OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation

OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation | 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 OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation Qi-ming Wang, Yun-lu Tian, Ke-yi Chen, Shan-shan Zhu, Ye-hui Xiong, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3873582/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2024 Read the published version in Plant Molecular Biology → Version 1 posted 4 You are reading this latest preprint version Abstract Plant lipid transfer proteins (LTPs) are distinguished by their capacity to facilitate lipid transport in vitro between membranes. This includes the transportation of lipid constituents from the tapetum to the microspore, thereby playing a pivotal role in the synthesis and construction of the pollen wall, encompassing the formation of the pollen aperture. However, our understanding of LTPs and their role in pollen aperture formation in rice remains limited. In this study, we have isolated and characterized a male sterile rice mutant named as pollen aperture defect 1 ( Ospad1 ). When compared to the wild type, Ospad1 mutant plants exhibit pollen grain abortion due to the absence of the fibrillar-granular layer, ultimately leading to the leakage of contents from the malformed aperture. OsPAD1 encodes a non-specific LTP and is specifically expressed in the tapetum and microspore during male development. Subsequently, in vitro lipid binding assays reveal that the recombinant OsPAD1 protein has the capability to bind to a broad spectrum of lipids. The malfunction of OsPAD1 results in disrupted lipid metabolism and compromised pollen aperture, ultimately leading to male sterility. Furthermore, yeast two-hybrid, bimolecular fluorescent complementation and pull-down assays all demonstrate that OsPAD1 can directly interact with OsINP1, an orthologue of a crucial aperture factor in Arabidopsis, together regulating rice aperture development. These findings offer new insights into the molecular mechanisms that underlie the function of LTPs in rice pollen aperture formation. This research holds potential implications not only for rice but also for other cereal crops. Sterile Fibrillar-granular layer Operculum Lipid binding Oriza sativa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In flowering plants, the successful accomplishment of double fertilization, involving the entry of sperm cells into the ovule, depends on the intricate process of guiding pollen tubes. The key players in this process are the apertures strategically positioned on the pollen wall. These apertures serve as entry points through which pollen tubes germinate. It is widely acknowledged that the formation of a robust pollen wall is a fundamental prerequisite for ensuring pollen viability. The pollen wall consists of essential components, namely, the exine, inner intine, and tryphine. The exine, in particular, is further subdivided into the outer sexine and the inner nexine. This inner nexine is itself divided into two distinct layers: the footlayer (Nexine I) and the endexine (Nexine II) (Murphy 2006; Blackmore et al. 2007; Jiang et al. 2013; Zhang and Yang 2014; Shi et al. 2015). Among the intricate elements of exine patterning, the pollen aperture stands out as a well-defined feature where exhibit patterns formed by the gaps in exine deposition (Furness and Rudall 2004). Much like the diverse patterns found on the surface of pollen grains, aperture patterns exhibit remarkable species-specific variations. These distinctions manifest in terms of shape, number, size, and margin characteristics among different species groups. For instance, monocots like rice typically possess a single polar aperture, while eudicots such as Arabidopsis typically exhibit three apertures (Wang and Dobritsa 2018). The pollen aperture in rice is a single-pore stucture located at the distal polar region, primarily composed of the annulus and the operculum. The annulus is a ring-like protrusion formed by a circle of thickened exine, while the operculum is a small, independent circular exine (Zhang et al. 2020). Similar to the formation of pollen exine, the initial indications of the aperture pattern in rice become discernible during the late tetrad stage. At this juncture, the plasma membrane polarizes to create an aperture plasma membrane protrusion that prevents the deposition of pre-exine or exine in these areas. By stage 9, the operculum and annulus commence their development, primarily comprising soropollenin derived from the tapetum. During stages 10 and 11, the fibrillar-granular layer and the Zwischenkörper layer, composed of well-developed callose/pectin substances, are formed to provide support to the operculum (El-Ghazaly and Jensen 1986; Zhang et al. 2020). Nonetheless, the mechanisms behind the formation of these pollen aperture structures and the genes responsible for regulating these processes remain intriguing and poorly understood questions. The first identified gene related to pollen aperture development in rice is OsDAF1 ( DEFECTIVE IN APERTURE FORMATION1 ). OsDAF1 encodes a legume-lectin kinase, playing an indispensable role in annulus formation. Notably, OsDAF1 has the capacity to interact with OsINP1 (INAPERTURATE POLLEN1), the rice counterpart of a pivotal aperture factor in Arabidopsis (Dobritsa and Coerper 2012; Zhang et al. 2020). Another key player in the aperture formation process in rice is DEAP1, which interacts with OsD6PKL3s, homologs of a well-known Arabidopsis aperture protein (Zhou et al. 2022). It is worth mentioning that research on aperture development in Arabidopsis has made significant strides. INP1, for instance, stands as the inaugural gene recognized for its involvement in pollen aperture formation, with a loss-of-function mutation resulting in the absence of pollen apertures in Arabidopsis (Dobritsa and Coerper 2012). INP1 plays a pivotal role in maintaining specific membrane domains within the callose wall during meiosis, thus preventing exine formation (Dobritsa et al. 2018). Although it has been demonstrated that pollen ploidy plays a crucial role in determining the number of pollen apertures, there is no doubt that INP1 serves as a marker for the positioning and quantification of future aperture development (Reeder et al. 2016). It's worth noting that the amino acid sequences of INP1 homologs in different species exhibit significant differences and are not interchangeable (Li et al. 2018). Recently, an INP2 protein was identified in Arabidopsis, and it was found to interact with INP1. These two proteins share strikingly similar structural features, expression patterns, and mutant phenotypes. When co-expressed in a heterologous system, the two proteins were able to restore the normal phenotype, whereas the expression of either one alone could not (Lee et al. 2021). In the realm of Arabidopsis, the protein kinase D6PKL3 also plays a direct role in pollen aperture formation. Similar to INP1 and INP2, D6PKL3 resides on the plasma membrane and binds to specific phosphoinositides to demarcate the site of pollen aperture formation (Lee et al. 2018). Nonetheless, the intricate molecular mechanisms governing pollen aperture formation remain a subject of ongoing investigation and continue to elude complete elucidation. Lipid transfer proteins (LTPs) with an N-terminal signal peptide play important roles in mediating lipid transfer across the cytoplasm (Kader 1996; Yeats and Rose 2008; Edqvist et al. 2018). These proteins, characterized by cysteine residues linked by disulfide bonds, fold into either a single large or two smaller cavity structures, which effectively bind lipids (Liu et al. 2015). LTPs serve crucial functions at various stages of plant growth, including responding to both biotic and abiotic stress (Wang et al. 2004; Sels et al. 2008), regulating processes like seed germination and seedling emergence (Eklund and Edqvist 2003), influencing reproduction (Wan et al. 2020; Tao et al. 2021), contributing to cell wall growth (Nieuwland et al. 2005), and participating in wax metabolism (Debono et al. 2009). For example, in rice, OsC6 , responsible for encoding a LTP, exhibits expression within the tapetum and is subject to modulation by TAPETUM DEGENERATION RETARDATION (TDR) (Zhang et al. 2010). In Arabidopsis, type III LTPs not only engage in lipid transport but also form an integral part of the exine structure (Huang et al. 2013). Non-specific lipid transfer proteins (nsLTPs) are another class of LTPs, characterized by their small size (6.5–10.5 kDa), and they are widely distributed throughout the plant kingdom. For example, dysfunction of OsLTP47 results in disrupted lipid metabolism and the formation of defective pollen walls, ultimately leading to male sterility (Chen et al. 2022). Similarly, mutations in glycosylphosphatidylinositol-anchored nsLTPs in Arabidopsis induce developmental defects in pollen walls, resulting in male sterility (Edstam and Edqvist 2014). These findings illuminate the significant roles that LTPs play in pollen development. However, despite these valuable insights, the intricate molecular mechanisms governing the participation of LTPs in pollen aperture formation and patterning remain poorly understood. In this study, we present our findings on the isolation and characterization of the rice gene, OsPAD1 . This gene encodes a nsLTP and serves a distinct function in the development of the fibrillar-granular layer within pollen grains. Notably, in the Ospad1 mutant, pollen grains exhibit a conspicuous absence of fibrillar-granular layer, resulting in the disruption of attachment between the operculum and the pollen. Ultimately, this leads to the rupture of the internal plasma membrane at the pollen aperture, causing the leakage of intracellular contents and leaving behind only the empty shell of the pollen exine. Furthermore, our investigation has revealed the interaction between OsPAD1 and OsINP1. This interaction highlights that OsPAD1 becomes distributed on the pollen surface when recruited as part of an OsINP-OsPAD1 complex, which is instrumental in the formation of the fibrillar-granular layer. These results present innovative insights into the precise deposition of each layer during the development of pollen walls, offering new avenues for further research in this field. Materials and methods Plant materials and conditions The rice ( Oryza sativa L.) materials were grown in the experimental fields of Nanjing Agricultural University (118°E, 31°N), Hainan province (110°E, 18°N) and Chinese Academy of Agricultural Sciences (116°E, 40°N). The Ospad1 mutant was a natural mutant from japonica variety Ningjing 7. The F 2 mapping population was generated from a cross between the Ospad1 mutant and N22 (ssp. indica ). I 2 -KI staining and aniline blue staining Freshly matured anthers were meticulously fixed in Carnoy’s solution (ethanol:glacial acetic = 3:1) and subsequently stored at a temperature of 4°C until observation. For detailed microscopic examination, pollen grains were gently released from the anthers using fine-tipped tweezers. Subsequently, these pollen grains were stained with a 1% iodine-potassium iodide (I 2 -KI) solution and meticulously captured using a Nikon AZ100 stereomicroscope. In the case of aniline blue staining, the spikelets from flowering rice were immersed in Carnoy's