Key genes and molecular mechanisms responsible for male sterility revealed by transcriptome analysis in cotton

Key genes and molecular mechanisms responsible for male sterility revealed by transcriptome analysis in cotton | 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 Key genes and molecular mechanisms responsible for male sterility revealed by transcriptome analysis in cotton Haili Qiu, Hongyu Dou, Kang Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7281172/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 Identification and characterization of genic male-sterility (GMS) genes is crucial for unraveling molecular mechanisms controlling anther and pollen development, and enable the development biotechnology-based male-sterility (BMS) systems for heterosis utilization and commercial hybrid seed production in crops. Here, we report a combined cytological and transcription analysis of the anther of a single-gene recessive GMS line Nan A and its near-isogenic male fertile line Nan B, and further verified the functions of two male sterility candidate genes. Nan A developed shorter stamen filaments, produced sterile pollens characterized by shriveled starch grains inside, delayed nexin deposition, no spikes on exine surface, and failure in dehiscence. A number of anther-preferentially expressed genes were unexpectedly up-regulated in Nan A, whereas loss-of-function mutants of their homologous genes in other plant species exhibit male sterility. By contrast, a number of stress-related transcription activation protein genes are down-regulated in Nan A. Either silencing the anther specifically expressed GhCYP450 that down-regulated in Nan A or overexpressing GhPHD-D that up-regulated in Nan A can convert wild-type into male sterility. Our results indicate that timely expression of anther and/or pollen developmental genes are pivotal for male fertility. Cotton Genic male sterility RNA-seq VIGS VOX Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message A novel male sterility line Nan A forms normal microspore tetrads, but defective pollen development. Dysregulated fertility/stress-related genes in anthers cause cotton male sterility. Introduction Cotton ( Gossypium spp.) is a major cash crop in the world supplying approximately a quarter of global textile fibers, and cottonseed is an important source of oil a vital source of protein for human consumption as byproducts. Cotton displays strong heterosis, the heterozygous first filial (F 1 ) generation typically generates 15–30% more fiber than traditional types, big seed size, early flowering, high fiber quality, biotic and abiotic stress tolerances over its parents (Zhang, et al. 2023 ). Therefore, breeding for cotton hybrid varieties is a promising way to tremendously improve cotton productivity. Currently, most commercially used F 1 hybrid cotton seeds are produced by hand-emasculation and pollination, the strong heterosis combinations were screened from extensive cross combinations of pure line parents without need to introduce the male sterility and restorer genes into the parents, which simplifies the breeding process and reduces the breeding cost. Additionally, using F 2 heterosis can reduce the cost of seed production by up to 10 times. However, the F 2 vigor is much lower than that of F 1 because of inbreeding depression. Reducing the production cost of F1 seeds and utilizing F1 hybrid vigor is an important direction for cotton breeding. The application of a male sterility line can facilitate large-scale production of crop hybrids. The application of male sterile line can eliminate the need for hand emasculation, facilitate breeding hybrid varieties, and greatly reduce the production cost of hybrid seeds. Male sterility can be generated by either cytoplasmic or nuclear genes. Cytoplasmic male sterility (CMS) is caused by mitochondrial genes together with nuclear genes and has been used in commercial hybrid cotton production (Li, et al. 2023 ), but this method could suffer from unreliable restoration or marginal heterosis. Genic male sterility (GMS) is caused by nuclear gene alone. The use of GMS can overcome these drawbacks, but it is difficult to segregate sterility lines from maintainer lines. The advent of seed production technology (SPT) has significantly advanced the field of maize hybrid seed production by overcoming the challenges associated with genic male sterility (Wu, et al. 2016 ). Identify and utilize GMS genes for developing efficient biotechnology-based male sterility systems in crops including cotton will revolute harnessing heterosis. 19 GMS genes (loci) have been genetically identified in cotton (Lan, et al. 2006 ), ms 2 (1355A), ms14 (Dong-A) and ms5ms6 have been utilized to produce hybrid seed. Gene responsible for ms 2 was identified as a mutation of GhNSP through map-based cloning and confirmed by genetic analysis. GhNSP encodes a polygalacturonase protein, biochemical analysis showed that de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation in the mutant (Wu, et al. 2022 ). Ms5 and Ms6 loci were identified through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 genome editing. Ms5 and Ms6 encode the cytochrome P450 monooxygenases CYP703A2-A and CYP703A2-D, the duplicate mutations of CYP703A2 genes encoding a cytochrome P450 protein essential for pollen exine formation and pollen development resulted in producing male sterility for the duplicate ms5ms6 line (Ma, et al. 2022 ; Mao, et al. 2023 ). The detailed description and elucidation of the cellular biology process of microspore development in Arabidopsis, from the formation of stamen primordia to the final maturation, dehiscence, and pollen dispersal (Schiefthaler, et al. 1999 ), provides a reference for the analysis of microspore development in other plants, including cotton (Xu, et al. 2014 ). The molecular, genetic, and biochemical pathways regulating anther and pollen development and information about GMS in plants provide opportunities to identify and utilize male sterility in economically important crops. MS 1 encodes a PHD (plant homeodomain) transcription factor in Arabidopsis, the ms 1 mutant showed abnormal vacuolization of tapetal cells and delayed PCD, leading to male sterility, but over expression of PHD resulted in partial fertility (Ito, et al. 2007 ; Yang, et al. 2007 ). The maize genic male sterility gene Ms 5 encoded a GDSL lipase, mutation of this gene disrupts lipid metabolism process and appropriate degradation the middle layer and tapetum of the anther which leads to pollen production (Huo, et al. 2020 ). Similarly, a lipid transfer protein that is exclusively produced in tapetum cells is encoded by the maize genic male sterility gene Ms 44 . Changes in the individual amino acids of the Ms 44 protein cause disruptions in the processing of proteins, which in turn leads to aberrant development of microspore mother cell and the prevention of tapetum cell secreting proteins into the pollen chambers (Fox, et al. 2017 ). In other studies, the UDT1 -encoded bHLH transcription factor in rice, the OsTDF1 -encoded R2R3 MYB transcription factor, and the OsGPAT3 -encoded glycerol 3-phosphate acyltransferase are required for normal development and degradation of the tapetal layer, mutation of any of them ultimately leads to sterility (Jung, et al. 2005 ; Men, et al. 2017 ). Developed anthers release pollen grains, normal pollen wall development serves as essential for pollen fertility (Ariizumi and Toriyama 2011 ). Sporobollenin is the primary constituent of the exine of pollen, and its comparatively steady physicochemical qualities account for the robust resistance and protection of the pollen wall. Phenylpropane-like compounds and aliphatic derivatives make up the majority of sporopollenin, whose synthesis influences pollen grain fertility. Cotton CYP703A2-A and CYP703A2‐D , maize Ms 10 and Ms 26 , etc. encode cytochrome P450 mono‐oxygenase which plays a central role in sporopollenin formation (Chen, et al. 2017 ; Ma, et al. 2022 ). The ATP-binding cassette G (ABCG) transporter protein, which is encoded by maize Ms 2 , is primarily involved in the transmembrane transport of substances during anther development. Lack of function in this protein leads to a decrease in lipid levels in the anthers and interferes with the normal formation of pollen grains (Xu, et al. 2021 ). Maize Ms 33 encodes a glycerol‑3‑phosphate acyltransferase (GPAT), which is expressed preferentially in immature anthers and root tissues, and is involved in fatty acid metabolism processes in tapetum layer, thereby regulating the synthesis and metabolism of maize anther cuticle substances and the formation of sporopollenin substances in exine (Zhu, et al. 2020 ). Maize Ms 8 encodes a β-1,3-galactosyltransferase protein, which is involved in the synthesis of important substances during anther development, the metabolism of substances in mitochondrial organelles, and the regulation of programmed cell death (PCD) in tapetal cells during anther development. Mutations in this protein cause abnormal development of the microspore mother cell (Wang, et al. 2013 ). OsACOS12 (Li, et al. 2016 ), OsPKS1 (Zou, et al. 2017 ), OsPKS2 (Zhu, et al. 2017 ), OsTKPR1 (Xu, et al. 2019 ), CYP703A3 (Yang, et al. 2014 ), CYP704B (Li, et al. 2010 ), and NP1 (Liu, et al. 2017 ) proteins in rice form sporopollenin precursors in the endoplasmic reticulum and efficiently produce lipid and phenolic precursors for sporopollenin biosynthesis. Nan A ( ms a ) is a spontaneous mutant characterized by male sterility controlled by a single recessive nuclear genetic locus in upland cotton, which provides us a good material to investigate genetic, cytological, and molecular mechanism underlying pollen development and male sterility in cotton. Here, we first employed optical and election microscopy to examine the mutant pollen development to show the cytological and structural alterations responsible for aberrant pollen formation, then we compared the transcriptome differences between the anthers of Nan A ( ms a ms a ) and its isogenic fertile line Nan B ( Ms a ms a ) to discover genes crucial for normal micropore development. And finally, two differential expression genes were selected for function analysis by using virus-induced gene silencing (VIGS) assay and virus-mediated gene overexpression assay (VOX), respectively. This study provides a new clue for understanding the molecular basis of male sterility and exploring male sterility genes in cotton. Results The key period for male sterility of Nan a is after tetrad stage Nan A and Nan B were planted adjacent to each other in the breeding nursery to minimize the impact of environmental differences on plant phenotype. We found no visible differences in the plant architecture, growth, flowering, and boll setting between Nan A and Nan B (Fig. 1A), except that the size of the filaments and anthers of the former is significantly smaller than that of the latter (Fig. 1D). When we imaged the anthers using the MVX10 microscope, we found that the anthers of Nan B dehisced normally and gave off plump pollens stained brown black with I 2 -KI (Fig. 1B, 1E), while the anthers of Nan A remained unopened, the wrapped pollens can hardly stain with I 2 -KI (Fig. 1C, 1F). To pinpoint the critical period and characteristics of male sterility in ms a ms a of cotton, we examined the microstructures of the anthers from 2 mm to 10 mm flower buds of male sterile and fertile plants using Carbolfuchsin staining followed by microscopy. The results showed that the developmental dynamics did not differ significantly at the meiosis stage corresponding to 4–5 mm buds and the tetrad stage corresponding to 5–6 mm buds, both Nan A and Nan B appear to be able to form normal tetrads inlaid in callus wall (Fig. 1G, 1H, and Fig. 1J, Fig. 1K). Thereafter, the callus around the tetrad began to disintegrate, when buds grow to 6–7 mm in length, the fertile anthers of Nan B released well-stacked pollen with spikes on exine (Fig. 1I), instead, the sterile anthers from Nan A generated unfilled pollens without spines on outer surfaces that cannot release form the anther chambers (Fig. 1L). Therefore, male sterility of Nan A may result from distorted microspore development and pollen maturation. Ms a is required for anther development and pollen formation in cotton To further characterize the cytological defects in ms a anthers, we performed scanning electron microscopy (SEM), transverse section, to compare the anthers at 1 d before anthesis, the results showed that there were a large number of prickly pollens scattered inside the vacuolated anther chamber of Nan B (Fig. 2A, B, and C). In contrast, there were very few deformed pollen grains without spikes on smooth exine clinging together to the inner wall of the relatively much smaller anther chamber (Fig. 2D, E, and F). These results confirm that ms a disrupts anther development and pollen formation. In order to further analyze the causes of the developmental defects of pollen wall in Nan A, anthers of flower buds 6–7 mm in length and 1 d before anthesis were taken for transmission electron microscopy (TEM) observation, respectively. In 6-7mm long bud, i.e., at stage 8, the construction of the outer wall of Nan B pollen commenced with the production of a substantial intine, noticeable spikes, and ample, well-developed starch grains in the lumen (Fig. 2G, H). In contrast, the nexine of Nan A pollen was less pronounced than that of Nan B, and the exine lacked any