis of the functional annotation and sequence comparison showed that Jin A CMS plants had 29 genes annotated with single nucleotide polymorphisms compared to \u003cem\u003eGossypium hirsutum\u003c/em\u003e plants, including ATP synthase subunits, NAD(P) H-quinone redox reductase subunits, and photosystem complex subunit genes. Compared to those of Jin B maintainer plants, the relative expression of \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, and \u003cem\u003eatpF\u003c/em\u003e significantly decreased in the anthers of Jin A CMS plants at the microspore abortion stage. The relative expression of these genes in the three-line hybrids F1 significantly increased compared with that in the Jin A CMS plants. The ROS levels in the leaves increased in the \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e silenced cotton plants.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, our study showed that \u003cem\u003eatpE\u003c/em\u003e and \u003cem\u003eatpF\u003c/em\u003e of ATP synthase subunits gene were closely related to ROS metabolism. These results provide a basic information for the analysis of ATP synthase function in cotton.\u003c/p\u003e","manuscriptTitle":"Effect of chloroplast ATP synthase on reactive oxygen species metabolism in the cotton cytoplasmic male sterile line Jin A","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 06:45:51","doi":"10.21203/rs.3.rs-4662060/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc172482-4154-45e3-a0dc-308635f115fe","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-15T08:08:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 06:45:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4662060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4662060","identity":"rs-4662060","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}