solution for an extended duration of more than 24 h and similarly stored at 4°C prior to utilization. The stigmas, once released, underwent a sequential treatment process, involving exposure to 70%, 50%, and 30% alcohol solutions. After a rehydration step in distilled water, they were immersed in 10 N NaOH at a temperature of 60°C for 10 min, followed by a thorough rinsing with distilled water. Subsequently, the stigmas were stained with aniline blue (0.1% in 0.1M K 3 PO 4 ) and photographed with ZEISS Imager A2 fluorescence microscope. Cytological observations Fresh rice spikelets and anthers at various developmental stages underwent immersion in FAA solution, which is composed of an 18:1:1 (v/v) mixture of formalin, 70% ethanol, and acetic acid, and also in 2.5% glutaraldehyde for a duration exceeding 24 h. Semi-thin cross-sections of the anthers were meticulously prepared, following established methods as previously reported (Yu et al. 2018). For SEM, anthers from both the wild-type (WT) and mutant samples were extracted from the glutaraldehyde solution and underwent a thorough washing process with distilled water. Subsequently, they were subjected to dehydration using a series of ethanol solutions and were subsequently fixed in a 1% OsO 4 solution for a period of 2 h. After this fixation, further dehydration was carried out, followed by critical point drying utilizing CO 2 . The anthers were then coated with a thin layer of ion-sputtered gold using an E-100 ion sputtering device and were observed using a scanning electron microscope (S3400; Hitachi). For TEM, the anthers were immersed in a solution containing 1% glutaraldehyde and 1% OsO 4 for 1 h, followed by dehydration using an ethanol series. Subsequently, they were embedded in Spurr's medium before undergoing thin sectioning. These sections were double-stained with a 2% uranyl acetate solution and a 2.6% aqueous lead citrate solution, after which they were observed at an acceleration voltage of 80 kV with a H7700 transmission electron microscope (Hitachi). TUNEL assays A Dead End Fluorometric TUNEL Kit (Promega) was employed to conduct TUNEL assays. Initially, fresh anthers at varying developmental stages were immersed in FAA for a duration exceeding 24 h. Subsequently, they underwent dehydration using an alcohol:xylene mixture and were then embedded in paraffin (Paraplast Plus, Sigma). The paraffin-embedded samples were meticulously sectioned, followed by the requisite dewaxing and rehydration steps to prepare the samples for the TUNEL assay. Throughout the experimental procedures, we adhered to the guidelines provided by the manufacturer of the kit. The excitation/emission spectra of the green fluorescence (representing the TUNEL signal) and the red fluorescence of propidium iodide were observed using a laser confocal microscope, with excitation/emission wavelengths of 488 nm/510 nm and 530 nm/640 nm, respectively. Quantitative real-time reverse transcription–PCR (qRT–PCR) Total RNA from the plant was extracted utilizing the RNeasy Plant Mini Kit (Qiagen). Subsequently, first-strand cDNA was synthesized, starting with 1 µg of RNA, employing the QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative RT-PCR assays were conducted using gene-specific primers in conjunction with SYBR Premix ExTaq reagent (Takara). These experiments were carried out using the ABI 7500 Real-Time PCR System (Applied Biosystems) in strict accordance with the manufacturer's guidelines. Each sample was subjected to PCR in triplicate as part of three independent biological replicates, with the rice ubiquitin gene serving as an internal control. RNA in situ hybridization Fresh rice anthers at various developmental stages were immersed in RNase-free FAA for an extended period of over 24 h, and then embedded in paraffin after dehydration. According to a previous publication (Li et al. 2006), OsPAD1 cDNA as template was used to prepare sense and antisense probes with a DIG Northern Starker Kit (Cat. no. 2039672, Roche). RNA hybridization and immunological detection followed the Kit instructions. Vector Construction Functional complementation and gene knockout experiments were devised to validate the role of OsPAD1 . The complete sequence from the WT, encompassing a 2,864 bp promoter region, a 2,089-bp gene region, and a 138-bp downstream region, was seamlessly integrated into the binary vector pCUbi1390 through in-fusion cloning using the In-Fusion HD Cloning Kit. Callus derived from the seeds of Ospad1 /WT heterozygotes served as the starting material for Agrobacterium-mediated transformation, following the method established by Hiei et al. in 1994. Twenty-base-pair gene-specific spacer sequences derived from OsPAD1 were inserted into the entry vector pOs-sgRNA, and subsequently, they were subcloned into the destination vector containing Cas9 using the Gateway LR Clonase II Enzyme mix (Invitrogen) method, as described previously (Miao et al. 2013). Callus that had been induced from WT seeds underwent Agrobacterium-mediated transformation. The specific primers used for PCR in this process are listed in Supplemental Table 1. To generate fully functional transgenic plants expressing YFP-tagged OsPAD1, we conducted a genomic complementation assay. In this approach, the YFP coding sequence, lacking a stop codon, was positioned upstream of the OsPAD1 cDNA. Subsequently, the YFP-OsPAD1 construct was integrated into the pCAMBIA1390 vector using in-fusion cloning. The fragment was placed under the control of the native UBI promoter present on the vector. This resulted in the creation of the pUBI:YFP-OsPAD1cDNA construct (Tan et al. 2014). Following the construction of pUBI:YFP-OsPAD1cDNA, this construct was introduced into the Ospad1 homozygous plants. Remarkably, this led to the restoration of pollen fertility, serving as compelling evidence that the YFP-tagged protein is indeed fully functional. Subcellular localization of OsPAD1 The OsPAD1 coding sequence (CDS) lacking stop codons was fused with green fluorescent protein (GFP) and subsequently inserted into the pAN580-GFP vector (primer details provided in Supplemental Table 1). The extracted plasmids were transfected into rice protoplasts, followed by an overnight incubation period in a dark environment at 28°C. Fluorescent images were acquired using a confocal laser scanning microscope (Zeiss LSM780). Lipid binding activity Lipid binding assays were conducted using PIP-strip membranes (Echelon Biosciences; P-6001) in accordance with the manufacturer's instructions. Initially, the PIP-strip membranes were immersed in blocking buffer for a duration of 1–2 h. Subsequently, the purified protein was introduced at a concentration of 5 µg/mL into the blocking buffer supplemented with 0.1% (v/v) Tween 20®, and this mixture was incubated with the membranes for an additional 1–2 h. Following this incubation period, the membranes were meticulously washed three times with PBST and then subjected to further incubation with the appropriate antibodies. This secondary incubation was conducted in blocking buffer containing 0.1% (v/v) Tween 20 for another 1–2 h. Lastly, the membranes were rinsed three times with PBST to conclude the assay. Pollen lipids determination Approximately 100 mg of anthers at the stage 10 from both the WT and Ospad1 mutant were swiftly frozen by submerging them in liquid nitrogen. These frozen anthers were then crushed with forceps and introduced into a preheated inactivation solvent consisting of 0.01% (w/v) butylated hydroxytoluene (BHT) dissolved in isopropanol at a temperature of 75°C. The mixture was thoroughly blended and subsequently placed in a water bath maintained at 75°C for an 18 min duration. After this, the pollen contained within the anthers was separated by filtering the mixture through a 100-mesh cell sieve to eliminate any impurities, thereby allowing for subsequent lipidomic analysis. Lipid determination was conducted following microscopic examination to ensure the correct pollen stage and the absence of excessive impurities. After inactivation, an extraction solvent consisting of chloroform : methanol: 300 mM ammonium acetate (30:41.5:3.5) (v/v/v) was introduced into the samples. Subsequently, these samples were incubated at room temperature for 24 h while being agitated at 150 rpm. Upon completion of the incubation, the samples were subjected to centrifugation, and the resulting clear supernatant was carefully transferred to fresh tubes. The inactivation and extraction steps were repeated once, and lipid extracts from both rounds of extraction were combined and subsequently desiccated in a SpeedVac system (Genevac, UK). The lipid extracts were then stored at -80℃ until they were ready for LCMS analyses. Phospholipids, sterol and neutral lipids were analyzed using an Agilent 1260 HPLC system (Agilent Technologies) coupled to a 5500 QTRAP instrument running analyst v.1.6.3 (Sciex). Sphingolipids were analyzed using a Shimadzu Nexera 20AD-HPLC/ExionLC-AD system connected to a Sciex QTRAP 6500 PLUS instrument, as previously described. For the analysis of polar lipids in normal phase, individual species were separated using a TUP-HB silica column (i.d. 150x2.1 mm, 3 µm) under the following precise conditions: mobile phase A consisted of a mixture of chloroform, methanol, and ammonium hydroxide (89.5: 10: 0.5), while mobile phase B was composed of chloroform, methanol, ammonium hydroxide, and water (55: 39: 0.5: 5.5). In the case of reverse-phase LC/MS analysis, lipids were assessed utilizing a modified version of reverse-phase (RP)-HPLC/ESI/MS/MS, as previously documented. In summary, the separation of the aforementioned lipids was executed on a Phenomenex Kinetex column with a 2.6 µm C18 packing material (internal diameter 4.6×100 mm). An isocratic mobile phase consisting of chloroform, methanol, and 0.1M ammonium acetate (100:100:4) was employed at a flow rate of 300 µl/min for a duration of 10 min. Free cholesterols and cholesteryl esters were analyzed in atmospheric pressure chemical ionization (APCI) mode using an Agilent 1260 HPLC system from Agilent Technologies, connected to a 5500 QTRAP instrument running analyst v.1.6.3 from Sciex. The analysis incorporated the use of d6-cholesterol and d6-C18:0 cholesteryl ester (CE) as internal standards, with the latter being sourced from CDN isotopes (Shui et al. 2011). Three biological replicates were performed. Quantification of individual lipid species was accomplished by referencing spiked internal standards, including d9-PC32:0(16:0/16:0), d7-PE33:1(15:0/18:1), d31-PS(d31-16:0/18:1), d7-PA33:1(15:0/18:1), d7-PG33:1(15:0/18:1), C17-SL, d5-CL72:8(18:2)4, C17-LPA, d7-LPC18:1, d7-LPE18:1, DMPS, DMPA, DMPG, MGDG 34:0, DGDG 36:0, Cer d18:1/15:0-d7, GluCer d18:1/12:0, d17:1 Sph, d17:1 S1P, D-ribo-phytoSph C17, d5-DAG16:0/16:0, and d5-DAG18:1/18:1 obtained from Avanti Polar Lipids (Alabaster, AL) and LIPID MAPS. Dioctanoyl phosphatidylinositol (PI) (16:0-PI) was procured from Echelon Biosciences, Inc. (Salt Lake City, UT) and utilized in conjunction with d7-PI33:1(15:0/18:1) (Avanti Polar Lipids) for PI quantitation. Triacylglycerols (TAGs) were quantified using TAG(14:0)3-d5, TAG(16:0)3-d5, and TAG(18:0)3-d5 obtained from CDN isotopes. Free fatty acids were quantified using d31-16:0 (Sigma-Aldrich). Yeast two-hybrid (Y2H) assays Protein interactions were evaluated through Y2H analysis employing