obvious protrusions, and the starch grains in the lumen being small and closely packed (Fig. 2J, K). The exine may be further elaborated with additional sporopollenin deposition, e.g., production of inner-column-shaped baculae and the surface decoration tectum. At 1d pre-anthesis, the nexine of Nan B pollen was remarkably thinner than that observed at stage 8, although a clear intine layer could be discerned (Fig. 2I), while Nan A pollen at the same stage displayed a thickened nexine but no intine layer was visible (Fig. 2L). These discrepancies imply that the hindered synthesis of pollen wall components and the inability to develop and form a typical pollen wall might be the root cause of pollen sterility in Nan A. Critical genes and networks involved in male sterility in Nan A To elucidate the genes and molecular mechanisms critical for male sterility in Nan A, we extracted RNA from the anthers of 6 mm long buds for RNA-seq analysis. A total of 569 differentially expressed genes (DEGs) in Nan A and Nan B buds were identified according to the method of reads per kilobase per million mapped reads (RPKM) on the basis of the applied criteria of q-value 1. Among those genes, compared with the Nan B buds, 249 (44%) were upregulated and 320 (56%) were downregulated in Nan A buds during microspore development (Supplementary Table S2 ). The housekeeping gene upland cotton His3 (histone 3) was used as an internal control for data normalization, 24 DEGs were chosen for qRT-PCR analysis to verify their expression levels (Fig. 3A). The figure shows that the differential expression of 19 genes determined by RNA-seq or qRT-PCR was consistent, account for 79.2% of the tested genes, but five genes (20.8%), i.e., GH_A02G0833 , GH_D02G0848 , GH_A05G0056 , GH_A08G2216 , and GH_A03G1602 , were significantly down-regulated in the anthers of the sterile line, which is opposite to RNA-seq results. This part of the inconsistency may be due to the sampling batch of different bud size corresponding to the development period is not completely consistent. Pearson’s correlation coefficient between the qRT-PCR data and RNA-seq data is 0.46, reaching moderate level of correlation, thus our RNA-seq data were are acceptable and conducive to identification of critical genes involved in anther development. To understand how DEGs specific to ms a line affect the pollen sterility process in Nan A cotton, GO class enrichment analyses of down- and up-regulated genes for Nan A were conducted, respectively. 249 up-regulated genes were significantly enriched in ‘transporter activity’ molecular function (MF), ‘response to endogenous stimulus’ biological process (BP), and ‘reproductive process’ BP (Fig. 3C, Supplementary Table S3 ). Among the 21 upregulated genes enriched in GO terms related to the reproductive process, nine gens including GH_D13G1652 (NAC), GH_D04G1012 (NFYA2), GH_A12G2483 (PHD, MALE STERILITY 1), GH_D12G2497 (PHD, MALE STERILITY 1), GH_D02G1428 (CRABS CLAW, CRC), GH_A12G1852 (TCP15), GH_D12G1848 (TCP15), GH_A05G1589 (WOS9, WUSCHEL-related homeobox 9) and GH_D05G1617 (WOS9) encode 6 transcription factors or transcription activators; six genes i.e. GH_D09G1973 (β-1,6-galactosyltransferase, GALT31A), GH_D12G0737 (glycerol-3-phosphate acyltransferase 1, GPAT1), GH_A12G1040 (GPAT1), GH_D02G2614 (polygalacturonase3, QRT3), GH_D04G1012 ( O -Glycosyl hydrolase), and GH_D02G0220 ( O -Glycosyl hydrolase) encode 4 proteins involved in metabolism; other three genes, GH_D12G2099 encodes a HVA22-like protein, GH_D11G0805 encodes a casein kinase subunit (CKB4), and GH_A10G0810 encodes a Rop guanine nucleotide exchange factor 12 (ROPGEF12). The homologs of these genes in other plants have been proved to be male sterility genes or related to pollen fertility. It is also worth noting that ‘reproductive process’ and ‘response to endogenous stimulus’ have eight genes in common, while ‘reproductive process’ has no intersection with ‘transporter activity’, suggesting that endogenous signaling might be involved in ms a -caused male sterility. 320 down-regulated genes are significantly enriched in four functionally related MFs of ‘DNA-binding transcription factor activity’, ‘transcription regulator activity’, ‘DNA binding’, and ‘nucleic acid binding’ in which genes are highly overlapped, with an additional ‘signaling receptor binding’ containing five unique genes. The most dominant biological processes of these genes are six ‘response’ related terms, in which genes are in a high degree of overlap. Three metabolism-related BPs also have a high proportion of identical genes. All of these genes are significantly enriched in nucleus (Fig. 3C, Supplementary Table S3 ). Remarkably, the enriched MF term of ‘transcription regulator activity’ and the BP term of ‘response to stress’ share 30 common genes, which account for 75% of the genes enriched in ‘transcription regulator activity’, moreover, 71 genes in ‘response to stress’ can be significantly enriched in ‘DNA-binding transcription factor’ per contra . So we may conclude that most proteins with transcriptional activation activity that are suppressed in ms a play major roles in stress response. In response to stress, ethylene (12 ERFs, 2 MBF1Cs, 1 ARB1, and 1 ACS6), ABA (3 DREB1Ds, 1 F23F1.6, 1 PYL2, 1 EDL3, and 1RDUF1), jasmonate (3 WRKY53s, 2 CYP94C1s, 1 TIFY9, 1 ZAT10, and 1 AOC4) may perform major signaling roles (Fig. 4). This may be because 7–8 stage is characterized by anthers that are more susceptible to high temperature in Nan A. Jasmonate mediated signaling have been reported to be relevant to male fertility in Arabidopsis . To further pursue the genes fatal for cotton male fertility, all the DEGs identified between Nan A and Nan B anthers in the present study were subjected for spatial expression analysis by querying the Cotton RNA-seq Database ( http://ipf.sustech.edu.cn/pub/cottonrna/ ), 22 DEGs were found specifically or preferentially in anthers, and their expression levels in Nan A and Nan B were showed in Fig. 5. Intriguingly, nineteen of the 22 DEGs have been proved to play an important role in male sterility or microspore development in cotton, Arabidopsis thaliana, rice and other plants. However, these genes related to male sterility caused by loss of function mutation were up-regulated in the sterile anther of Nan A, except GH_A12G1289 which was down-regulated in the anther of NanA (Fig. 5, Table 1). Silencing GhCYP450 expression in WT lead to sterile pollens GH_A12G1289 is only one anther preferentially expressed gene that is significantly suppressed in Nan A. The homologous GH_A12G1289 gene in Arabidopsis encodes a cytochrome P450 enzyme catalyzing the hydroxylation of lauric acid, which provides the raw material for sporopollenin synthesis and thus affects the synthesis of exine. The loss of P450 mutant causes male infertility in maize and rice. Therefore, we hypothesized that GH_A12G1289 is a candidate sterility gene for Ms a . We cloned a 500-bp fragment of the GhCYP450 CDS and inserted it into pCLCrVA vector for VIGS assay to silence the expression of GH_A12G1289 in TM-1. The typical photobleaching phenotype generated by silencing the cotton magnesium chelatase subunit I ( CHLI ) gene was employed to monitor the silencing efficiency of endogenous gene as a positive control (Fig. 6A). The expression of GhCYP450 was almost completely silenced in the anthers of the cotton plants infected with CLCrV- GhCYP450 plants compared to that of TM-1 infected with empty CLCrV vector (Fig. 6B). GhCYP450 -silenced plants showed no significant alterations in most aspects of phenotype compared with the empty control, but on the day of flowering, the filaments of the silenced plants became much shorter (Fig. 6H), the anthers cannot dehisce, and there were no pollen grains on the outer surface of the anthers (Fig. 6I), in contrast, the control plants developed long filaments and plump anthers which dehisced to disperse a great number of pollen grains (Fig. 6D, E). Consequently, the control plants produced fertile pollens that were stained dark brown with I 2 -KI (Fig. 6F), and germinated in vitro. at an average rate of 37.6%, while the silenced plants produced shriveled and deformed pollen with spikes on outer surface, which can only weakly stain with I 2 -KI and unable to germinate on culture medium (Fig. 6J, K). In summary, silencing GhCYP450 expression gave up to male sterility, but the sterile pollen coated with a layer of spiky sexine, this is different from those without spikes produced in Nan A. It seems that merely silencing GhCYP450 is not sufficient to achieve the effects of the ms a mutation gene, namely ms a has multiple molecular functions in regulating cotton microspore development. Overexpression of GhPHD-D induced male sterility in upland cotton GH_A12G2483 and GH_D12G2497 two anther preferentially expressed genes sharing 98.12% (37 of 1968 bp) similarity in CDS sequence encoding a PHD- finger transcription factor. They were significantly up-regulated in Nan A by more than 200 folds (Fig. 5). However, its homologous genes were reported to be required for the formation of exine and the development of tapetum cells in Arabidopsis, Chinese cabbage, rice, and maize, but premature expression of ZmMs7 (encoding a PHD-finger transcription factor) disrupts tapetum and pollen development and results in male sterility in maize (An, et al. 2020 ). These results raise a question about the effect of over expression of GH_A12G2483 or GH_D12G2497 on cotton pollen fertility. Therefore, we cloned the CDS sequence of GH_D12G2497 , and inserted it into pCaBS-γ2 vector to generate pCaBS-γ2- GhPHD-D for BSMV-VOX assay. The results showed that GhPHD-D expression level in the anthers of the VOX TM-1 plants soared more than 200-fold (Fig. 7A). The GhPHD-D -overexpressed plants exhibited exactly the same vegetative growth as normal plants. On the day of flowering, there was no significant difference in filament length between overexpressed plants and normal plants, but the basal filament tube of the monomeric stamen was relatively short, and the filaments were concentrated at the base of the columella so that the anthers were far from the stigma (Fig. 7C, G). Notably, the anthers of overexpressed plants cannot dehisce and release pollen as normal plants (Fig. 7D, H). The pollen grains produced by GhPHD-D -overexpressed cotton were smaller in size, deformed, with spikes on the outer wall of pollen, and can be lightly stained brown with I 2 -KI, which cannot germinate in vitro culture (Fig. 7J, K). In contrast, the anthers of the empty vector control plants developed normally, producing plump pollen grains that could be stained brown black by I 2 -KI and could germinate in vitro in the culture medium (Fig. 7F, 7G). Together, either overexpression of GhPHD-D or silencing GhCYP450 can lead to male sterility in cotton, and the sterile pollen grains are similar in shape coated with spikes. These results further confirm that ms a may regulate the development and maturation of cotton microspores through a complex molecular pathway to determine pollen fertility. Materials and methods Plant materials The cotton genic male sterile mutant was originally discovered in a cotton cultivar Sumian 22 and named Nan A. The male sterile mutant was continuously backcrossed with Sumian 22 for more than 12 generations, and developed a pair of isogenic lines Nan A (male sterility) and Nan B (male fertility). All plants were planted in the breeding nursery of Nanjing Agricultural University, Jiangsu province under regular field management. Optical microscopy, scanning and transmission electron microscopy Flower observation was conducted using a Digital microscope DVM6a (Leica, Germany). 