the DUALhunter system, a product of Dualsystems Biotech. To facilitate this analysis, the coding fragment of the target gene was merged with either the Cub fragment contained within the pXGY17 vector or the Nub fragment present in the pXGY18 vector. This led to the generation of pXGY17-OsPAD1 and pXGY18-INP1 constructs, respectively. The specific primers used for Y2H assays can be found in Supplemental Table 1. Pull-down assays Transgenic plants were used to extract the OsPAD1 protein carrying the FLAG tag. The coding sequence of OsINP1 was cloned into the pGEX-4T-2 vector to obtain GST-OsINP1 fusion protein. The experimental procedure was performed following a published method (Miernyk and Thelen 2008). Immunoassays were performed using anti-FLAG (Medical Biological Laboratories, 1:2000) and anti-GST (Medical Biological Laboratories, 1:2000). Bimolecular fluorescence complementation (BiFC) analysis The coding sequences of OsPAD1 and OsINP1 were cloned into the pYN1 or pYC1 vectors (a gift of Joh A. Lindbo, OARDC, Ohio State University, Wooster) to construct Y N -OsPAD1 and Y C -INP1 (primers used in BiFC assays are listed in Supplemental Table 1). The recombinant plasmid was introduced into Agrobacterium strain EHA105 and used to infiltrate Nicotiana benthamiana leaves, as described previously (Waadt and Kudla 2008). Fluorescent images were captured using a Zeiss LSM700 laser scanning confocal microscope. Results Characterization of the Ospad1 mutant Under natural growing conditions, the phenotypes of the Ospad1 mutant and WT are identical during the vegetative period (Fig. 1 A). The first visual difference is the appearance of white colored anthers in the mutant (Fig. 1 B, C). Ospad1 mutant anthers and pollen failed to stain by iodine potassium iodide (I 2 -KI), indicating the inability to accumulate starch (Fig. 1 D-G). Magenta acetate staining indicated that the mutant pollen developed normally until the microspore development stage, after which there was no further development (Supplementary Fig. S1 ). Applicatiom of WT pollen to Ospad1 mutant stigmas resulted in normal seed production, indicating that the mutation did not affect the female reproductive organs. To further investigate the cellular defects of the Ospad1 mutant anthers, we compared transverse sections of mutant and WT anthers at various stages of pollen development. No difference was observed prior to microspore development stage 9 (Fig. 1 H, I, M, N), after which the tapetum layer of WT anthers began to shrink and staining intensified, the middle layer began to narrow and gradually disappear, and the microspores gradually became spherical (Fig. 1 J). However, the tapetum layer of Ospad1 mutant was not concentrated, and the middle layer remained still clearly visible (Fig. 1 O). After stage 10, Ospad1 mutant microspores began to shrink and ceased development (Fig. 1 P). At stages 11 and 12, degradation of the WT tapetum layer was completed, and the middle layer disappeared (Fig. 1 K, L). In contrast, the tapetum layer and middle layer in the mutant remained visible (Fig. 1 Q). Degradation of the tapetum is considered to result from programmed cell death (PCD), which can be detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. At stage 7, no TUNEL signal was observed in the WT or mutant samples, indicating that the tapetum had not initiated the degradation process (Fig. 1 R, V). At the stage 8, TUNEL signals were evident in the tapetum layer of the WT, but not in the mutant (Fig. 1 S, W). In stage 9, intense signals of PCD were observed in the WT tapetum (Fig. 1 T), while the mutant displayed only a weak signal (Fig. 1 X). In stage 10, the WT exhibited no signal, indicating that the completion of PCD and full degradation of the tapetum (Fig. 1 U). Conversely, the Ospad1 muatant tapetum exhibited a persistent intense signal (Fig. 1 Y). These results suggested that the PCD in the Ospad1 mutant was delayed compared to the WT. The Ospad1 mutant pollen is deficient in pollen aperture Delayed degradation of the tapetum often results in the developmental abortion of the pollen wall (Shi et al., 2015). To observe whether there are defects in the mutant pollen wall, scanning electron microscopy (SEM) was employed to observe the development of pollen walls in WT and Ospad1 mutant. Further insights from SEM revealed a significant abnormal in mature mutant pollen: the absence of the operculum, coupled with the lack of a distinct annulus. These observed structural deficiencies may directly contribute to pollen abortion (Fig. 2 A-D). To thoroughly explore the phenotype of the mutant at the aperture, we employed transmission electron microscopy (TEM) to examine the pollen aperture at different developmental stages in both the WT and Ospad1 mutant. At stage 10, the WT initiated the formation of the pollen aperture, resulting in the inner wall of the annulus forming a deeply stained multilayered structure. Additionally, a lightly stained layer, known as the fibrillar-granular layer, developed between the pollen apertures and the plasma membrane (Fig. 2 E, F). Conversely, at stage 10, the mutant displayed the operculum detaching from the pollen, with no deeply stained multilayer structure present on the inner wall of the annulus, and a notable absence of the fibrillar-granular layer between the operculum and the plasma membrane (Fig. 2 I, J). Subsequently, the plasma membrane of the Ospad1 mutant pollen ruptured through the aperture, resulting in the leakage of the internal cellular structure into the locule (Fig. 2 K, L). In contrast, the fibrillar-granular layer in WT pollen continued to thicken (Fig. 2 G, H). These results suggested that the absence of the operculum in the Ospad1 mutant did not stem from its failure to form, but rather resulted from the absence of the fibrillar-granular layer. In the absence of this essential layer, the operculum lacked proper attachment to the aperture, consequently leading to its shedding during the later stages of development. Ultimately, this detachment resulted in pollen abortion. Identification of Ospad1 mutation To determine the molecular lesion of the Ospad1 mutant, we used a map-based cloning approach to isolate the OsPAD1 allele. In an F 2 population from cross between Ospad1 and N22, fertile and sterile plants segregated in a 3:1 ratio (340 fertile vs 137 sterile, χ 2 3:1 = 3.33 0.05), indicatting that the Ospad1 defects were caused by a recessive mutation in a single nuclear gene. Initially, we mapped the OsPAD1 locus to a region flanked by markers M-17 and RM-5 on chromosome 1. Subsequently, we refined this region to a 96-kb physical interval positioned between markers M-29 and M-30 . This interval encompasses 17 putative open reading frames (ORFs), as annotated by the Rice Genome Annotation Project Database ( http://rice.plantbiology.msu.edu/ ) (Fig. 3 A). Upon conducting sequencing and comparative analyses, a 30-base pair (bp) deletion is identified in the first exon of ORF16 ( LOC_Os01g42210 ) in the Ospad1 mutant. This deletion affects 10 amino acids within a region of LOC_Os01g42210 that is conserved across multiple species (Supplementary Fig. S2 A). To identify its homologs in other species, we performed BLASTP searches using amino acid sequences and subsequently constructed a phylogenetic tree (Supplementary Fig. S2 B). The results revealed the existence of OsPAD1 homologs in numerous plant species, many of which remain unstudied. Interestingly, LOC_Os01g42210 is the OsLTP47 gene (Chen et al. 2022). Notably, while a mutation in this gene led to male sterility, no report was provided regarding the phenotype of pollen aperture defection. Consequently, LOC_Os01g42210 was the most promising candidate gene. To identify the causal mutation, we firstly conducted a functional complementation experiment. Transgenic plants were generated by introducing a WT genomic fragment into Ospad1 mutant callus. This approach aimed to confirm that the observed pollen aperture defect in the Ospad1 mutant was indeed attributed to the disruption of LOC_Os01g42210 . Positive transgenic plants exhibited normal pollen aperture development, characterized by yellow anthers and restored pollen fertility (Fig. 3 B, F, D, H; Supplementary Fig. S3A, C, E). Furthermore, we employed CRISPR/Cas9-targeted mutagenesis to generate a homozygous mutant, known as Cri-Ospad1 . These Cri-Ospad1 mutant plants exhibited an 848-bp deletion, situated 78-bp upstream of the ATG start codon, and displayed white anthers with non-fertile pollen, mirroring the phenotype of the Ospad1 mutant (Fig. 3 C, E; Supplementary Fig. S3B-E). TEM analysis revealed that pollen grains from the knockout plants lacked an operculum, similar to those observed in the Ospad1 mutant (Fig. 3 G, I). Seed setting of the transgenic plants were sterile as expected (Supplementary Fig. S3F). In summary, our findings conclusively establish that LOC_Os01g42210 corresponds to OsPAD1 , confirming its pivotal role in pollen aperture development. Subcellular localization of OsPAD1 and expression pattern To investigate the subcellular localization of the OsPAD1 protein, a transient expression experiment was conducted in rice protoplasts. We utilized a green fluorescent protein (GFP)-tagged OsPAD1 fusion protein, driven by the cauliflower mosaic virus (CaMV) 35S promoter. Our observations revealed that GFP signals were detected within two distinct subcellular compartments: the plasma membrane (PM) and the nucleus. Importantly, these signals colocalized with the PM marker SCAMP-mCherry and the nuclear marker D53-mCherry, respectively (Fig. 4 A, B) (Cai et al. 2011; Zhou et al. 2013). A closer examination of the OsPAD1 amino acid sequence revealed the presence of a transmembrane domain spanning amino acids 52nd to 74th (Supplementary Fig. S4A). This domain effectively divided the protein into two segments: an N-terminal region (residues 1–51) and a C-terminal region (residues 75–268). Intriguingly, when the N-terminal region (OsPAD1 1–51 -GFP) and the C-terminal region (OsPAD1 75–268 -GFP) were individually controlled by the CaMV-35S promoter, their subcellular locations within the protoplast were not entirely identical. OsPAD1 1–51 -GFP exhibited a presence in both the PM and the nucleus, mirroring the behavior of the full-length OsPAD1-GFP (Fig. 4 C, D). In contrast, OsPAD1 75–268 -GFP was exclusively localized to the PM (Fig. 4 E, F). These findings collectively indicate that the N-terminal region is necessary for nuclear localization. In its absence, the protein still tends to localize to the PM, suggesting that the N-terminal region is not a prerequisite for PM localization. In addition, we also conducted transgenic experiments to investigate the protein localization of OsPAD1 on pollen grains at various developmental stages in OsPAD1-YFP transgenic plants. To begin with, we generated OsPAD1-YFP transgenic plants in the Ospad1 mutant background. It is worth noting that pollen fertility was successfully restored in all positive transgenic plants, thus confirming the functionality of the transgene (Supplementary Fig. S4B-I). Subsequently, we meticulously examined the localization pattern of the OsPAD1 protein within pollen grains at different developmental stages. Our results revealed that starting from stage 9, the fluorescence signal of the OsPAD1 protein began to emerge at the pollen aperture site. As the developmental