4-5mm, 5-6mm, and 6-7mm long buds were collected between 6:00 am to 8:00 am, and immediately put into Carnot's fixative and stored at 4°C for miosis slide preparation. The anthers were rinsed with ddH 2 O, dissociated in 0.1M HCl for 8 min, then washed with water three times, and placed onto a slide. A drop of carbolfuchsin was added to stain the anthers on slides, and then placed a coverslip on the sample, the anthers were crushed by gently tapping the coverslip with a needle, and was subsequently observed with fluorescence microscope BX53 (Olympus, Japan) under light field. For scanning electron microscopy, anthers at different developmental stages were peeled off the stamens and immediately put into the fixative containing 2.5% (v/v) glutaraldehyde and 1% (v/v) paraformaldehyde, vacuumed so that the anthers were fully submerged, and fixed overnight at 4°C. Next, the anthers were washed and postfixed in 1% osmium tetraoxide in 0.1 M sodium phosphate buffer (PBS, pH 7.2) for 2h, followed by washing with Milli-Q water for 10 min. Samples were then dehydrated in increasing concentrations of ethanol solution (30, 50, 70, 95 and 100%), and dried in 100% ethanol with liquid CO 2 . Finally, the dried anthers were carefully cut into pieces using a razor blade and placed on the sample stage using conductive adhesive, followed by sputtering with gold palladium for 300 s at 25 mA, and visualized using scanning electron microscope (Regulus 8100, Hitachi, Japan). The above fixed anthers were washed three times (5 min for each) with 0.1M PBS, postfixed in 1% (w/v) OsO 4 for 2 h, and washed with PBS three times, 5 min for each. Samples were then dehydrated as described above, treated with propylene oxide, and embedded in Spurr’s resin. Thin sections (70 nm) were taken using the Leica UC6 cryoultramicrotome. Sliced sections were placed on 100-mesh copper grids and sequentially stained with uranylacetate (30 min) and lead citrate (Sato’s Lead; 15 min). Transmission electron microscopy was performed using a Hitachi HT7800 transmission electron microscope. RNA extraction, RNA-seq, and data analysis Six mm long anthers from Nan A and Nan B buds were collected, the anthers were then ground into powder in liquid nitrogen, total RNA was extracted using the Plant Total RNA Isolation Kit (Novozymes, China) following the manufacturer’s protocol. The total RNA quantity and purity were analyzed on NANODROP ONE (Thermo Scientific, USA) and evaluated by agarose gel electrophoresis. Six RNA samples represent three biological repeats were entrusted to the Nanjing Personalbio Technology Company for paired-end cDNA library construction, and the libraries were subjected to sequencing on Illumina HiSeq 4000 platform according to the protocol recommended by the manufacturer. The raw data were first filtered to produce clean data, HISAT2 was used to map reads to the reference genome ( http://cotton.zju.edu.cn/index.htm ). The mapped reads of each sample were assembled using StringTie. The final transcriptome was generated using Perl scripts. DESeq was used to analyze the differentially expressed genes (DEGs). DEGs were selected with | \(\:{\text{log}}_{2}\text{f}\text{o}\text{l}\text{d}\:\text{c}\text{h}\text{a}\text{n}\text{g}\text{e}\) |>1 coupled with statistical significance ( p -value < 0.05). GO enrichment and KEGG pathway enrichment analysis was performed on the DEGs using the TBtools-II software(Chen, et al. 2023 ), the corrected p-value ≤ 0.05 was set as the threshold and rich factor. The tempo-spatial expression characteristics of the DEGs were analyzed using the Cotton RNA-seq Database ( http://ipf.sustech.edu.cn/pub/cottonrna/ ) to screen anther preferentially expressed genes. Protein-protein interaction networks were generated using STRING (version 12.0, https://cn.string-db.org/cgi/input?sessionId= bsDGvjLcMSbg&input_page_show_search = off), the network was visualized with Cytoscape (Version 3.10.2). qRT-PCR analysis Total RNA was extracted from the anthers of 6 mm long buds of Nan A and Nan B using the same method described in 2.6, the first strand cDNA synthesized with HiScript® Ⅲ 1st Strand cDNA Synthesis Kit (+ gDNA wiper) reverse transcription kit (Novozymes, China). Primers for qPCR were designed using online software PrimerQuest ( http://www.idtdna.com/Primerquest/ ) and Primer-BLAST ( http://www.ncbi.nlm.nih.gov/tools/ primer- blast/), synthesized commercially, and are shown in Supplementary Table S1 . His3 gene was used as the internal control and three biological replicates were used for each sample. Gene relative expression levels were calculated using the 2 −ΔΔCt method. Virus‑induced gene silencing (VIGS) assays We used cotton leaf crumple virus (CLCrV)-based vectors viz pCLCrVA and CLCrVB for VIGS experiments. The gene-specific 500 bp fragments for magnesium chelatase subunit I ( GhCHLI ) and GhPHD-D and were amplified by PCR from TM-1 cDNAs and inserted into pCLCrVA respectively(Gu, et al. 2014 ). pCLCrVA, pCLCrVA- GhCHLI , pCLCrVA- GhPHD-D , and pCLCrVB were individually transformed into Agrobacterium tumefaciens strain GV3101. Agrobacterium cultures were grown overnight in YEP medium containing rifampicin (50 mg⋅L − 1 ) and kanamycin (50 mg⋅L − 1 ) at 28°C. The Agrobacterium cultures were pelleted and resuspended (OD600 = 1) individually in an infiltration buffer containing 10 mM MgCl 2 ,10 mM 2-(4-Morpholino) ethanesulfonic acid and 200 µM acetosyringone. After 3h incubation at room temperature, Agrobacterium harboring pCLCrVA or one of its derivatives was mixed with an equal volume of Agrobacterium harboring pCLCrVB. The mixed Agrobacterium solutions were infiltrated into the abaxial side of cotyledons of 2-week-old cotton seedlings using needleless syringes. BSMV-mediated gene overexpressing (VOX) assay VOX assay was conducted according to a previous study with minor modifications (Chen, et al. 2022 ). The CDS of GhPHD-D was amplified from the cDNA of TM-1 by using PCR and inserted into pCaBS-γ2 via Gibson assembly, which allow to express the inserted gene driven by CaMV35S promoter. The pCaBS-α, pCaBS-β, pCaBS-γ1, and pCaBS-γ2 harboring the interested gene were transformed into Agrobacterium tumefaciens GV3101, respectively. Four Agrobacterium strains (OD600 = 0.80) were mixed in equal volumes and infiltrated into the leaves of N. benthamiana with a syringe, the tobacco was incubated for 24 hours after the injection in dark at 26°C, then grew at 26°C for six days. 0.5 g of the infected leaves were harvested and ground in 1mL of 20 mM phosphate buffer (pH 7.2). The homogenate was stored at -20°C for later or direct viral inoculation. The virus homogenate was diluted 30 times before injected into the cotyledons 10-day-old cotton seedlings as described above in VIGS. The wound leaf surface was sprayed with ddH 2 O, the seedlings were inoculated in a dark hood with 70% humidity for 1 h at ambient temperature, and transferred into artificial climate chamber to grow at 26°C, 8 h light / 16 h dark photoperiod. In vitro pollen germination and pollen KI-I2 staining Pollen grains from crushed anthers (Nan A, Nan B, GhCYP450 -silenced, GhPHD-D -overexpressed plants) were placed on petri dishes at 30°C for 4 h in a pollen germination medium consisting of 0.04% (w/v) calcium nitrate, 0.01% (w/v) glutamic acid, 0.07% (w/v) manganese sulfate, 0.01% (w/v) lysine, 0.01% (w/v) serine, 0.01% (w/v) proline, 0.02% (w/v) boric acid, and 40% (w/v) sucrose. The relative humidity was maintained at above 80%. The 1% (w/v) iodium potassium-iodide solution was used for pollen fertility staining. The germination pollen grains and KI-I 2 stained pollens were observed with a microscope (Olympus, BX53) in bright-field illumination. Three biological repeats, five fields per sample were photographed for statistics. Discussion Harnessing hybrid heterosis is an important way to increase cotton yield, improve fiber quality and enhance disease resistance. Male sterile lines are widely used in many crops to effectively facilitate hybrid breeding and seed production (Zhang, et al. 2023 ). But it is difficult to obtain a pure and large-scale increase of male-sterile female lines through self-pollination when GMS is used. Biotechnology-based male sterility (BMS) system can overcome this limitation and significantly improve the efficiency of hybrid seed production, and is expected to be an important tool for the efficient use of hybrid vigor in cotton and other crops. Mining male sterility genes and understanding their mechanisms are important prerequisites for the creation and application of BMS systems in cotton. In this study, we identified a GMS line Nan A controlled by a pair of recessive genes, this mutant can develop normal tetraploids, but fail in microspore development, transcriptome analysis indicate that ill-time overexpression of anther preferentially expressed genes, most of which are required for male fertility. A large set of genes encoding transcription activation proteins involved in stress response were downregulated in the GMS line. These genes are thus potential targets for developing male sterile lines through genetic engineering for the exploitation of cotton hybrid vigor. Nan A is mutated in pollen dehiscence and development Nan A is a spontaneous mutant fortuitously discovered from cotton cultivar Sumian 22 in our breeding nursery. This mutant was preliminarily identified to be controled by a single recessive gene. Although its accurate chromosome localization awaits further genetic mapping, the near isogenic fertile (Nan B) and sterile plants (Nan A) generated by backcrossing Nan A with its original cultivar Sumian 22 provide ideal isogenic lines for joint phenotypical, cytological and molecular analysis to dissect mechanisms underlying male sterility of Nan A. Nan A has normal vegetative phenotypes very similar to Nan B, but developed much shorter filaments, non-dehiscent anthers without disperse pollens, and pollen grains can be slightly stained using I 2 -KI, and the isolated pollen grains cannot germinate on culture medium. Optical microscopy and SEM analysis showed that Nan A formed normal tetrads at stage 7, but could not release normal microspores with spines on exine at stage 8 as observed in the WT, Nan B. This suggests that sterility in Nan A likely occurred between Stage 7 and Stage 8. At Stage 8, the pollens produced by Nan A are significantly different from those produced by Nan B in formation of nexine, exine together with spines. These phenotypes are highly similar to those observed in the sterile lines ZK-A ( ms 5 ms 6 ) (Ma, et al. 2022 ; Mao, et al. 2023 ) and 1355A ( ghnsp ) (Wu, et al. 2022 ; Wu, et al. 2015 ), their male sterility all occur during S7 to S8 of anther development, indicating that this period is critical for microspore development and pollen wall construction that determining anther dehiscence and pollen dispersal, and consequently controlling pollen fertility in upland cotton. From these results, we can also speculate that there may be many genes involved in this development stage, and the mutation of any key gene of them may cause abnormal microspore development or abnormal pollen maturation process. Transcriptomics unravels genes and molecular mechanisms underlying male sterility in cotton RNA-seq analysis of Nan A and Nan B anthers from S7 to S8 showed 569 DEGs. GO and KEGG enrichment analyses indicated that the 249 up-regulated genes were significantly enriched in ‘transporter activity’ MF, ‘response to endogenous stimulus’, and ‘reproductive process’ BPs, and there are 21 genes enriched in GO term of ‘reproductive process’. On the other hand, we identified 22 DEGs specifically or dominantly expressed in cotton anther. GH_D02G2614 (QRT3), GH_A12G2483/GH_D12G2497 (PHD), and GH_A02G0201 / GH_D02G0220 / GH_A12G1628 (GHL 17) are anther preferentially expressed genes related to ‘reproductive process’. These genes were all up-regulated in Nan A, and their homologs have been reported to be causative genes for male sterility in various plant species (Table 1). Among the ‘reproductive process’ proteins, GH_D13G1652 is a NAC transcription factor, its homologs in A rabidopsis , NST1 and NST2 regulate secondary wall thickenings and are required for anther dehiscence, double mutant nst1 nst2 exhibited male sterility (Mitsuda, et al. 2005 ); GH_A12G1040 / GH_D12G0737 (GPAT1) are involved in biosynthetic process of CDP-diacylglycerol, which is a critical intermediate in lipid metabolism, their homologs in rice (Men, et al. 2017 ), maize (Xie, et al. 2018 ), Arabidopsis (Zheng, et al. 2003 ) etc. are essential for male fertility; GH_A10G0810 encodes a Rop guanine nucleotide exchange factor 12 (ROPGEF12) which is required for pollen tube and control polarized pollen tube growth. As shown in Table 1, most anther predominantly expressed DEGs are related male fertility. Collectively, a substantial number of genes, whose analogs known as the causative genes for male sterility or required for male fertility, are up-regulated in Nan A. Paradoxically, all of these genes are recessive male sterility genes, namely loss-function of them confers male sterility, but they are up-regulated in Nan A, a recessive genic male sterility. For example, mutation of PHD-finger proteins cause male sterility in soybean (Thu, et al. 2019 ), maize (Zhang, et al. 2018 ), rice(Yang, et al. 2019 ), Chinese cabbage(Dong, et al. 2022 ), etc. which usually lead to pollen degeneration after microspore release and the abnormal tapetum vacuolation as observed in Arabidopsis (Ito, et al. 2007 ). But we confirm that constitutively over-expressed the anther specifically expressed PHD gene (GH_D12G2497) driven by double CaMV 35S promoter can convert wild type cotton to be male sterility. This result is very similar to an early study (An, et al. 2020 ), thus we may infer that over expression of these genes at wrong time can lead to abnormal development of anther or pollen, resulting in male sterility, even though they are essential for pollen development and dispersal. However, more examinations are needed to extend to other anther preferentially expressed genes that were upregulated in Nan A. 320 down-regulated genes are significantly enriched in MF of ‘DNA-binding transcription factor activity’, or ‘response’ related BP terms (Fig. 3b, Supplementary Table S4 ). Intriguingly, 75% of the genes enriched in ‘transcription regulator activity’ are responsible to stress, 71 genes enriched in ‘response to stress’ are DNA-binding transcription factors. So ms a compromised expression of a large number of transcriptional activators involved in stress response, among which proteins involved in ethylene, jasmonate, and ABA signaling pathways take a high proportion. Jasmonic acid (JA) has been proven to be a key plant hormone that promote anther dehiscence, recently, transcriptional repressor OsTIE1 and transcription factor OsTCP1 were found to tightly regulates JA biosynthesis during anther development and dehiscence(Fang, et al. 2024 ). A lot of studies revealed that the analogs of WRKY53(Zhang, et al. 2022 ), CYP94C1s(Heitz, et al. 2012 ), TIFY9(Virag, et al. 2024 ), AOC4(Acosta and Przybyl 2019 ) are involved in regulation of JA biosynthesis or signaling during floral development and response to stress, but their