stages progressed to 10 and 11, the fluorescence signals exhibited more pronounced characteristics in proximity to the pollen aperture site. Consequently, during stages 12 and 13, the fluorescence signals gradually diminished in the vicinity of the pollen aperture site (Supplementary Fig. S5). To investigate the expression patterns of OsPAD1 in both anthers and vegetative tissues of WT plants, we employed a combination of reverse-transcription quantitative PCR (RT-qPCR) and RNA in situ hybridization. Our results revealed that OsPAD1 expression levels were notably higher in anthers compared to roots, leaves, leaf sheaths, and stems. Furthermore, upon closer examination of anthers at different developmental stages, we observed a positive correlation between the maturation of anthers and the expression of OsPAD1 (Fig. 4 G). We also conducted spatial and temporal analysis of OsPAD1 expression within the anther using RNA in situ hybridization on sections of WT anthers. Initially, OsPAD1 exhibited very low levels of expression in the tapetum and microspores during stages 7 and 8 (Fig. 4 H, I). By stage 9, OsPAD1 expression was significantly upregulated in the tapetum, with a comparatively lower expression in the microspores (Fig. 4 J). Notably, during stage 10, when the tapetum had undergone substantial degradation, OsPAD1 expression was absent in the microspores (Fig. 4 K). However, at stage 11, OsPAD1 was once again expressed in the microspores (Fig. 4 L). These dynamic expression patterns strongly support the role of OsPAD1 in tapetum development and the formation of pollen apertures. Dysfunction of OsPAD1 leads to alterations in the lipid content of pollen grains LTPs are typically characterized as small, 9-kDa proteins found in abundance within higher plants (Kader, 1996). However, it is noteworthy that the molecular weight of OsPAD1 is significantly larger, at 27 kDa, compared to the conventional LTP proteins. Despite this difference, OsPAD1 exhibits key structural features consistent with LTPs, including the presence of four conserved disulfide bridges and an eight-Cys motif, which serves as the hallmark signature of LTP proteins (Supplementary Fig. S6A). This structural arrangement forms the molecular basis through which LTPs transport lipids. Given that OsC6 , another gene in rice, encodes an LTP and has been shown to bind lipid molecules in vitro (Zhang et al. 2010), we hypothesized that OsPAD1 may similarly exhibit lipid-binding activity. Since we did not have access to purified full-length OsPAD1, to investigate this possibility, we isolated and purified the AAI domain within OsPAD1, a region predicted to interact with lipids, and assessed its ability to bind various phospholipids (Supplementary Fig. S6B). The results demonstrated that His-SUMO-AAI exhibited binding affinity to a variety of phospholipids, with a predominant binding to phosphatidylinositol (PI) and its mono/bisphosphates (PI(3)P, PI(4)P, PI(5)P, PI(3,5)P 2 , and PI(4,5)P 2 ). In addition, there was notable binding to phosphatidic acid (PA) (Fig. 5 A). To further validate whether the loss of lipid transport function in the Ospad1 mutant contributes to the male sterility phenotype, we conducted measurements of lipid content in pollen grains at maturity for both mutant and WT plants (Supplementary Fig. S7). At maturity, significant reductions in the levels of various lipids were observed in the Ospad1 mutant pollen grains compared to the WT, including PI, PA, lysophosphatidic acids, cardiolipins, phosphatidylglycerols, diacylglycerols, phosphatidylethanolamines, digalactosyl diacylglycerols, phosphatidylcholines, and monogalactosyl diacylglycerols (Fig. 5 B, Supplementary Fig. S8, 9). Remarkably, the decreases in the levels of PI and PA, these lipids that OsPAD1 can bind to, have caught our attention. Therefore, we speculate that in the Ospad1 mutant hinders the normal transport of these lipids, ultimately resulting pollen aperture defection. OsPAD1 interacts with OsINP1 to affect the pollen aperture formation At present, two genes, OsINP1 and OsDAF1 , associated with the aperture, were both found to be localized at the aperture (Zhang et al. 2020). An experiment using a yeast two-hybrid system confirmed the interaction between OsPAD1 and OsINP1 (Fig. 6 A). Further bimolecular fluorescence complementation (BiFC) and pull-down assays showed direct the interaction between OsPAD1 and OsINP1 (Fig. 6 B, C). To delve deeper into the significance of this interaction, we generated the Osinp1 mutant using CRISPR/Cas9 technology on the WT background. In additon, a double mutant ( Ospad1/Osinp1 ) was obtained by knocking out the OsINP1 allele with CRISPR/Cas9 technology in the Ospad1 mutant. Under natural growing conditions, the phenotypes of Ospad1/Osinp1 double mutant plants showed no apparent differences from the WT, Ospad1 , and Osinp1 mutants (Fig. 7 A-D). Of interest, SEM revealed that, in comparison to the WT, the Osinp1 mutant exhibited no pollen aperture phenotype (Fig. 7 E-G), consistent with findings from previous studies (Zhang et al. 2020). However, the double mutant displayed heightened pollen sterility, characterized by complete shrinkage of pollen grains (Fig. 7 H). Further TEM analysis indicated that the Osinp1 mutant pollen grains solely lacked the aperture phenotype but showed an accumulation of starch contents (Fig. 7 I-K). In contrast, the double mutant pollen grains not only lacked the aperture phenotype but also exhibited no accumulation of starch contents (Fig. 7 L). These results suggest that OsPAD1 may play a role in the final stages of pollen development, contributing to the completion of pollen starch and nutrient filling, as well as participating in pollen aperture formation. Discussion Lipid transport plays a pivotal role in the development of plant reproductive tissues, particularly in the anther cuticle and pollen wall (Shi et al. 2015; Wan et al. 2020). Disruptions in lipid transport during anther and pollen development frequently result in male sterility. For instance, under the positive regulation of TDR, OsC6 facilitates lipid transport from tapetum cells to microspores, thereby promoting pollen wall formation (Zhang et al. 2010). Type III nsLTPs not only transport lipids from tapetum cells to microspores but also actively participate in pollen wall formation as essential components (Huang et al. 2013). EAT1, through its binding to the promoter of OsLTPL94 , activates this lipid transporter, enabling its secretion from microspores and tapetum to the pollen wall, further enhancing pollen wall formation (Tao et al. 2021). Notably, OsLTP47 encodes a grass-specific, membrane-localized nsLTP, and its dysfunction results in disrupted lipid metabolism in the anther and defective pollen walls (Chen et al. 2022). While nsLTPs have been studied in pollen grain development, research on pollen aperture development remains limited. In this study, we observed that the Ospad1 mutant lacks the fibrillar-granular layer, while the rest of the pollen wall remains intact (Fig. 2 ). The absence of the fibrillar-granular layer in the pollen wall prevents the operculum from attaching, resulting in the rupture of the internal plasma membrane (Fig. 2 I-L). Our results introduce novel insights into the mechanisms of lipid transporter proteins participating in the formation of the rice pollen aperture. An intriguing finding from our research is the phylogenetic relationship analysis, which reveals that OsPAD1 and some of its homologous proteins are classified within the monocotyledonous branch, while homologous proteins found in other species, such as willow, rose, pomegranate, and Arabidopsis, are categorized within the dicotyledonous branch. Notably, most of the species closely related to OsPAD1 belong to the Poaceae family, which typically possess only one aperture in their pollen. This suggests a conserved role for OsPAD1 related to aperture development. Additionally, OsPAD1 homologs are exclusively found in both monocotyledonous and dicotyledonous plants, indicating that OsPAD1 likely emerged during the later stages of plant evolution and might play a crucial role in the evolution of angiosperms (Supplementary Fig. S2 B). These unique phenotypes not only distinguish themselves from those previously reported in male sterile mutants (Zhang et al. 2010; Wan et al. 2020; Tao et al. 2021; Chen et al. 2022), but also open up new avenues for investigating pollen aperture formation, particularly in relation to the formation of the fibrillar-granular layer in grasses. In Arabidopsis, INP1 plays a critical role in regulating aperture formation by preventing sporopollenin deposition at future aperture sites (Dobritsa and Coerper 2012). Another key Arabidopsis protein, D6PKL3, also involved in aperture regulation, does not directly interact with INP1. Nevertheless, these two proteins mutually influence each other's localization (Lee et al. 2018). Importantly, both INP1 and D6PKL3 interact with various PI, including PI(4)P and PI(4,5)P 2 , known to accumulate in the future aperture region of Arabidopsis. This accumulation suggests their potential role in recruiting proteins related to aperture development to these specific regions (Lee et al. 2018). Similarly, our lipid-binding experiments have demonstrated that OsPAD1 can interact with PI and its mono/bisphosphates, including PI(4)P and PI(4,5)P 2 (Fig. 5 A). This suggests that OsPAD1 might be recruited by PI(4)P and PI(4,5)P 2 to carry out its function. OsINP1, a homolog of INP1 in rice, serves a similar role to its Arabidopsis counterpart, accumulating at the future aperture position during the late tetrad stage. OsDAF1, another protein, interacts with OsINP1, and both proteins localize to the aperture. However, the polarized accumulation of OsDAF1 at the aperture depends on its interaction with OsINP1, whereas the polarization of OsINP1 occurs independently of OsDAF1 (Zhang et al. 2020). In our current study, the pollen aperture of the Ospad1 mutant was found to lack an operculum. TEM revealed that the absence of an operculum in the mutant was due to the absence of the fibrillar-granular layer (Fig. 2 I, J). Since OsINP1 aggregates at the future aperture position during the late stage 8 and converges to the fibrillar-granular layer during pollen wall formation, it is plausible that during the development of the pollen outer wall at stage 9, OsPAD1 accumulates unevenly on the pollen surface under the guidance of OsINP1. This accumulation likely involves the utilization of lipids from the tapetum to form the fibrillar-granular layer. These findings shed light on the specific molecular function of OsINP1 in aperture formation and provide new insights for the study of aperture formation mechanisms. Notably, the double mutant's pollen displayed a typical abortion phenotype, even in the absence of an aperture (Fig. 7 H, L). The result suggests that OsPAD1 plays a crucial role not only in lipid transport for the fibrillar-granular layer formation but also potentially contributes to starch accumulation in pollen during late flower development. Declarations Statements and Declarations Acknowledgements We thank Wenhua Zhang (Nanjing Agricultural University) for useful comments. Author Contributions J.M.W. and Z.G.Z. directed the project. J.M.W., Z.G.Z., and Q.M.W. designed the experiments and wrote the paper. Y.L.T. provided the Ospad1 mutant material. Q.M.W. and K.Y.C. performed most of the experiments. S.S.Z., Y.H.X., C.L.W, X.W.Y., W.T.B., H.Z., and S.M.Y. helped to analyze data. Y.H., D.K.L., A.Q.J., J.Y.L., and H.Y. helped to generate the transgenic plants. X.Z., Y.L.R., C.L.L., Z.J.C., Q.B.L., and L.J. provided technical support. All authors read the final manuscript and approved of its content. Funding This research was supported by the National Key Research and Development Program of China (2022YFD1201504, 2022YFD1200801), the National Nature Science Foundation of China (U2002202, 31991224, 31971909), the Key Research and Development Program of Jiangsu Province (BE2021360), the Key Laboratory of Biology, Genetics and Breeding of Japonica Rice in the Mid-lower Yangtze River, and the Jiangsu Collaborative Innovation Center for Modern Crop Production. Data Availability Enquiries about data availability should be directed to the authors. Competing interests The authors have no relevant financial or non-financial interests to disclose. References Blackmore S, Wortley AH, Skvarla JJ, and Rowley JR (2007) Pollen wall development in flowering plants. New Phytol 174: 483-498. Cai Y, Jia T, Lam SK, Ding Y, Gao C, San MW, Pimpl P, Jiang L (2011) Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER-Golgi-TGN-PM pathway. 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Cai-lin","middleName":"","lastName":"Lei","suffix":""},{"id":275066380,"identity":"ebbb0866-1b96-4242-bbbc-eaf1ab578f8d","order_by":18,"name":"Zhi-Jun Cheng","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences Institute of Agro-food Science and Technology: Chinese Academy of Agricultural Sciences Institute of Food Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Jun","middleName":"","lastName":"Cheng","suffix":""},{"id":275066381,"identity":"3688ac7c-0db3-49b1-901b-2566c5a391a6","order_by":19,"name":"Qi-bing Lin","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences Institute of Crop Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qi-bing","middleName":"","lastName":"Lin","suffix":""},{"id":275066382,"identity":"dcfd0539-d48e-48ef-9905-2fa3ec6185bc","order_by":20,"name":"Ling Jiang","email":"","orcid":"","institution":"Nanjing Agricultural University State Key Laboratory of Crop Genetics and Germplasm Enhancement","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Jiang","suffix":""},{"id":275066383,"identity":"3e3e8ebb-0965-4188-8b78-b1b046f4c337","order_by":21,"name":"Zhi-gang Zhao","email":"","orcid":"","institution":"Nanjing Agricultural University State Key Laboratory of Crop Genetics and Germplasm Enhancement","correspondingAuthor":false,"prefix":"","firstName":"Zhi-gang","middleName":"","lastName":"Zhao","suffix":""},{"id":275066384,"identity":"6a5f2ed8-6044-4aa9-9178-a33bbf30d888","order_by":22,"name":"Jian Min Wan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYJACA4YKBmYwi4d4LWdI1cLA2AZlEKXF4PjZA0U3591h55+RwPjgbRuDvDlBLWfyEoxztz1jlriRwGw4t43BcGcDAS1mB3IMgFoOMzPcSGCT5m1jSDA4QEjL+TdALXMOM8vfSGD/TZyWGyBbGg4zGwBtYSZKi/0NoC05xw4zG5552Cw555yE4QZCWiT7c8yMc2oOJ8sdTz744U2ZjTxBW4CAzQBIJANjpwFISxBWDwTMD4CEHVFKR8EoGAWjYGQCAJxGPx0OnQsWAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7813-4362","institution":"1.State Key Laboratory for Crop Genetics ;Germplasm Enhancement, Jiangsu Provincial Center of Plant Gene Engineering, Nanjing Agricultural University; 2.Institute of Crop Science, Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"Min","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2024-01-17 18:58:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3873582/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3873582/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11103-024-01531-z","type":"published","date":"2024-12-22T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51776826,"identity":"a3d61a2c-6789-45ef-af7a-495163fb1dd8","added_by":"auto","created_at":"2024-02-28 21:00:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":732943,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of WT and \u003cem\u003eOspad1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e WT and \u003cem\u003eOspad1\u003c/em\u003emutant plants. Scale bar = 10 cm. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e WT (\u003cstrong\u003eB\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eC\u003c/strong\u003e) flowers after removal of the lemma and palea. Scale bars = 1.5 mm. \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE \u003c/strong\u003eAnthers of WT (\u003cstrong\u003eD\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eE\u003c/strong\u003e) stained with iodine potassium iodide solution. Scale bars = 1 mm. \u003cstrong\u003eF\u003c/strong\u003e and \u003cstrong\u003eG \u003c/strong\u003ePollen of WT (\u003cstrong\u003eF\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eG\u003c/strong\u003e) stained with iodine potassium iodide solution. Scale bars = 200 µm. \u003cstrong\u003eH-Q\u003c/strong\u003e Semi-thin sections of WT and \u003cem\u003eOspad1\u003c/em\u003e mutant anthers.\u003cstrong\u003e \u003c/strong\u003eT, tapetum. Scale bars = 50 µm. \u003cstrong\u003eR-Y \u003c/strong\u003eTUNEL assay on anthers of WT (\u003cstrong\u003eR\u003c/strong\u003e-\u003cstrong\u003eU\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eV\u003c/strong\u003e-\u003cstrong\u003eY\u003c/strong\u003e) at different developmental stages. Scale bars = 50 µm.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/b43ab41a99b0cb26ba756402.jpg"},{"id":51776323,"identity":"4737b01e-1433-45ea-8b96-8324bed02ff9","added_by":"auto","created_at":"2024-02-28 20:52:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":840550,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype of WT and \u003cem\u003eOspad1\u003c/em\u003e mutant at the aperture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e Comparison of mature pollen grains by SEM for WT(\u003cstrong\u003eA, B\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e (\u003cstrong\u003eC, D\u003c/strong\u003e) mutant plants. Scale bars = 5µm. \u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003e TEM observation of the WT (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e) and \u003cem\u003eOspad1\u003c/em\u003e (\u003cstrong\u003eI\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003e) mutant pollen aperture at stage 10 and 11. Scale bars = 5 µm. An, annulus; FG, fibrillar-granular layer; Op, operculum; PM, plasma membrane.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/b7d9d55d892d35bba8582cad.jpg"},{"id":51776322,"identity":"dd083633-0593-42c5-b27b-676d3796e267","added_by":"auto","created_at":"2024-02-28 20:52:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":995044,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation of the \u003cem\u003eOsPAD1\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eFine mapping of the \u003cem\u003eOsPAD1\u003c/em\u003e locus. Physical distances, names and locations of molecular markers are shown. \u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eE\u003c/strong\u003e Phenotypic comparison of WT (\u003cstrong\u003eB\u003c/strong\u003e), \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eC\u003c/strong\u003e), CL (pOsPAD1-gOsPAD1 complementation line) (\u003cstrong\u003eD\u003c/strong\u003e) and a \u003cem\u003eCri-Ospad1\u003c/em\u003emutant (\u003cstrong\u003eE\u003c/strong\u003e) created by CRISPR-Cas9. Scale bars = 10 cm. \u003cstrong\u003eF\u003c/strong\u003e-\u003cstrong\u003eI\u003c/strong\u003e TEM of the aperture in WT (\u003cstrong\u003eF\u003c/strong\u003e), \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eG\u003c/strong\u003e), CL (pOsPAD1-gOsPAD1 complementation line) (\u003cstrong\u003eH\u003c/strong\u003e) and a \u003cem\u003eCri-Ospad1\u003c/em\u003emutant (\u003cstrong\u003eI\u003c/strong\u003e) created by CRISPR-Cas9. Ap, aperture; FG, fibrillar-granular layer; Op, operculum. Scale bars = 2 µm.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/242cdcd8e5fa6cd7aa6680ac.jpg"},{"id":51776325,"identity":"c155dc95-4032-4222-8518-8283f6ec98d5","added_by":"auto","created_at":"2024-02-28 20:52:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546000,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal expression pattern of \u003cem\u003eOsPAD1\u003c/em\u003e and subcellular localization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eF\u003c/strong\u003e Transient expression of p35S::OsPAD1::GFP, SCAMP-mCherry (\u003cstrong\u003eA\u003c/strong\u003e), p35S::OsPAD1::GFP, D53-mCherry (\u003cstrong\u003eB\u003c/strong\u003e), p35S::OsPAD1\u003csup\u003e1-51\u003c/sup\u003e::GFP, SCAMP-mCherry (\u003cstrong\u003eC\u003c/strong\u003e), p35S::OsPAD1\u003csup\u003e1-51\u003c/sup\u003e::GFP, D53-mCherry (\u003cstrong\u003eD\u003c/strong\u003e), p35S::OsPAD1\u003csup\u003e76-268\u003c/sup\u003e::GFP, SCAMP-mCherry (\u003cstrong\u003eE\u003c/strong\u003e), p35S::OsPAD1\u003csup\u003e76-268\u003c/sup\u003e::GFP and D53-mCherry (\u003cstrong\u003eF\u003c/strong\u003e) in rice protoplasts and observed using laser confocal. Scale bars = 20 µm. \u003cstrong\u003eG\u003c/strong\u003e Relative expression of \u003cem\u003eOsPAD1\u003c/em\u003e in various rice tissues. Error bars indicate SDs from three biological replicates. \u003cstrong\u003eH\u003c/strong\u003e-\u003cstrong\u003eM\u003c/strong\u003e \u003cem\u003eOsPAD1\u003c/em\u003e transcripts detected by in situ hybridization of WT anthers at different developmental stages. A sense probe was used as the negative control (\u003cstrong\u003eM\u003c/strong\u003e). Scale bars = 30 µm.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/6b931ded9aad0710b9b759e2.jpg"},{"id":51776330,"identity":"d091fca0-141b-420f-8bf6-4e1afd7d28be","added_by":"auto","created_at":"2024-02-28 20:52:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":301990,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro recombinant OsPAD1 exhibits lipid-binding activity and differences of lipid components in mature pollen between WT and \u003cem\u003eOspad1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eIn vitro lipid binding assays with a panel of phospholipids and purified His-SUMO, His-SUMO-AAI. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol and its mono/bis/trisphosphates; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. \u003cstrong\u003eB\u003c/strong\u003e Differences of major lipid components in mature pollen between WT and \u003cem\u003eOspad1\u003c/em\u003emutant. Each value is the mean of three biological replicates ± SD. *, P \u0026lt;0.05; **, P \u0026lt;0.01; ***, P \u0026lt;0.001. Compounds: PS, phosphatidylserines; LPA, lyso-PA; PA, phosphatidic acids; CL, cardiolipins; FFA, free fatty acids; PI, phosphatidylinositols; PG, phosphatidylglycerols; DAG, diacylglycerols; PE, phosphatidylethanolamines; DGDG, digalactosyl diacylglycerols; TAG, triacylglycerols; PC, phosphatidylcholines; MGDG, monogalactosyl diacylglycerols.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/6aee7c6270b65a3fa8f69c01.jpg"},{"id":51776328,"identity":"144d1b78-08c1-4a2f-ad85-8cd543c11ef1","added_by":"auto","created_at":"2024-02-28 20:52:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":637817,"visible":true,"origin":"","legend":"\u003cp\u003eOsPAD1 interacts with OsINP1 to regulate rice pollen aperture formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eYeast two-hybrid assay demonstrating that OsPAD1 interacts with OsINP1. Triangle denotes 10-fold dilution. \u003cstrong\u003eB\u003c/strong\u003e BiFC analysis verifies interaction of OsPAD1 with OsINP1. Scale bars = 100 µm. \u003cstrong\u003eC\u003c/strong\u003e Pull-down assay shows that OsPAD1 interacts with OsINP1.