roles in male fertility remain unknown. In this study. Up to sixteen proteins involved in ethylene biosynthesis and signaling including twelve ERFs, two MBF1Cs, one ARB1, and one ACS6 are down-regulated in Nan A, as there complicated synergistic and antagonistic interactions between ET and JA, ethylene may play important roles in male fertility, although little direct evidence is available so far (Ishimaru, et al. 2006 ). Unexpectedly, among the 320 downregulated genes, GH_A12G1289 (CYP703A2) is the only one specifically expressed in anthers (Fig. 5, Table 1). Silencing GH_A12G1289 resulted in anther indehiscence and dramatic reduction in pollen viability, these phenotypes are very similar to those of the Nan A and VOX- GhPHD-D cotton. However, either silencing GhCYP450 or overexpressing GhPHD-D cannot eliminate the spikes on the surface of cotton pollen, namely changing a single gene cannot fully achieve the phenotype of Nan A pollen, indicating that the mutated ms a gene perform multiple biological functions in pollen development. Conclusion In this study, cytological analysis of the anther of a single-gene recessive genic male sterility system of male sterile line Nan A and its near-isogenic male fertile line Nan B showed that abortive pollen dehiscence, distorted wall biosynthesis, and compromised starch granule formation may be responsible for the male sterility trait. Down-regulated expression of stress responsive genes involving ethylene, JA, and ABA biosynthesis of signaling, and inappropriate overexpression of proteins even essential for reproductive development maybe the molecular mechanisms conferred by the recessive male sterility gene ms a . Either silencing the down-regulated GhCYP450 or over expression of the over-regulated GhPHD-D in the wild-type cotton can lead to male sterility. Our study provides novel clues for understanding the molecular mechanism of male sterility and candidate genes for BMS in cotton. Abbreviations DPA Days post anthesis Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Data and materials in the manuscript were in the methods section. Other data provided in Supplementary Materials. Funding This work was supported by the National Natural Science Foundation of China (No. 30671120, No. 32172083). Author contributions Haili Qiu: The primary experimenter, Writing the original draft. Hongyu Dou: Data analysis. Kang Liu: Project administration, Funding acquisition, Conceptualization, manuscript revision. Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements We are grateful to Dr Gongyao Shi, College of Agronomy, Zhengzhou University, Henan province, China, for providing pCaBS-α, pCaBS-β, pCaBS-γ1, and pCaBS-γ2 vectors of BSMV system. References Acosta IF, Przybyl M (2019) Jasmonate Signaling during Arabidopsis Stamen Maturation. 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Rice (N Y) 10:53 Supplementary Files TableS1.xlsx Table S1Primers used in this research TableS2.xlsx Table S2 Differentially expressed genes between Nan A and Nan B anthers at stage 8 TableS3.xlsx Table S3 GO enrichment of the genes up-regulated in male sterility line of Nan A TableS4.xlsx Table S4 GO enrichment of the genes down-regulated in male sterility line of Nan A 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-7281172","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496918553,"identity":"44a463b7-b623-4d31-b14f-e06484889654","order_by":0,"name":"Haili Qiu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haili","middleName":"","lastName":"Qiu","suffix":""},{"id":496918554,"identity":"9b2360d1-4ee1-456e-869a-8c0c04291abd","order_by":1,"name":"Hongyu Dou","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Dou","suffix":""},{"id":496918555,"identity":"02b572d6-4943-4e76-8fdb-5c42200db3bc","order_by":2,"name":"Kang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3RuwrCMBSA4VMEXQquR4r6CpFCEfoy6WKWKoKLg0OmOrr6GDqJWyDQLi2ujk7i4NDJScS03WPcBPMPuUA+AgmAzfaDYTVcCAwARLVsGRJKwP+WAES82RuQ3rqQNzqX7ODIK8IyjHinEFriubPJmBI5PfI0QMhZxN0Z1ZIBxAGpyE6IAJ1ERhxdoifde00YEdkDnZcB8TD2L4pQInJ1Czcgve09UI/MRjuRL8Y0ZX7ixnqCp9gvy2c4JOdsfy5XYX/TyfVE1cYGi/qDoP3pvKpV1lOXG5y12Wy2v+wNTShHhyIrd1AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7205-4030","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Kang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-08-03 04:09:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7281172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7281172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88902670,"identity":"d0d2c0f4-50a8-484d-b0dd-fbdd2f2c2e9d","added_by":"auto","created_at":"2025-08-12 13:55:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3179743,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotyping and microsporogenesis of male sterility line Nan A and its near isogenic male fertile line Nan B (A) Plants of Nan B (left) and Nan A (right). (B) Nan B anthers dehisce normally. (C) Nan A anthers can’t dehisce. (D) The stamen filaments of Nan A (right) are much shorter than Nan B (left). (E) The wild-type pollen grains of Nan B were dyed brown by I\u003csub\u003e2\u003c/sub\u003e-KI. (F) The infertile pollen grains produced by Nan A cannot effectively stained by I\u003csub\u003e2\u003c/sub\u003e-KI. (G-I), Meiosis of pollen mother cells at stage 6, a tetrad observed at stage 7, and normal pollen with spikes on the surface in Nan B. (J-L) Meiosis at stage 6 and tetrad formation at stage 7 in Nan A are as normal as observed in Nan B, distorted pollens observed at stage 8 in Nan A. Scales: Marked on each image.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/75002bdb87887c5b6201182d.png"},{"id":88901337,"identity":"6366b998-65a3-43b9-b6f1-bad57f0a5b7e","added_by":"auto","created_at":"2025-08-12 13:47:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1839608,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis of pollen grains of Nan A and Nan B (A-C) The pollen grains in Nan B anther were well scattered, full and covered with spikes on the surface. (D-F) The pollen grains of A adhere together and are distributed in the corners of the pollen chamber, with no spikes on the surface of the atrophied pollens. (G-H) TEM imaging of the pollen grains and their wall structures of Nan B at stage 8. (I) TEM imaging of pollen wall of Nan B at 1 d before anthesis, the thinned nexine and intine layers are present.\u003cstrong\u003e \u003c/strong\u003e(J-K) TEM imaging of the pollen grains and their wall structures of Nan A at stage 8. (L) TEM imaging of pollen wall of Nan A at the 1 d before anthesis, thickened nexine layer and no intine are bespoken. Msp, microspores; DMsp, degenerated microspores; In, intine; Ne, nexine; Ba, bacula; Tc, tectum; Sg, Starch granule. Scales: Marked on each image.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/795ef1bb53d494f4b2d03daf.png"},{"id":88902671,"identity":"fa0d02c7-530f-4e3e-a15b-8e4a933e5b2d","added_by":"auto","created_at":"2025-08-12 13:55:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":196240,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic analysis of the anthers of Nan A and Nan B at stage 8 using RNA-seq (A) Relative expression levels of 24 DEGs identified by RNA-seq were verified by qRT-PCR. (B) GO enrichment analysis of significantly down-regulated DEGs in Nan A. (C) GO enrichment analysis of significantly up-regulated DEGs in Nan A. Error bars represent the standard deviation of three biological replicates, ** indicates significance at p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/fdb24fab44d1fab66467ed1e.png"},{"id":88902674,"identity":"60b6217d-7c19-4d2c-aa23-c30e726a8611","added_by":"auto","created_at":"2025-08-12 13:55:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":826735,"visible":true,"origin":"","legend":"\u003cp\u003eThe interaction networks of the DEGs that down-regulated in Nan A and enriched in ‘response to endogenous stimulus’ biological process.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/a2356375415f8d4e69f68b1e.png"},{"id":88903188,"identity":"3fec3a11-1108-4f49-821a-57ae180e90fb","added_by":"auto","created_at":"2025-08-12 14:03:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71822,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of 22 anther preferentially expressed genes in Nan A and Nan B at stage 8 based on RNA-seq analysis data. Error bars represent the standard deviation of three biological replicates, ** indicates significance at p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/604053503eb9fc9d126d8b4a.png"},{"id":88903189,"identity":"a86e2cd1-f9e6-4347-a584-5b83b9b99544","added_by":"auto","created_at":"2025-08-12 14:03:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1165184,"visible":true,"origin":"","legend":"\u003cp\u003eVirus induced silencing GhCYP450 lead to male sterility in cotton (A) GhCHLI was used as a visible marker to monitor the efficiency of VIGS, photobleaching phenotype produced at around 14 d post agro-infiltration with the CLCrVB and CLCrV : GhCHLI. (B) Expression of GhCYP450 was effectively silenced through VIGS. (C) The germination rate of pollens was nearly reduced to zero in GhCYP450-silenced plants. (D) Wild type stamen with relatively long filaments. (H) Stamen filaments were shortened in VIGS plants. (E) Anther dehiscence in wild type plant. (I) Loss of anther dehiscence in VIGS plants. (F) The pollens were dyed brown with I2-KI. (J) Shrinking and deformed pollen grains of GhCYP450-silenced cotton can hardly be dyed with I2-KI, weak spikes are indicated. (G) Pollen grains of the wild type plants germinated in vitro. (K) Pollens from the GhCYP450-silenced plants can poorly germinate in vitro. Error bars represent the standard deviation of three biological replicates for gene expression analysis, and at least 100 pollen grains under 6 fields of view of an optical microscope, respectively, * indicates significance at p\u0026lt; 0.05, ** indicates significance at p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/6518602f499fe7a1826b21d5.png"},{"id":88901346,"identity":"7e636c93-0e9d-47e4-95d9-e423d38b24b0","added_by":"auto","created_at":"2025-08-12 13:47:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":757971,"visible":true,"origin":"","legend":"\u003cp\u003eVirus-mediated gene overexpression (VOX) assay of GhPHD-D gave rise to male sterility in cotton (A) The expression of GhPHD-D was increased more than 200 folds by using BSMV mediated VOX. (B) The germination rate of pollens was nearly reduced to zero in VOX plants. (C) Wild type stamens evenly distributed on the stigma. (G) The stamens are relatively clustered and distributed at the base of the stigma in VOX plants. (D) Anther dehiscence in wild type plant. (H) Loss of anther dehiscence in VOX plants. (E) The pollens in the wild type plants were dyed brown with I2-KI. (I) Shrinking and deformed pollen grains from VOX plants can hardly be dyed with I2-KI, weak spikes are indicated. (F) Pollen grains of the wild type plants germinated in vitro. (J) Pollens from the VOX plants can poorly germinate in vitro. Error bars represent the standard deviation of three biological replicates for gene expression analysis, and at least 100 pollen grains under 6 fields of view of an optical microscope, respectively, ** indicates significance at p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/3607c91941f80448757441f5.png"},{"id":90184069,"identity":"d7f57aa2-e7cc-4a0a-8ba9-4578bc7d2edf","added_by":"auto","created_at":"2025-08-29 14:13:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9275064,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/7eaa7f9e-553a-4e6d-889d-2b18c5ca0a33.pdf"},{"id":88901339,"identity":"6e509248-4970-4d1f-8817-c5fc58d7fc87","added_by":"auto","created_at":"2025-08-12 13:47:05","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003ePrimers used in this research\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/65cc80429f42c84213aa48a3.xlsx"},{"id":88901340,"identity":"eac11bf1-e372-495b-9a2b-ad73c97a4c69","added_by":"auto","created_at":"2025-08-12 13:47:05","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":123159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e Differentially expressed genes between Nan A and Nan B anthers at stage 8\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/c3822dc1eb099a7a14de4989.xlsx"},{"id":88901344,"identity":"ea079c8a-bdb6-44c8-a2f2-95cb463c9294","added_by":"auto","created_at":"2025-08-12 13:47:05","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3\u003c/strong\u003e GO enrichment of the genes up-regulated in male sterility line of Nan A\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/868ace6b0827383e1a1e4b5d.xlsx"},{"id":88902676,"identity":"212e2a46-8e8b-4f39-9fb1-b3d0833d17a7","added_by":"auto","created_at":"2025-08-12 13:55:05","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4\u003c/strong\u003e GO enrichment of the genes down-regulated in male sterility line of Nan A\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7281172/v1/0a29baf7bf74b7d733f549b4.xlsx"}],"financialInterests":"","formattedTitle":"Key genes and molecular mechanisms responsible for male sterility revealed by transcriptome analysis in cotton","fulltext":[{"header":"Key Message","content":"\u003cp\u003eA novel male sterility line Nan A forms normal microspore tetrads, but defective pollen development. Dysregulated fertility/stress-related genes in anthers cause cotton male sterility.