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/40478418bc100b92d3a91439.jpg"},{"id":51776326,"identity":"b75e11a2-c195-4471-8804-2f42c5b45d1e","added_by":"auto","created_at":"2024-02-28 20:52:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":834366,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the WT, \u003cem\u003eOspad1\u003c/em\u003e mutant, \u003cem\u003eOsinp1\u003c/em\u003e mutant and double mutant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D \u003c/strong\u003ePhenotypic comparison of WT (\u003cstrong\u003eA\u003c/strong\u003e), \u003cem\u003eOspad1\u003c/em\u003emutant (\u003cstrong\u003eB\u003c/strong\u003e), \u003cem\u003eOsinp1\u003c/em\u003e mutant (\u003cstrong\u003eC\u003c/strong\u003e) and double mutant (\u003cstrong\u003eD\u003c/strong\u003e). \u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e SEM observation of WT (\u003cstrong\u003eE\u003c/strong\u003e), \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eF\u003c/strong\u003e), \u003cem\u003eOsinp1\u003c/em\u003e mutant (\u003cstrong\u003eG\u003c/strong\u003e) and double mutant (\u003cstrong\u003eH\u003c/strong\u003e) pollen. Scale bars = 10 µm. \u003cstrong\u003eI\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003eTEM observation of WT (\u003cstrong\u003eI\u003c/strong\u003e), \u003cem\u003eOspad1\u003c/em\u003e mutant (\u003cstrong\u003eJ\u003c/strong\u003e), \u003cem\u003eOsinp1\u003c/em\u003emutant (\u003cstrong\u003eK\u003c/strong\u003e) and double mutant (\u003cstrong\u003eL\u003c/strong\u003e) pollen. Scale bars = 5 µm.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/60694cb8f65e19f1c89427bb.jpg"},{"id":72201951,"identity":"a90210c1-5b52-4474-b2fc-8877e91aa6be","added_by":"auto","created_at":"2024-12-23 16:12:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5639730,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/53dd90a4-5a5b-43a6-9847-e4230f96f137.pdf"},{"id":51776329,"identity":"e0532c00-90cc-401b-b08e-9598e7a55405","added_by":"auto","created_at":"2024-02-28 20:52:24","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":18079,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/2dded5ed3370b3a8ab113f1a.docx"},{"id":51776331,"identity":"8ea7b888-9bd2-456a-b912-2f6199f5f05f","added_by":"auto","created_at":"2024-02-28 20:52:24","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":8194089,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-3873582/v1/dfdc1b983bf63b549dc7d100.docx"}],"financialInterests":"","formattedTitle":"OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn flowering plants, the successful accomplishment of double fertilization, involving the entry of sperm cells into the ovule, depends on the intricate process of guiding pollen tubes. The key players in this process are the apertures strategically positioned on the pollen wall. These apertures serve as entry points through which pollen tubes germinate. It is widely acknowledged that the formation of a robust pollen wall is a fundamental prerequisite for ensuring pollen viability. The pollen wall consists of essential components, namely, the exine, inner intine, and tryphine. The exine, in particular, is further subdivided into the outer sexine and the inner nexine. This inner nexine is itself divided into two distinct layers: the footlayer (Nexine I) and the endexine (Nexine II) (Murphy 2006; Blackmore et al. 2007; Jiang et al. 2013; Zhang and Yang 2014; Shi et al. 2015). Among the intricate elements of exine patterning, the pollen aperture stands out as a well-defined feature where exhibit patterns formed by the gaps in exine deposition (Furness and Rudall 2004). Much like the diverse patterns found on the surface of pollen grains, aperture patterns exhibit remarkable species-specific variations. These distinctions manifest in terms of shape, number, size, and margin characteristics among different species groups. For instance, monocots like rice typically possess a single polar aperture, while eudicots such as Arabidopsis typically exhibit three apertures (Wang and Dobritsa 2018).\u003c/p\u003e \u003cp\u003eThe pollen aperture in rice is a single-pore stucture located at the distal polar region, primarily composed of the annulus and the operculum. The annulus is a ring-like protrusion formed by a circle of thickened exine, while the operculum is a small, independent circular exine (Zhang et al. 2020). Similar to the formation of pollen exine, the initial indications of the aperture pattern in rice become discernible during the late tetrad stage. At this juncture, the plasma membrane polarizes to create an aperture plasma membrane protrusion that prevents the deposition of pre-exine or exine in these areas. By stage 9, the operculum and annulus commence their development, primarily comprising soropollenin derived from the tapetum. During stages 10 and 11, the fibrillar-granular layer and the Zwischenk\u0026ouml;rper layer, composed of well-developed callose/pectin substances, are formed to provide support to the operculum (El-Ghazaly and Jensen 1986; Zhang et al. 2020). Nonetheless, the mechanisms behind the formation of these pollen aperture structures and the genes responsible for regulating these processes remain intriguing and poorly understood questions.\u003c/p\u003e \u003cp\u003eThe first identified gene related to pollen aperture development in rice is \u003cem\u003eOsDAF1\u003c/em\u003e (\u003cem\u003eDEFECTIVE IN APERTURE FORMATION1\u003c/em\u003e). \u003cem\u003eOsDAF1\u003c/em\u003e encodes a legume-lectin kinase, playing an indispensable role in annulus formation. Notably, OsDAF1 has the capacity to interact with OsINP1 (INAPERTURATE POLLEN1), the rice counterpart of a pivotal aperture factor in Arabidopsis (Dobritsa and Coerper 2012; Zhang et al. 2020). Another key player in the aperture formation process in rice is DEAP1, which interacts with OsD6PKL3s, homologs of a well-known Arabidopsis aperture protein (Zhou et al. 2022). It is worth mentioning that research on aperture development in Arabidopsis has made significant strides. INP1, for instance, stands as the inaugural gene recognized for its involvement in pollen aperture formation, with a loss-of-function mutation resulting in the absence of pollen apertures in Arabidopsis (Dobritsa and Coerper 2012). INP1 plays a pivotal role in maintaining specific membrane domains within the callose wall during meiosis, thus preventing exine formation (Dobritsa et al. 2018). Although it has been demonstrated that pollen ploidy plays a crucial role in determining the number of pollen apertures, there is no doubt that INP1 serves as a marker for the positioning and quantification of future aperture development (Reeder et al. 2016). It's worth noting that the amino acid sequences of INP1 homologs in different species exhibit significant differences and are not interchangeable (Li et al. 2018). Recently, an INP2 protein was identified in Arabidopsis, and it was found to interact with INP1. These two proteins share strikingly similar structural features, expression patterns, and mutant phenotypes. When co-expressed in a heterologous system, the two proteins were able to restore the normal phenotype, whereas the expression of either one alone could not (Lee et al. 2021). In the realm of Arabidopsis, the protein kinase D6PKL3 also plays a direct role in pollen aperture formation. Similar to INP1 and INP2, D6PKL3 resides on the plasma membrane and binds to specific phosphoinositides to demarcate the site of pollen aperture formation (Lee et al. 2018). Nonetheless, the intricate molecular mechanisms governing pollen aperture formation remain a subject of ongoing investigation and continue to elude complete elucidation.\u003c/p\u003e \u003cp\u003eLipid transfer proteins (LTPs) with an N-terminal signal peptide play important roles in mediating lipid transfer across the cytoplasm (Kader 1996; Yeats and Rose 2008; Edqvist et al. 2018). These proteins, characterized by cysteine residues linked by disulfide bonds, fold into either a single large or two smaller cavity structures, which effectively bind lipids (Liu et al. 2015). LTPs serve crucial functions at various stages of plant growth, including responding to both biotic and abiotic stress (Wang et al. 2004; Sels et al. 2008), regulating processes like seed germination and seedling emergence (Eklund and Edqvist 2003), influencing reproduction (Wan et al. 2020; Tao et al. 2021), contributing to cell wall growth (Nieuwland et al. 2005), and participating in wax metabolism (Debono et al. 2009). For example, in rice, \u003cem\u003eOsC6\u003c/em\u003e, responsible for encoding a LTP, exhibits expression within the tapetum and is subject to modulation by TAPETUM DEGENERATION RETARDATION (TDR) (Zhang et al. 2010). In Arabidopsis, type III LTPs not only engage in lipid transport but also form an integral part of the exine structure (Huang et al. 2013). Non-specific lipid transfer proteins (nsLTPs) are another class of LTPs, characterized by their small size (6.5\u0026ndash;10.5 kDa), and they are widely distributed throughout the plant kingdom. For example, dysfunction of OsLTP47 results in disrupted lipid metabolism and the formation of defective pollen walls, ultimately leading to male sterility (Chen et al. 2022). Similarly, mutations in glycosylphosphatidylinositol-anchored nsLTPs in Arabidopsis induce developmental defects in pollen walls, resulting in male sterility (Edstam and Edqvist 2014). These findings illuminate the significant roles that LTPs play in pollen development. However, despite these valuable insights, the intricate molecular mechanisms governing the participation of LTPs in pollen aperture formation and patterning remain poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we present our findings on the isolation and characterization of the rice gene, \u003cem\u003eOsPAD1\u003c/em\u003e. This gene encodes a nsLTP and serves a distinct function in the development of the fibrillar-granular layer within pollen grains. Notably, in the \u003cem\u003eOspad1\u003c/em\u003e mutant, pollen grains exhibit a conspicuous absence of fibrillar-granular layer, resulting in the disruption of attachment between the operculum and the pollen. Ultimately, this leads to the rupture of the internal plasma membrane at the pollen aperture, causing the leakage of intracellular contents and leaving behind only the empty shell of the pollen exine. Furthermore, our investigation has revealed the interaction between OsPAD1 and OsINP1. This interaction highlights that OsPAD1 becomes distributed on the pollen surface when recruited as part of an OsINP-OsPAD1 complex, which is instrumental in the formation of the fibrillar-granular layer. These results present innovative insights into the precise deposition of each layer during the development of pollen walls, offering new avenues for further research in this field.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and conditions\u003c/h2\u003e \u003cp\u003eThe rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) materials were grown in the experimental fields of Nanjing Agricultural University (118\u0026deg;E, 31\u0026deg;N), Hainan province (110\u0026deg;E, 18\u0026deg;N) and Chinese Academy of Agricultural Sciences (116\u0026deg;E, 40\u0026deg;N). The \u003cem\u003eOspad1\u003c/em\u003e mutant was a natural mutant from \u003cem\u003ejaponica\u003c/em\u003e variety Ningjing 7. The F\u003csub\u003e2\u003c/sub\u003e mapping population was generated from a cross between the \u003cem\u003eOspad1\u003c/em\u003e mutant and N22 (ssp. \u003cem\u003eindica\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eI\u003csub\u003e2\u003c/sub\u003e-KI staining and aniline blue staining\u003c/h2\u003e \u003cp\u003eFreshly matured anthers were meticulously fixed in Carnoy\u0026rsquo;s solution (ethanol:glacial acetic\u0026thinsp;=\u0026thinsp;3:1) and subsequently stored at a temperature of 4\u0026deg;C until observation. For detailed microscopic examination, pollen grains were gently released from the anthers using fine-tipped tweezers. Subsequently, these pollen grains were stained with a 1% iodine-potassium iodide (I\u003csub\u003e2\u003c/sub\u003e-KI) solution and meticulously captured using a Nikon AZ100 stereomicroscope. In the case of aniline blue staining, the spikelets from flowering rice were immersed in Carnoy's solution for an extended duration of more than 24 h and similarly stored at 4\u0026deg;C prior to utilization. The stigmas, once released, underwent a sequential treatment process, involving exposure to 70%, 50%, and 30% alcohol solutions. After a rehydration step in distilled water, they were immersed in 10 N NaOH at a temperature of 60\u0026deg;C for 10 min, followed by a thorough rinsing with distilled water. Subsequently, the stigmas were stained with aniline blue (0.1% in 0.1M K\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and photographed with ZEISS Imager A2 fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCytological observations\u003c/h2\u003e \u003cp\u003eFresh rice spikelets and anthers at various developmental stages underwent immersion in FAA solution, which is composed of an 18:1:1 (v/v) mixture of formalin, 70% ethanol, and acetic acid, and also in 2.5% glutaraldehyde for a duration exceeding 24 h. Semi-thin cross-sections of the anthers were meticulously prepared, following established methods as previously reported (Yu et al. 2018). For SEM, anthers from both the wild-type (WT) and mutant samples were extracted from the glutaraldehyde solution and underwent a thorough washing process with distilled water. Subsequently, they were subjected to dehydration using a series of ethanol solutions and were subsequently fixed in a 1% OsO\u003csub\u003e4\u003c/sub\u003e solution for a period of 2 h. After this fixation, further dehydration was carried out, followed by critical point drying utilizing CO\u003csub\u003e2\u003c/sub\u003e. The anthers were then coated with a thin layer of ion-sputtered gold using an E-100 ion sputtering device and were observed using a scanning electron microscope (S3400; Hitachi). For TEM, the anthers were immersed in a solution containing 1% glutaraldehyde and 1% OsO\u003csub\u003e4\u003c/sub\u003e for 1 h, followed by dehydration using an ethanol series. Subsequently, they were embedded in Spurr's medium before undergoing thin sectioning. These sections were double-stained with a 2% uranyl acetate solution and a 2.6% aqueous lead citrate solution, after which they were observed at an acceleration voltage of 80 kV with a H7700 transmission electron microscope (Hitachi).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL assays\u003c/h2\u003e \u003cp\u003eA Dead End Fluorometric TUNEL Kit (Promega) was employed to conduct TUNEL assays. Initially, fresh anthers at varying developmental stages were immersed in FAA for a duration exceeding 24 h. Subsequently, they underwent dehydration using an alcohol:xylene mixture and were then embedded in paraffin (Paraplast Plus, Sigma). The paraffin-embedded samples were meticulously sectioned, followed by the requisite dewaxing and rehydration steps to prepare the samples for the TUNEL assay. Throughout the experimental procedures, we adhered to the guidelines provided by the manufacturer of the kit. The excitation/emission spectra of the green fluorescence (representing the TUNEL signal) and the red fluorescence of propidium iodide were observed using a laser confocal microscope, with excitation/emission wavelengths of 488 nm/510 nm and 530 nm/640 nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time reverse transcription\u0026ndash;PCR (qRT\u0026ndash;PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from the plant was extracted utilizing the RNeasy Plant Mini Kit (Qiagen). Subsequently, first-strand cDNA was synthesized, starting with 1 \u0026micro;g of RNA, employing the QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative RT-PCR assays were conducted using gene-specific primers in conjunction with SYBR Premix ExTaq reagent (Takara). These experiments were carried out using the ABI 7500 Real-Time PCR System (Applied Biosystems) in strict accordance with the manufacturer's guidelines. Each sample was subjected to PCR in triplicate as part of three independent biological replicates, with the rice ubiquitin gene serving as an internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA in situ hybridization\u003c/h2\u003e \u003cp\u003eFresh rice anthers at various developmental stages were immersed in RNase-free FAA for an extended period of over 24 h, and then embedded in paraffin after dehydration. According to a previous publication (Li et al. 2006), \u003cem\u003eOsPAD1\u003c/em\u003e cDNA as template was used to prepare sense and antisense probes with a DIG Northern Starker Kit (Cat. no. 2039672, Roche). RNA hybridization and immunological detection followed the Kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVector Construction\u003c/h2\u003e \u003cp\u003eFunctional complementation and gene knockout experiments were devised to validate the role of \u003cem\u003eOsPAD1\u003c/em\u003e. The complete sequence from the WT, encompassing a 2,864 bp promoter region, a 2,089-bp gene region, and a 138-bp downstream region, was seamlessly integrated into the binary vector pCUbi1390 through in-fusion cloning using the In-Fusion HD Cloning Kit. Callus derived from the seeds of \u003cem\u003eOspad1\u003c/em\u003e/WT heterozygotes served as the starting material for Agrobacterium-mediated transformation, following the method established by Hiei et al. in 1994.\u003c/p\u003e \u003cp\u003eTwenty-base-pair gene-specific spacer sequences derived from \u003cem\u003eOsPAD1\u003c/em\u003e were inserted into the entry vector pOs-sgRNA, and subsequently, they were subcloned into the destination vector containing Cas9 using the Gateway LR Clonase II Enzyme mix (Invitrogen) method, as described previously (Miao et al. 2013). Callus that had been induced from WT seeds underwent Agrobacterium-mediated transformation. The specific primers used for PCR in this process are listed in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eTo generate fully functional transgenic plants expressing YFP-tagged OsPAD1, we conducted a genomic complementation assay. In this approach, the YFP coding sequence, lacking a stop codon, was positioned upstream of the OsPAD1 cDNA. Subsequently, the YFP-OsPAD1 construct was integrated into the pCAMBIA1390 vector using in-fusion cloning. The fragment was placed under the control of the native UBI promoter present on the vector. This resulted in the creation of the pUBI:YFP-OsPAD1cDNA construct (Tan et al. 2014). Following the construction of pUBI:YFP-OsPAD1cDNA, this construct was introduced into the \u003cem\u003eOspad1\u003c/em\u003e homozygous plants. Remarkably, this led to the restoration of pollen fertility, serving as compelling evidence that the YFP-tagged protein is indeed fully functional.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eSubcellular localization of OsPAD1\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eOsPAD1\u003c/em\u003e coding sequence (CDS) lacking stop codons was fused with green fluorescent protein (GFP) and subsequently inserted into the pAN580-GFP vector (primer details provided in Supplemental Table\u0026nbsp;1). The extracted plasmids were transfected into rice protoplasts, followed by an overnight incubation period in a dark environment at 28\u0026deg;C. Fluorescent images were acquired using a confocal laser scanning microscope (Zeiss LSM780).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLipid binding activity\u003c/h2\u003e \u003cp\u003eLipid binding assays were conducted using PIP-strip membranes (Echelon Biosciences; P-6001) in accordance with the manufacturer's instructions. Initially, the PIP-strip membranes were immersed in blocking buffer for a duration of 1\u0026ndash;2 h. Subsequently, the purified protein was introduced at a concentration of 5 \u0026micro;g/mL into the blocking buffer supplemented with 0.1% (v/v) Tween 20\u0026reg;, and this mixture was incubated with the membranes for an additional 1\u0026ndash;2 h. Following this incubation period, the membranes were meticulously washed three times with PBST and then subjected to further incubation with the appropriate antibodies. This secondary incubation was conducted in blocking buffer containing 0.1% (v/v) Tween 20 for another 1\u0026ndash;2 h. Lastly, the membranes were rinsed three times with PBST to conclude the assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePollen lipids determination\u003c/h2\u003e \u003cp\u003eApproximately 100 mg of anthers at the stage 10 from both the WT and \u003cem\u003eOspad1\u003c/em\u003e mutant were swiftly frozen by submerging them in liquid nitrogen. These frozen anthers were then crushed with forceps and introduced into a preheated inactivation solvent consisting of 0.01% (w/v) butylated hydroxytoluene (BHT) dissolved in isopropanol at a temperature of 75\u0026deg;C. The mixture was thoroughly blended and subsequently placed in a water bath maintained at 75\u0026deg;C for an 18 min duration. After this, the pollen contained within the anthers was separated by filtering the mixture through a 100-mesh cell sieve to eliminate any impurities, thereby allowing for subsequent lipidomic analysis. Lipid determination was conducted following microscopic examination to ensure the correct pollen stage and the absence of excessive impurities.\u003c/p\u003e \u003cp\u003eAfter inactivation, an extraction solvent consisting of chloroform : methanol: 300 mM ammonium acetate (30:41.5:3.5) (v/v/v) was introduced into the samples. Subsequently, these samples were incubated at room temperature for 24 h while being agitated at 150 rpm. Upon completion of the incubation, the samples were subjected to centrifugation, and the resulting clear supernatant was carefully transferred to fresh tubes. The inactivation and extraction steps were repeated once, and lipid extracts from both rounds of extraction were combined and subsequently desiccated in a SpeedVac system (Genevac, UK). The lipid extracts were then stored at -80℃ until they were ready for LCMS analyses. Phospholipids, sterol and neutral lipids were analyzed using an Agilent 1260 HPLC system (Agilent Technologies) coupled to a 5500 QTRAP instrument running analyst v.1.6.3 (Sciex). Sphingolipids were analyzed using a Shimadzu Nexera 20AD-HPLC/ExionLC-AD system connected to a Sciex QTRAP 6500 PLUS instrument, as previously described.