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCotton (\u003cem\u003eGossypium\u003c/em\u003e spp.) is a major cash crop in the world supplying approximately a quarter of global textile fibers, and cottonseed is an important source of oil a vital source of protein for human consumption as byproducts. Cotton displays strong heterosis, the heterozygous first filial (F\u003csub\u003e1\u003c/sub\u003e) generation typically generates 15\u0026ndash;30% more fiber than traditional types, big seed size, early flowering, high fiber quality, biotic and abiotic stress tolerances over its parents (Zhang, et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, breeding for cotton hybrid varieties is a promising way to tremendously improve cotton productivity. Currently, most commercially used F\u003csub\u003e1\u003c/sub\u003e hybrid cotton seeds are produced by hand-emasculation and pollination, the strong heterosis combinations were screened from extensive cross combinations of pure line parents without need to introduce the male sterility and restorer genes into the parents, which simplifies the breeding process and reduces the breeding cost. Additionally, using F\u003csub\u003e2\u003c/sub\u003e heterosis can reduce the cost of seed production by up to 10 times. However, the F\u003csub\u003e2\u003c/sub\u003e vigor is much lower than that of F\u003csub\u003e1\u003c/sub\u003e because of inbreeding depression. Reducing the production cost of F1 seeds and utilizing F1 hybrid vigor is an important direction for cotton breeding.\u003c/p\u003e\u003cp\u003eThe application of a male sterility line can facilitate large-scale production of crop hybrids. The application of male sterile line can eliminate the need for hand emasculation, facilitate breeding hybrid varieties, and greatly reduce the production cost of hybrid seeds. Male sterility can be generated by either cytoplasmic or nuclear genes. Cytoplasmic male sterility (CMS) is caused by mitochondrial genes together with nuclear genes and has been used in commercial hybrid cotton production (Li, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), but this method could suffer from unreliable restoration or marginal heterosis. Genic male sterility (GMS) is caused by nuclear gene alone. The use of GMS can overcome these drawbacks, but it is difficult to segregate sterility lines from maintainer lines. The advent of seed production technology (SPT) has significantly advanced the field of maize hybrid seed production by overcoming the challenges associated with genic male sterility (Wu, et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Identify and utilize GMS genes for developing efficient biotechnology-based male sterility systems in crops including cotton will revolute harnessing heterosis. 19 GMS genes (loci) have been genetically identified in cotton (Lan, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e (1355A), \u003cem\u003ems14\u003c/em\u003e (Dong-A) and \u003cem\u003ems5ms6\u003c/em\u003e have been utilized to produce hybrid seed. Gene responsible for \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e was identified as a mutation of \u003cem\u003eGhNSP\u003c/em\u003e through map-based cloning and confirmed by genetic analysis. \u003cem\u003eGhNSP\u003c/em\u003e encodes a polygalacturonase protein, biochemical analysis showed that de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation in the mutant (Wu, et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eMs5\u003c/em\u003e and \u003cem\u003eMs6\u003c/em\u003e loci were identified through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 genome editing. \u003cem\u003eMs5\u003c/em\u003e and \u003cem\u003eMs6\u003c/em\u003e encode the cytochrome P450 monooxygenases CYP703A2-A and CYP703A2-D, the duplicate mutations of \u003cem\u003eCYP703A2\u003c/em\u003e genes encoding a cytochrome P450 protein essential for pollen exine formation and pollen development resulted in producing male sterility for the duplicate \u003cem\u003ems5ms6\u003c/em\u003e line (Ma, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mao, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe detailed description and elucidation of the cellular biology process of microspore development in Arabidopsis, from the formation of stamen primordia to the final maturation, dehiscence, and pollen dispersal (Schiefthaler, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), provides a reference for the analysis of microspore development in other plants, including cotton (Xu, et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The molecular, genetic, and biochemical pathways regulating anther and pollen development and information about GMS in plants provide opportunities to identify and utilize male sterility in economically important crops. \u003cem\u003eMS\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e encodes a PHD (plant homeodomain) transcription factor in Arabidopsis, the \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e mutant showed abnormal vacuolization of tapetal cells and delayed PCD, leading to male sterility, but over expression of PHD resulted in partial fertility (Ito, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yang, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The maize genic male sterility gene \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sub\u003e encoded a GDSL lipase, mutation of this gene disrupts lipid metabolism process and appropriate degradation the middle layer and tapetum of the anther which leads to pollen production (Huo, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, a lipid transfer protein that is exclusively produced in tapetum cells is encoded by the maize genic male sterility gene \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e44\u003c/em\u003e\u003c/sub\u003e. Changes in the individual amino acids of the \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e44\u003c/em\u003e\u003c/sub\u003e protein cause disruptions in the processing of proteins, which in turn leads to aberrant development of microspore mother cell and the prevention of tapetum cell secreting proteins into the pollen chambers (Fox, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In other studies, the \u003cem\u003eUDT1\u003c/em\u003e-encoded bHLH transcription factor in rice, the \u003cem\u003eOsTDF1\u003c/em\u003e-encoded R2R3 MYB transcription factor, and the \u003cem\u003eOsGPAT3\u003c/em\u003e-encoded glycerol 3-phosphate acyltransferase are required for normal development and degradation of the tapetal layer, mutation of any of them ultimately leads to sterility (Jung, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Men, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDeveloped anthers release pollen grains, normal pollen wall development serves as essential for pollen fertility (Ariizumi and Toriyama \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Sporobollenin is the primary constituent of the exine of pollen, and its comparatively steady physicochemical qualities account for the robust resistance and protection of the pollen wall. Phenylpropane-like compounds and aliphatic derivatives make up the majority of sporopollenin, whose synthesis influences pollen grain fertility. Cotton \u003cem\u003eCYP703A2-A\u003c/em\u003e and \u003cem\u003eCYP703A2‐D\u003c/em\u003e, maize \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e10\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e26\u003c/em\u003e\u003c/sub\u003e, etc. encode cytochrome P450 mono‐oxygenase which plays a central role in sporopollenin formation (Chen, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ma, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ATP-binding cassette G (ABCG) transporter protein, which is encoded by maize \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e, is primarily involved in the transmembrane transport of substances during anther development. Lack of function in this protein leads to a decrease in lipid levels in the anthers and interferes with the normal formation of pollen grains (Xu, et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Maize \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e33\u003c/em\u003e\u003c/sub\u003e encodes a glycerol‑3‑phosphate acyltransferase (GPAT), which is expressed preferentially in immature anthers and root tissues, and is involved in fatty acid metabolism processes in tapetum layer, thereby regulating the synthesis and metabolism of maize anther cuticle substances and the formation of sporopollenin substances in exine (Zhu, et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Maize \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003e8\u003c/em\u003e\u003c/sub\u003e encodes a β-1,3-galactosyltransferase protein, which is involved in the synthesis of important substances during anther development, the metabolism of substances in mitochondrial organelles, and the regulation of programmed cell death (PCD) in tapetal cells during anther development. Mutations in this protein cause abnormal development of the microspore mother cell (Wang, et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). OsACOS12 (Li, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), OsPKS1 (Zou, et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), OsPKS2 (Zhu, et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), OsTKPR1 (Xu, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), CYP703A3 (Yang, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), CYP704B (Li, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and NP1 (Liu, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) proteins in rice form sporopollenin precursors in the endoplasmic reticulum and efficiently produce lipid and phenolic precursors for sporopollenin biosynthesis.\u003c/p\u003e\u003cp\u003eNan A (\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e) is a spontaneous mutant characterized by male sterility controlled by a single recessive nuclear genetic locus in upland cotton, which provides us a good material to investigate genetic, cytological, and molecular mechanism underlying pollen development and male sterility in cotton. Here, we first employed optical and election microscopy to examine the mutant pollen development to show the cytological and structural alterations responsible for aberrant pollen formation, then we compared the transcriptome differences between the anthers of Nan A (\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e) and its isogenic fertile line Nan B (\u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e) to discover genes crucial for normal micropore development. And finally, two differential expression genes were selected for function analysis by using virus-induced gene silencing (VIGS) assay and virus-mediated gene overexpression assay (VOX), respectively. This study provides a new clue for understanding the molecular basis of male sterility and exploring male sterility genes in cotton.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe key period for male sterility of Nan a is after tetrad stage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNan A and Nan B were planted adjacent to each other in the breeding nursery to minimize the impact of environmental differences on plant phenotype. We found no visible differences in the plant architecture, growth, flowering, and boll setting between Nan A and Nan B (Fig.\u0026nbsp;1A), except that the size of the filaments and anthers of the former is significantly smaller than that of the latter (Fig.\u0026nbsp;1D). When we imaged the anthers using the MVX10 microscope, we found that the anthers of Nan B dehisced normally and gave off plump pollens stained brown black with I\u003csub\u003e2\u003c/sub\u003e-KI (Fig.\u0026nbsp;1B, 1E), while the anthers of Nan A remained unopened, the wrapped pollens can hardly stain with I\u003csub\u003e2\u003c/sub\u003e-KI (Fig.\u0026nbsp;1C, 1F).\u003c/p\u003e\u003cp\u003eTo pinpoint the critical period and characteristics of male sterility in \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e of cotton, we examined the microstructures of the anthers from 2 mm to 10 mm flower buds of male sterile and fertile plants using Carbolfuchsin staining followed by microscopy. The results showed that the developmental dynamics did not differ significantly at the meiosis stage corresponding to 4\u0026ndash;5 mm buds and the tetrad stage corresponding to 5\u0026ndash;6 mm buds, both Nan A and Nan B appear to be able to form normal tetrads inlaid in callus wall (Fig.\u0026nbsp;1G, 1H, and Fig.\u0026nbsp;1J, Fig.\u0026nbsp;1K). Thereafter, the callus around the tetrad began to disintegrate, when buds grow to 6\u0026ndash;7 mm in length, the fertile anthers of Nan B released well-stacked pollen with spikes on exine (Fig.\u0026nbsp;1I), instead, the sterile anthers from Nan A generated unfilled pollens without spines on outer surfaces that cannot release form the anther chambers (Fig.\u0026nbsp;1L). Therefore, male sterility of Nan A may result from distorted microspore development and pollen maturation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMs\u003c/b\u003e\u003csub\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eis required for anther development and pollen formation in cotton\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further characterize the cytological defects in \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e anthers, we performed scanning electron microscopy (SEM), transverse section, to compare the anthers at 1 d before anthesis, the results showed that there were a large number of prickly pollens scattered inside the vacuolated anther chamber of Nan B (Fig.\u0026nbsp;2A, B, and C). In contrast, there were very few deformed pollen grains without spikes on smooth exine clinging together to the inner wall of the relatively much smaller anther chamber (Fig.\u0026nbsp;2D, E, and F). These results confirm that \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e disrupts anther development and pollen formation. In order to further analyze the causes of the developmental defects of pollen wall in Nan A, anthers of flower buds 6\u0026ndash;7 mm in length and 1 d before anthesis were taken for transmission electron microscopy (TEM) observation, respectively. In 6-7mm long bud, i.e., at stage 8, the construction of the outer wall of Nan B pollen commenced with the production of a substantial intine, noticeable spikes, and ample, well-developed starch grains in the lumen (Fig.\u0026nbsp;2G, H). In contrast, the nexine of Nan A pollen was less pronounced than that of Nan B, and the exine lacked any obvious protrusions, and the starch grains in the lumen being small and closely packed (Fig.\u0026nbsp;2J, K). The exine may be further elaborated with additional sporopollenin deposition, e.g., production of inner-column-shaped baculae and the surface decoration tectum. At 1d pre-anthesis, the nexine of Nan B pollen was remarkably thinner than that observed at stage 8, although a clear intine layer could be discerned (Fig.\u0026nbsp;2I), while Nan A pollen at the same stage displayed a thickened nexine but no intine layer was visible (Fig.\u0026nbsp;2L). These discrepancies imply that the hindered synthesis of pollen wall components and the inability to develop and form a typical pollen wall might be the root cause of pollen sterility in Nan A.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCritical genes and networks involved in male sterility in Nan A\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the genes and molecular mechanisms critical for male sterility in Nan A, we extracted RNA from the anthers of 6 mm long buds for RNA-seq analysis. A total of 569 differentially expressed genes (DEGs) in Nan A and Nan B buds were identified according to the method of reads per kilobase per million mapped reads (RPKM) on the basis of the applied criteria of q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and log\u003csub\u003e2\u003c/sub\u003e (fold-change)\u0026thinsp;\u0026gt;\u0026thinsp;1. Among those genes, compared with the Nan B buds, 249 (44%) were upregulated and 320 (56%) were downregulated in Nan A buds during microspore development (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The housekeeping gene upland cotton \u003cem\u003eHis3\u003c/em\u003e (histone 3) was used as an internal control for data normalization, 24 DEGs were chosen for qRT-PCR analysis to verify their expression levels (Fig.\u0026nbsp;3A). The figure shows that the differential expression of 19 genes determined by RNA-seq or qRT-PCR was consistent, account for 79.2% of the tested genes, but five genes (20.8%), i.e., \u003cem\u003eGH_A02G0833\u003c/em\u003e, \u003cem\u003eGH_D02G0848\u003c/em\u003e, \u003cem\u003eGH_A05G0056\u003c/em\u003e, \u003cem\u003eGH_A08G2216\u003c/em\u003e, \u003cem\u003eand GH_A03G1602\u003c/em\u003e, were significantly down-regulated in the anthers of the sterile line, which is opposite to RNA-seq results. This part of the inconsistency may be due to the sampling batch of different bud size corresponding to the development period is not completely consistent. Pearson\u0026rsquo;s correlation coefficient between the qRT-PCR data and RNA-seq data is 0.46, reaching moderate level of correlation, thus our RNA-seq data were are acceptable and conducive to identification of critical genes involved in anther development.\u003c/p\u003e\u003cp\u003eTo understand how DEGs specific to \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e line affect the pollen sterility process in Nan A cotton, GO class enrichment analyses of down- and up-regulated genes for Nan A were conducted, respectively. 249 up-regulated genes were significantly enriched in \u0026lsquo;transporter activity\u0026rsquo; molecular function (MF), \u0026lsquo;response to endogenous stimulus\u0026rsquo; biological process (BP), and \u0026lsquo;reproductive process\u0026rsquo; BP (Fig.\u0026nbsp;3C, Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Among the 21 upregulated genes enriched in GO terms related to the reproductive process, nine gens including GH_D13G1652 (NAC), GH_D04G1012 (NFYA2), GH_A12G2483 (PHD, MALE STERILITY 1), GH_D12G2497 (PHD, MALE STERILITY 1), GH_D02G1428 (CRABS CLAW, CRC), GH_A12G1852 (TCP15), GH_D12G1848 (TCP15), GH_A05G1589 (WOS9, WUSCHEL-related homeobox 9) and GH_D05G1617 (WOS9) encode 6 transcription factors or transcription activators; six genes i.e. GH_D09G1973 (β-1,6-galactosyltransferase, GALT31A), GH_D12G0737 (glycerol-3-phosphate acyltransferase 1, GPAT1), GH_A12G1040 (GPAT1), GH_D02G2614 (polygalacturonase3, QRT3), GH_D04G1012 (\u003cem\u003eO\u003c/em\u003e-Glycosyl hydrolase), and GH_D02G0220 (\u003cem\u003eO\u003c/em\u003e-Glycosyl hydrolase) encode 4 proteins involved in metabolism; other three genes, GH_D12G2099 encodes a HVA22-like protein, GH_D11G0805 encodes a casein kinase subunit (CKB4), and GH_A10G0810 encodes a Rop guanine nucleotide exchange factor 12 (ROPGEF12). The homologs of these genes in other plants have been proved to be male sterility genes or related to pollen fertility. It is also worth noting that \u0026lsquo;reproductive process\u0026rsquo; and \u0026lsquo;response to endogenous stimulus\u0026rsquo; have eight genes in common, while \u0026lsquo;reproductive process\u0026rsquo; has no intersection with \u0026lsquo;transporter activity\u0026rsquo;, suggesting that endogenous signaling might be involved in \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e-caused male sterility.\u003c/p\u003e\u003cp\u003e320 down-regulated genes are significantly enriched in four functionally related MFs of \u0026lsquo;DNA-binding transcription factor activity\u0026rsquo;, \u0026lsquo;transcription regulator activity\u0026rsquo;, \u0026lsquo;DNA binding\u0026rsquo;, and \u0026lsquo;nucleic acid binding\u0026rsquo; in which genes are highly overlapped, with an additional \u0026lsquo;signaling receptor binding\u0026rsquo; containing five unique genes. The most dominant biological processes of these genes are six \u0026lsquo;response\u0026rsquo; related terms, in which genes are in a high degree of overlap. Three metabolism-related BPs also have a high proportion of identical genes. All of these genes are significantly enriched in nucleus (Fig.\u0026nbsp;3C, Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Remarkably, the enriched MF term of \u0026lsquo;transcription regulator activity\u0026rsquo; and the BP term of \u0026lsquo;response to stress\u0026rsquo; share 30 common genes, which account for 75% of the genes enriched in \u0026lsquo;transcription regulator activity\u0026rsquo;, moreover, 71 genes in \u0026lsquo;response to stress\u0026rsquo; can be significantly enriched in \u0026lsquo;DNA-binding transcription factor\u0026rsquo; \u003cem\u003eper contra\u003c/em\u003e. So we may conclude that most proteins with transcriptional activation activity that are suppressed in \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e play major roles in stress response. In response to stress, ethylene (12 ERFs, 2 MBF1Cs, 1 ARB1, and 1 ACS6), ABA (3 DREB1Ds, 1 F23F1.6, 1 PYL2, 1 EDL3, and 1RDUF1), jasmonate (3 WRKY53s, 2 CYP94C1s, 1 TIFY9, 1 ZAT10, and 1 AOC4) may perform major signaling roles (Fig.\u0026nbsp;4). This may be because 7\u0026ndash;8 stage is characterized by anthers that are more susceptible to high temperature in Nan A. Jasmonate mediated signaling have been reported to be relevant to male fertility in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo further pursue the genes fatal for cotton male fertility, all the DEGs identified between Nan A and Nan B anthers in the present study were subjected for spatial expression analysis by querying the Cotton RNA-seq Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ipf.sustech.edu.cn/pub/cottonrna/\u003c/span\u003e\u003cspan address=\"http://ipf.sustech.edu.cn/pub/cottonrna/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), 22 DEGs were found specifically or preferentially in anthers, and their expression levels in Nan A and Nan B were showed in Fig.\u0026nbsp;5. Intriguingly, nineteen of the 22 DEGs have been proved to play an important role in male sterility or microspore development in cotton, Arabidopsis thaliana, rice and other plants. However, these genes related to male sterility caused by loss of function mutation were up-regulated in the sterile anther of Nan A, except GH_A12G1289 which was down-regulated in the anther of NanA (Fig.\u0026nbsp;5, Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSilencing\u003c/b\u003e \u003cb\u003eGhCYP450\u003c/b\u003e \u003cb\u003eexpression in WT lead to sterile pollens\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eGH_A12G1289\u003c/em\u003e is only one anther preferentially expressed gene that is significantly suppressed in Nan A. The homologous \u003cem\u003eGH_A12G1289\u003c/em\u003e gene in Arabidopsis encodes a cytochrome P450 enzyme catalyzing the hydroxylation of lauric acid, which provides the raw material for sporopollenin synthesis and thus affects the synthesis of exine. The loss of P450 mutant causes male infertility in maize and rice. Therefore, we hypothesized that \u003cem\u003eGH_A12G1289\u003c/em\u003e is a candidate sterility gene for \u003cem\u003eMs\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e. We cloned a 500-bp fragment of the \u003cem\u003eGhCYP450\u003c/em\u003e CDS and inserted it into pCLCrVA vector for VIGS assay to silence the expression of \u003cem\u003eGH_A12G1289\u003c/em\u003e in TM-1. The typical photobleaching phenotype generated by silencing the cotton magnesium chelatase subunit I (\u003cem\u003eCHLI\u003c/em\u003e) gene was employed to monitor the silencing efficiency of endogenous gene as a positive control (Fig.\u0026nbsp;6A). The expression of \u003cem\u003eGhCYP450\u003c/em\u003e was almost completely silenced in the anthers of the cotton plants infected with CLCrV- \u003cem\u003eGhCYP450\u003c/em\u003e plants compared to that of TM-1 infected with empty CLCrV vector (Fig.\u0026nbsp;6B). \u003cem\u003eGhCYP450\u003c/em\u003e-silenced plants showed no significant alterations in most aspects of phenotype compared with the empty control, but on the day of flowering, the filaments of the silenced plants became much shorter (Fig.\u0026nbsp;6H), the anthers cannot dehisce, and there were no pollen grains on the outer surface of the anthers (Fig.\u0026nbsp;6I), in contrast, the control plants developed long filaments and plump anthers which dehisced to disperse a great number of pollen grains (Fig.\u0026nbsp;6D, E). Consequently, the control plants produced fertile pollens that were stained dark brown with I\u003csub\u003e2\u003c/sub\u003e-KI (Fig.\u0026nbsp;6F), and germinated in vitro. at an average rate of 37.6%, while the silenced plants produced shriveled and deformed pollen with spikes on outer surface, which can only weakly stain with I\u003csub\u003e2\u003c/sub\u003e-KI and unable to germinate on culture medium (Fig.\u0026nbsp;6J, K). In summary, silencing \u003cem\u003eGhCYP450\u003c/em\u003e expression gave up to male sterility, but the sterile pollen coated with a layer of spiky sexine, this is different from those without spikes produced in Nan A. It seems that merely silencing \u003cem\u003eGhCYP450\u003c/em\u003e is not sufficient to achieve the effects of the \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e mutation gene, namely \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e has multiple molecular functions in regulating cotton microspore development.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eGhPHD-D\u003c/b\u003e \u003cb\u003einduced male sterility in upland cotton\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGH_A12G2483 and GH_D12G2497 two anther preferentially expressed genes sharing 98.12% (37 of 1968 bp) similarity in CDS sequence encoding a PHD- finger transcription factor. They were significantly up-regulated in Nan A by more than 200 folds (Fig.\u0026nbsp;5). However, its homologous genes were reported to be required for the formation of exine and the development of tapetum cells in Arabidopsis, Chinese cabbage, rice, and maize, but premature expression of \u003cem\u003eZmMs7\u003c/em\u003e (encoding a PHD-finger transcription factor) disrupts tapetum and pollen development and results in male sterility in maize (An, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results raise a question about the effect of over expression of GH_A12G2483 or GH_D12G2497 on cotton pollen fertility. Therefore, we cloned the CDS sequence of \u003cem\u003eGH_D12G2497\u003c/em\u003e, and inserted it into pCaBS-γ2 vector to generate pCaBS-γ2-\u003cem\u003eGhPHD-D\u003c/em\u003e for BSMV-VOX assay. The results showed that \u003cem\u003eGhPHD-D\u003c/em\u003e expression level in the anthers of the VOX TM-1 plants soared more than 200-fold (Fig.\u0026nbsp;7A). The \u003cem\u003eGhPHD-D\u003c/em\u003e-overexpressed plants exhibited exactly the same vegetative growth as normal plants. On the day of flowering, there was no significant difference in filament length between overexpressed plants and normal plants, but the basal filament tube of the monomeric stamen was relatively short, and the filaments were concentrated at the base of the columella so that the anthers were far from the stigma (Fig.\u0026nbsp;7C, G). Notably, the anthers of overexpressed plants cannot dehisce and release pollen as normal plants (Fig.\u0026nbsp;7D, H). The pollen grains produced by \u003cem\u003eGhPHD-D\u003c/em\u003e-overexpressed cotton were smaller in size, deformed, with spikes on the outer wall of pollen, and can be lightly stained brown with I\u003csub\u003e2\u003c/sub\u003e-KI, which cannot germinate in vitro culture (Fig.\u0026nbsp;7J, K). In contrast, the anthers of the empty vector control plants developed normally, producing plump pollen grains that could be stained brown black by I\u003csub\u003e2\u003c/sub\u003e-KI and could germinate in vitro in the culture medium (Fig.\u0026nbsp;7F, 7G). Together, either overexpression of \u003cem\u003eGhPHD-D\u003c/em\u003e or silencing \u003cem\u003eGhCYP450\u003c/em\u003e can lead to male sterility in cotton, and the sterile pollen grains are similar in shape coated with spikes. These results further confirm that \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e may regulate the development and maturation of cotton microspores through a complex molecular pathway to determine pollen fertility.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePlant materials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cotton genic male sterile mutant was originally discovered in a cotton cultivar Sumian 22 and named Nan A. The male sterile mutant was continuously backcrossed with Sumian 22 for more than 12 generations, and developed a pair of isogenic lines Nan A (male sterility) and Nan B (male fertility). All plants were planted in the breeding nursery of Nanjing Agricultural University, Jiangsu province under regular field management.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptical microscopy, scanning and transmission electron microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFlower observation was conducted using a Digital microscope DVM6a (Leica, Germany). 4-5mm, 5-6mm, and 6-7mm long buds were collected between 6:00 am to 8:00 am, and immediately put into Carnot's fixative and stored at 4\u0026deg;C for miosis slide preparation. The anthers were rinsed with ddH\u003csub\u003e2\u003c/sub\u003eO, dissociated in 0.1M HCl for 8 min, then washed with water three times, and placed onto a slide. A drop of carbolfuchsin was added to stain the anthers on slides, and then placed a coverslip on the sample, the anthers were crushed by gently tapping the coverslip with a needle, and was subsequently observed with fluorescence microscope BX53 (Olympus, Japan) under light field.\u003c/p\u003e\u003cp\u003eFor scanning electron microscopy, anthers at different developmental stages were peeled off the stamens and immediately put into the fixative containing 2.5% (v/v) glutaraldehyde and 1% (v/v) paraformaldehyde, vacuumed so that the anthers were fully submerged, and fixed overnight at 4\u0026deg;C. Next, the anthers were washed and postfixed in 1% osmium tetraoxide in 0.1 M sodium phosphate buffer (PBS, pH 7.2) for 2h, followed by washing with Milli-Q water for 10 min. Samples were then dehydrated in increasing concentrations of ethanol solution (30, 50, 70, 95 and 100%), and dried in 100% ethanol with liquid CO\u003csub\u003e2\u003c/sub\u003e. Finally, the dried anthers were carefully cut into pieces using a razor blade and placed on the sample stage using conductive adhesive, followed by sputtering with gold palladium for 300 s at 25 mA, and visualized using scanning electron microscope (Regulus 8100, Hitachi, Japan).\u003c/p\u003e\u003cp\u003eThe above fixed anthers were washed three times (5 min for each) with 0.1M PBS, postfixed in 1% (w/v) OsO\u003csub\u003e4\u003c/sub\u003e for 2 h, and washed with PBS three times, 5 min for each. Samples were then dehydrated as described above, treated with propylene oxide, and embedded in Spurr\u0026rsquo;s resin. Thin sections (70 nm) were taken using the Leica UC6 cryoultramicrotome. Sliced sections were placed on 100-mesh copper grids and sequentially stained with uranylacetate (30 min) and lead citrate (Sato\u0026rsquo;s Lead; 15 min). Transmission electron microscopy was performed using a Hitachi HT7800 transmission electron microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA extraction, RNA-seq, and data analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSix mm long anthers from Nan A and Nan B buds were collected, the anthers were then ground into powder in liquid nitrogen, total RNA was extracted using the Plant Total RNA Isolation Kit (Novozymes, China) following the manufacturer\u0026rsquo;s protocol. The total RNA quantity and purity were analyzed on \u003cem\u003eNANODROP ONE\u003c/em\u003e (Thermo Scientific, USA) and evaluated by agarose gel electrophoresis. Six RNA samples represent three biological repeats were entrusted to the Nanjing Personalbio Technology Company for paired-end cDNA library construction, and the libraries were subjected to sequencing on Illumina HiSeq 4000 platform according to the protocol recommended by the manufacturer. The raw data were first filtered to produce clean data, HISAT2 was used to map reads to the reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cotton.zju.edu.cn/index.htm\u003c/span\u003e\u003cspan address=\"http://cotton.zju.edu.cn/index.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The mapped reads of each sample were assembled using StringTie. The final transcriptome was generated using Perl scripts. DESeq was used to analyze the differentially expressed genes (DEGs). DEGs were selected with |\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{log}}_{2}\\text{f}\\text{o}\\text{l}\\text{d}\\:\\text{c}\\text{h}\\text{a}\\text{n}\\text{g}\\text{e}\\)\u003c/span\u003e\u003c/span\u003e|\u0026gt;1 coupled with statistical significance (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GO enrichment and KEGG pathway enrichment analysis was performed on the DEGs using the TBtools-II software(Chen, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the corrected p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was set as the threshold and rich factor. The tempo-spatial expression characteristics of the DEGs were analyzed using the Cotton RNA-seq Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ipf.sustech.edu.cn/pub/cottonrna/\u003c/span\u003e\u003cspan address=\"http://ipf.sustech.edu.cn/pub/cottonrna/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to screen anther preferentially expressed genes. Protein-protein interaction networks were generated using STRING (version 12.0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/cgi/input?sessionId=\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/cgi/input?sessionId=\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003ebsDGvjLcMSbg\u0026amp;input_page_show_search\u0026thinsp;=\u0026thinsp;off), the network was visualized with Cytoscape (Version 3.10.2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eqRT-PCR analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from the anthers of 6 mm long buds of Nan A and Nan B using the same method described in 2.6, the first strand cDNA synthesized with HiScript\u0026reg; Ⅲ 1st Strand cDNA Synthesis Kit (+\u0026thinsp;gDNA wiper) reverse transcription kit (Novozymes, China). Primers for qPCR were designed using online software PrimerQuest (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.idtdna.com/Primerquest/\u003c/span\u003e\u003cspan address=\"http://www.idtdna.com/Primerquest/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/tools/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/tools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e primer- blast/), synthesized commercially, and are shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u003cem\u003eHis3\u003c/em\u003e gene was used as the internal control and three biological replicates were used for each sample. Gene relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVirus‑induced gene silencing (VIGS) assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe used cotton leaf crumple virus (CLCrV)-based vectors viz pCLCrVA and CLCrVB for VIGS experiments. The gene-specific 500 bp fragments for magnesium chelatase subunit I (\u003cem\u003eGhCHLI\u003c/em\u003e) and \u003cem\u003eGhPHD-D\u003c/em\u003e and were amplified by PCR from TM-1 cDNAs and inserted into pCLCrVA respectively(Gu, et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). pCLCrVA, pCLCrVA-\u003cem\u003eGhCHLI\u003c/em\u003e, pCLCrVA-\u003cem\u003eGhPHD-D\u003c/em\u003e, and pCLCrVB were individually transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Agrobacterium cultures were grown overnight in YEP medium containing rifampicin (50 mg\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and kanamycin (50 mg\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 28\u0026deg;C. The Agrobacterium cultures were pelleted and resuspended (OD600\u0026thinsp;=\u0026thinsp;1) individually in an infiltration buffer containing 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e,10 mM 2-(4-Morpholino) ethanesulfonic acid and 200 \u0026micro;M acetosyringone. After 3h incubation at room temperature, \u003cem\u003eAgrobacterium\u003c/em\u003e harboring pCLCrVA or one of its derivatives was mixed with an equal volume of \u003cem\u003eAgrobacterium\u003c/em\u003e harboring pCLCrVB. The mixed Agrobacterium solutions were infiltrated into the abaxial side of cotyledons of 2-week-old cotton seedlings using needleless syringes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBSMV-mediated gene overexpressing (VOX) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVOX assay was conducted according to a previous study with minor modifications (Chen, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The CDS of \u003cem\u003eGhPHD-D\u003c/em\u003e was amplified from the cDNA of TM-1 by using PCR and inserted into pCaBS-γ2 via Gibson assembly, which allow to express the inserted gene driven by CaMV35S promoter. The pCaBS-α, pCaBS-β, pCaBS-γ1, and pCaBS-γ2 harboring the interested gene were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, respectively. Four \u003cem\u003eAgrobacterium strains\u003c/em\u003e (OD600\u0026thinsp;=\u0026thinsp;0.80) were mixed in equal volumes and infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e with a syringe, the tobacco was incubated for 24 hours after the injection in dark at 26\u0026deg;C, then grew at 26\u0026deg;C for six days. 0.5 g of the infected leaves were harvested and ground in 1mL of 20 mM phosphate buffer (pH 7.2). The homogenate was stored at -20\u0026deg;C for later or direct viral inoculation. The virus homogenate was diluted 30 times before injected into the cotyledons 10-day-old cotton seedlings as described above in VIGS. The wound leaf surface was sprayed with ddH\u003csub\u003e2\u003c/sub\u003eO, the seedlings were inoculated in a dark hood with 70% humidity for 1 h at ambient temperature, and transferred into artificial climate chamber to grow at 26\u0026deg;C, 8 h light / 16 h dark photoperiod.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro pollen germination and pollen KI-I2 staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePollen grains from crushed anthers (Nan A, Nan B, \u003cem\u003eGhCYP450\u003c/em\u003e-silenced, \u003cem\u003eGhPHD-D\u003c/em\u003e-overexpressed plants) were placed on petri dishes at 30\u0026deg;C for 4 h in a pollen germination medium consisting of 0.04% (w/v) calcium nitrate, 0.01% (w/v) glutamic acid, 0.07% (w/v) manganese sulfate, 0.01% (w/v) lysine, 0.01% (w/v) serine, 0.01% (w/v) proline, 0.02% (w/v) boric acid, and 40% (w/v) sucrose. The relative humidity was maintained at above 80%. The 1% (w/v) iodium potassium-iodide solution was used for pollen fertility staining. The germination pollen grains and KI-I\u003csub\u003e2\u003c/sub\u003e stained pollens were observed with a microscope (Olympus, BX53) in bright-field illumination. Three biological repeats, five fields per sample were photographed for statistics.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHarnessing hybrid heterosis is an important way to increase cotton yield, improve fiber quality and enhance disease resistance. Male sterile lines are widely used in many crops to effectively facilitate hybrid breeding and seed production (Zhang, et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). But it is difficult to obtain a pure and large-scale increase of male-sterile female lines through self-pollination when GMS is used. Biotechnology-based male sterility (BMS) system can overcome this limitation and significantly improve the efficiency of hybrid seed production, and is expected to be an important tool for the efficient use of hybrid vigor in cotton and other crops. Mining male sterility genes and understanding their mechanisms are important prerequisites for the creation and application of BMS systems in cotton. In this study, we identified a GMS line Nan A controlled by a pair of recessive genes, this mutant can develop normal tetraploids, but fail in microspore development, transcriptome analysis indicate that ill-time overexpression of anther preferentially expressed genes, most of which are required for male fertility. A large set of genes encoding transcription activation proteins involved in stress response were downregulated in the GMS line. These genes are thus potential targets for developing male sterile lines through genetic engineering for the exploitation of cotton hybrid vigor.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNan A is mutated in pollen dehiscence and development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNan A is a spontaneous mutant fortuitously discovered from cotton cultivar Sumian 22 in our breeding nursery. This mutant was preliminarily identified to be controled by a single recessive gene. Although its accurate chromosome localization awaits further genetic mapping, the near isogenic fertile (Nan B) and sterile plants (Nan A) generated by backcrossing Nan A with its original cultivar Sumian 22 provide ideal isogenic lines for joint phenotypical, cytological and molecular analysis to dissect mechanisms underlying male sterility of Nan A.\u003c/p\u003e\u003cp\u003eNan A has normal vegetative phenotypes very similar to Nan B, but developed much shorter filaments, non-dehiscent anthers without disperse pollens, and pollen grains can be slightly stained using I\u003csub\u003e2\u003c/sub\u003e-KI, and the isolated pollen grains cannot germinate on culture medium. Optical microscopy and SEM analysis showed that Nan A formed normal tetrads at stage 7, but could not release normal microspores with spines on exine at stage 8 as observed in the WT, Nan B. This suggests that sterility in Nan A likely occurred between Stage 7 and Stage 8. At Stage 8, the pollens produced by Nan A are significantly different from those produced by Nan B in formation of nexine, exine together with spines. These phenotypes are highly similar to those observed in the sterile lines ZK-A (\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sub\u003e\u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) (Ma, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mao, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and 1355A (\u003cem\u003eghnsp\u003c/em\u003e) (Wu, et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu, et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), their male sterility all occur during S7 to S8 of anther development, indicating that this period is critical for microspore development and pollen wall construction that determining anther dehiscence and pollen dispersal, and consequently controlling pollen fertility in upland cotton. From these results, we can also speculate that there may be many genes involved in this development stage, and the mutation of any key gene of them may cause abnormal microspore development or abnormal pollen maturation process.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptomics unravels genes and molecular mechanisms underlying male sterility in cotton\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA-seq analysis of Nan A and Nan B anthers from S7 to S8 showed 569 DEGs. GO and KEGG enrichment analyses indicated that the 249 up-regulated genes were significantly enriched in \u0026lsquo;transporter activity\u0026rsquo; MF, \u0026lsquo;response to endogenous stimulus\u0026rsquo;, and \u0026lsquo;reproductive process\u0026rsquo; BPs, and there are 21 genes enriched in GO term of \u0026lsquo;reproductive process\u0026rsquo;. On the other hand, we identified 22 DEGs specifically or dominantly expressed in cotton anther. GH_D02G2614 (QRT3), GH_A12G2483/GH_D12G2497 (PHD), and GH_A02G0201 / GH_D02G0220 / GH_A12G1628 (GHL 17) are anther preferentially expressed genes related to \u0026lsquo;reproductive process\u0026rsquo;. These genes were all up-regulated in Nan A, and their homologs have been reported to be causative genes for male sterility in various plant species (Table\u0026nbsp;1). Among the \u0026lsquo;reproductive process\u0026rsquo; proteins, GH_D13G1652 is a NAC transcription factor, its homologs in A\u003cem\u003erabidopsis\u003c/em\u003e, NST1 and NST2 regulate secondary wall thickenings and are required for anther dehiscence, double mutant \u003cem\u003enst1 nst2\u003c/em\u003e exhibited male sterility (Mitsuda, et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); GH_A12G1040 / GH_D12G0737 (GPAT1) are involved in biosynthetic process of CDP-diacylglycerol, which is a critical intermediate in lipid metabolism, their homologs in rice (Men, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), maize (Xie, et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003eArabidopsis\u003c/em\u003e (Zheng, et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) etc. are essential for male fertility; GH_A10G0810 encodes a Rop guanine nucleotide exchange factor 12 (ROPGEF12) which is required for pollen tube and control polarized pollen tube growth. As shown in Table\u0026nbsp;1, most anther predominantly expressed DEGs are related male fertility. Collectively, a substantial number of genes, whose analogs known as the causative genes for male sterility or required for male fertility, are up-regulated in Nan A. Paradoxically, all of these genes are recessive male sterility genes, namely loss-function of them confers male sterility, but they are up-regulated in Nan A, a recessive genic male sterility. For example, mutation of PHD-finger proteins cause male sterility in soybean (Thu, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), maize (Zhang, et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), rice(Yang, et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Chinese cabbage(Dong, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), etc. which usually lead to pollen degeneration after microspore release and the abnormal tapetum vacuolation as observed in \u003cem\u003eArabidopsis\u003c/em\u003e (Ito, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). But we confirm that constitutively over-expressed the anther specifically expressed PHD gene (GH_D12G2497) driven by double CaMV 35S promoter can convert wild type cotton to be male sterility. This result is very similar to an early study (An, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), thus we may infer that over expression of these genes at wrong time can lead to abnormal development of anther or pollen, resulting in male sterility, even though they are essential for pollen development and dispersal. However, more examinations are needed to extend to other anther preferentially expressed genes that were upregulated in Nan A.\u003c/p\u003e\u003cp\u003e320 down-regulated genes are significantly enriched in MF of \u0026lsquo;DNA-binding transcription factor activity\u0026rsquo;, or \u0026lsquo;response\u0026rsquo; related BP terms (Fig.\u0026nbsp;3b, Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Intriguingly, 75% of the genes enriched in \u0026lsquo;transcription regulator activity\u0026rsquo; are responsible to stress, 71 genes enriched in \u0026lsquo;response to stress\u0026rsquo; are DNA-binding transcription factors. So \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e compromised expression of a large number of transcriptional activators involved in stress response, among which proteins involved in ethylene, jasmonate, and ABA signaling pathways take a high proportion. Jasmonic acid (JA) has been proven to be a key plant hormone that promote anther dehiscence, recently, transcriptional repressor OsTIE1 and transcription factor OsTCP1 were found to tightly regulates JA biosynthesis during anther development and dehiscence(Fang, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A lot of studies revealed that the analogs of WRKY53(Zhang, et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), CYP94C1s(Heitz, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), TIFY9(Virag, et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), AOC4(Acosta and Przybyl \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) are involved in regulation of JA biosynthesis or signaling during floral development and response to stress, but their roles in male fertility remain unknown. In this study. Up to sixteen proteins involved in ethylene biosynthesis and signaling including twelve ERFs, two MBF1Cs, one ARB1, and one ACS6 are down-regulated in Nan A, as there complicated synergistic and antagonistic interactions between ET and JA, ethylene may play important roles in male fertility, although little direct evidence is available so far (Ishimaru, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnexpectedly, among the 320 downregulated genes, GH_A12G1289 (CYP703A2) is the only one specifically expressed in anthers (Fig.\u0026nbsp;5, Table\u0026nbsp;1). Silencing GH_A12G1289 resulted in anther indehiscence and dramatic reduction in pollen viability, these phenotypes are very similar to those of the Nan A and VOX-\u003cem\u003eGhPHD-D\u003c/em\u003e cotton. However, either silencing \u003cem\u003eGhCYP450\u003c/em\u003e or overexpressing \u003cem\u003eGhPHD-D\u003c/em\u003e cannot eliminate the spikes on the surface of cotton pollen, namely changing a single gene cannot fully achieve the phenotype of Nan A pollen, indicating that the mutated \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e gene perform multiple biological functions in pollen development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, cytological analysis of the anther of a single-gene recessive genic male sterility system of male sterile line Nan A and its near-isogenic male fertile line Nan B showed that abortive pollen dehiscence, distorted wall biosynthesis, and compromised starch granule formation may be responsible for the male sterility trait. Down-regulated expression of stress responsive genes involving ethylene, JA, and ABA biosynthesis of signaling, and inappropriate overexpression of proteins even essential for reproductive development maybe the molecular mechanisms conferred by the recessive male sterility gene \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e. Either silencing the down-regulated \u003cem\u003eGhCYP450\u003c/em\u003e or over expression of the over-regulated \u003cem\u003eGhPHD-D\u003c/em\u003e in the wild-type cotton can lead to male sterility. Our study provides novel clues for understanding the molecular mechanism of male sterility and candidate genes for BMS in cotton.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDPA Days post anthesis\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and materials in the manuscript were in the methods section. Other data provided in Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 30671120, No. 32172083).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaili Qiu: The primary experimenter, Writing the original draft. Hongyu Dou: Data analysis. Kang Liu: Project administration, Funding acquisition, Conceptualization, manuscript revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr Gongyao Shi, College of Agronomy, Zhengzhou University, Henan province, China, for providing pCaBS-α, pCaBS-β, pCaBS-γ1, and pCaBS-γ2 vectors of BSMV system.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcosta IF, Przybyl M (2019) Jasmonate Signaling during Arabidopsis Stamen Maturation. 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Rice (N Y) 10:53\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Cotton, Genic male sterility, RNA-seq, VIGS, VOX","lastPublishedDoi":"10.21203/rs.3.rs-7281172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7281172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIdentification and characterization of genic male-sterility (GMS) genes is crucial for unraveling molecular mechanisms controlling anther and pollen development, and enable the development biotechnology-based male-sterility (BMS) systems for heterosis utilization and commercial hybrid seed production in crops. Here, we report a combined cytological and transcription analysis of the anther of a single-gene recessive GMS line Nan A and its near-isogenic male fertile line Nan B, and further verified the functions of two male sterility candidate genes. Nan A developed shorter stamen filaments, produced sterile pollens characterized by shriveled starch grains inside, delayed nexin deposition, no spikes on exine surface, and failure in dehiscence. A number of anther-preferentially expressed genes were unexpectedly up-regulated in Nan A, whereas loss-of-function mutants of their homologous genes in other plant species exhibit male sterility. By contrast, a number of stress-related transcription activation protein genes are down-regulated in Nan A. Either silencing the anther specifically expressed \u003cem\u003eGhCYP450\u003c/em\u003e that down-regulated in Nan A or overexpressing \u003cem\u003eGhPHD-D\u003c/em\u003e that up-regulated in Nan A can convert wild-type into male sterility. Our results indicate that timely expression of anther and/or pollen developmental genes are pivotal for male fertility.\u003c/p\u003e","manuscriptTitle":"Key genes and molecular mechanisms responsible for male sterility revealed by transcriptome analysis in cotton","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 13:47:00","doi":"10.21203/rs.3.rs-7281172/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":"6faabe5a-9cba-4cf0-8d78-25102e207d64","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-02T02:07:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 13:47:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7281172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7281172","identity":"rs-7281172","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}