\u003c/p\u003e \u003cp\u003eFor the analysis of polar lipids in normal phase, individual species were separated using a TUP-HB silica column (i.d. 150x2.1 mm, 3 \u0026micro;m) under the following precise conditions: mobile phase A consisted of a mixture of chloroform, methanol, and ammonium hydroxide (89.5: 10: 0.5), while mobile phase B was composed of chloroform, methanol, ammonium hydroxide, and water (55: 39: 0.5: 5.5). In the case of reverse-phase LC/MS analysis, lipids were assessed utilizing a modified version of reverse-phase (RP)-HPLC/ESI/MS/MS, as previously documented. In summary, the separation of the aforementioned lipids was executed on a Phenomenex Kinetex column with a 2.6 \u0026micro;m C18 packing material (internal diameter 4.6\u0026times;100 mm). An isocratic mobile phase consisting of chloroform, methanol, and 0.1M ammonium acetate (100:100:4) was employed at a flow rate of 300 \u0026micro;l/min for a duration of 10 min.\u003c/p\u003e \u003cp\u003eFree cholesterols and cholesteryl esters were analyzed in atmospheric pressure chemical ionization (APCI) mode using an Agilent 1260 HPLC system from Agilent Technologies, connected to a 5500 QTRAP instrument running analyst v.1.6.3 from Sciex. The analysis incorporated the use of d6-cholesterol and d6-C18:0 cholesteryl ester (CE) as internal standards, with the latter being sourced from CDN isotopes (Shui et al. 2011).\u003c/p\u003e \u003cp\u003eThree biological replicates were performed. Quantification of individual lipid species was accomplished by referencing spiked internal standards, including d9-PC32:0(16:0/16:0), d7-PE33:1(15:0/18:1), d31-PS(d31-16:0/18:1), d7-PA33:1(15:0/18:1), d7-PG33:1(15:0/18:1), C17-SL, d5-CL72:8(18:2)4, C17-LPA, d7-LPC18:1, d7-LPE18:1, DMPS, DMPA, DMPG, MGDG 34:0, DGDG 36:0, Cer d18:1/15:0-d7, GluCer d18:1/12:0, d17:1 Sph, d17:1 S1P, D-ribo-phytoSph C17, d5-DAG16:0/16:0, and d5-DAG18:1/18:1 obtained from Avanti Polar Lipids (Alabaster, AL) and LIPID MAPS. Dioctanoyl phosphatidylinositol (PI) (16:0-PI) was procured from Echelon Biosciences, Inc. (Salt Lake City, UT) and utilized in conjunction with d7-PI33:1(15:0/18:1) (Avanti Polar Lipids) for PI quantitation. Triacylglycerols (TAGs) were quantified using TAG(14:0)3-d5, TAG(16:0)3-d5, and TAG(18:0)3-d5 obtained from CDN isotopes. Free fatty acids were quantified using d31-16:0 (Sigma-Aldrich).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid (Y2H) assays\u003c/h2\u003e \u003cp\u003eProtein interactions were evaluated through Y2H analysis employing the DUALhunter system, a product of Dualsystems Biotech. To facilitate this analysis, the coding fragment of the target gene was merged with either the Cub fragment contained within the pXGY17 vector or the Nub fragment present in the pXGY18 vector. This led to the generation of pXGY17-OsPAD1 and pXGY18-INP1 constructs, respectively. The specific primers used for Y2H assays can be found in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePull-down assays\u003c/h2\u003e \u003cp\u003eTransgenic plants were used to extract the OsPAD1 protein carrying the FLAG tag. The coding sequence of OsINP1 was cloned into the pGEX-4T-2 vector to obtain GST-OsINP1 fusion protein. The experimental procedure was performed following a published method (Miernyk and Thelen 2008). Immunoassays were performed using anti-FLAG (Medical Biological Laboratories, 1:2000) and anti-GST (Medical Biological Laboratories, 1:2000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation (BiFC) analysis\u003c/h2\u003e \u003cp\u003eThe coding sequences of \u003cem\u003eOsPAD1\u003c/em\u003e and \u003cem\u003eOsINP1\u003c/em\u003e were cloned into the pYN1 or pYC1 vectors (a gift of Joh A. Lindbo, OARDC, Ohio State University, Wooster) to construct Y\u003csup\u003eN\u003c/sup\u003e-OsPAD1 and Y\u003csup\u003eC\u003c/sup\u003e-INP1 (primers used in BiFC assays are listed in Supplemental Table\u0026nbsp;1). The recombinant plasmid was introduced into Agrobacterium strain EHA105 and used to infiltrate \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves, as described previously (Waadt and Kudla 2008). Fluorescent images were captured using a Zeiss LSM700 laser scanning confocal microscope.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of the\u003c/b\u003e \u003cb\u003eOspad1\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder natural growing conditions, the phenotypes of the \u003cem\u003eOspad1\u003c/em\u003e mutant and WT are identical during the vegetative period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The first visual difference is the appearance of white colored anthers in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). \u003cem\u003eOspad1\u003c/em\u003e mutant anthers and pollen failed to stain by iodine potassium iodide (I\u003csub\u003e2\u003c/sub\u003e-KI), indicating the inability to accumulate starch (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Magenta acetate staining indicated that the mutant pollen developed normally until the microspore development stage, after which there was no further development (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Applicatiom of WT pollen to \u003cem\u003eOspad1\u003c/em\u003e mutant stigmas resulted in normal seed production, indicating that the mutation did not affect the female reproductive organs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the cellular defects of the \u003cem\u003eOspad1\u003c/em\u003e mutant anthers, we compared transverse sections of mutant and WT anthers at various stages of pollen development. No difference was observed prior to microspore development stage 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I, M, N), after which the tapetum layer of WT anthers began to shrink and staining intensified, the middle layer began to narrow and gradually disappear, and the microspores gradually became spherical (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). However, the tapetum layer of \u003cem\u003eOspad1\u003c/em\u003e mutant was not concentrated, and the middle layer remained still clearly visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO). After stage 10, \u003cem\u003eOspad1\u003c/em\u003e mutant microspores began to shrink and ceased development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP). At stages 11 and 12, degradation of the WT tapetum layer was completed, and the middle layer disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, L). In contrast, the tapetum layer and middle layer in the mutant remained visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). Degradation of the tapetum is considered to result from programmed cell death (PCD), which can be detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. At stage 7, no TUNEL signal was observed in the WT or mutant samples, indicating that the tapetum had not initiated the degradation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR, V). At the stage 8, TUNEL signals were evident in the tapetum layer of the WT, but not in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS, W). In stage 9, intense signals of PCD were observed in the WT tapetum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eT), while the mutant displayed only a weak signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eX). In stage 10, the WT exhibited no signal, indicating that the completion of PCD and full degradation of the tapetum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eU). Conversely, the \u003cem\u003eOspad1\u003c/em\u003e muatant tapetum exhibited a persistent intense signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eY). These results suggested that the PCD in the \u003cem\u003eOspad1\u003c/em\u003e mutant was delayed compared to the WT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eOspad1\u003c/b\u003e \u003cb\u003emutant pollen is deficient in pollen aperture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDelayed degradation of the tapetum often results in the developmental abortion of the pollen wall (Shi et al., 2015). To observe whether there are defects in the mutant pollen wall, scanning electron microscopy (SEM) was employed to observe the development of pollen walls in WT and \u003cem\u003eOspad1\u003c/em\u003e mutant. Further insights from SEM revealed a significant abnormal in mature mutant pollen: the absence of the operculum, coupled with the lack of a distinct annulus. These observed structural deficiencies may directly contribute to pollen abortion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). To thoroughly explore the phenotype of the mutant at the aperture, we employed transmission electron microscopy (TEM) to examine the pollen aperture at different developmental stages in both the WT and \u003cem\u003eOspad1\u003c/em\u003e mutant. At stage 10, the WT initiated the formation of the pollen aperture, resulting in the inner wall of the annulus forming a deeply stained multilayered structure. Additionally, a lightly stained layer, known as the fibrillar-granular layer, developed between the pollen apertures and the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Conversely, at stage 10, the mutant displayed the operculum detaching from the pollen, with no deeply stained multilayer structure present on the inner wall of the annulus, and a notable absence of the fibrillar-granular layer between the operculum and the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Subsequently, the plasma membrane of the \u003cem\u003eOspad1\u003c/em\u003e mutant pollen ruptured through the aperture, resulting in the leakage of the internal cellular structure into the locule (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, L). In contrast, the fibrillar-granular layer in WT pollen continued to thicken (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). These results suggested that the absence of the operculum in the \u003cem\u003eOspad1\u003c/em\u003e mutant did not stem from its failure to form, but rather resulted from the absence of the fibrillar-granular layer. In the absence of this essential layer, the operculum lacked proper attachment to the aperture, consequently leading to its shedding during the later stages of development. Ultimately, this detachment resulted in pollen abortion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eOspad1\u003c/b\u003e \u003cb\u003emutation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the molecular lesion of the \u003cem\u003eOspad1\u003c/em\u003e mutant, we used a map-based cloning approach to isolate the \u003cem\u003eOsPAD1\u003c/em\u003e allele. In an F\u003csub\u003e2\u003c/sub\u003e population from cross between \u003cem\u003eOspad1\u003c/em\u003e and N22, fertile and sterile plants segregated in a 3:1 ratio (340 fertile vs 137 sterile, χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3:1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.33\u0026thinsp;\u0026lt;\u0026thinsp;χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e0.05,1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.84; P\u003csub\u003e1df\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicatting that the \u003cem\u003eOspad1\u003c/em\u003e defects were caused by a recessive mutation in a single nuclear gene. Initially, we mapped the \u003cem\u003eOsPAD1\u003c/em\u003e locus to a region flanked by markers \u003cem\u003eM-17\u003c/em\u003e and \u003cem\u003eRM-5\u003c/em\u003e on chromosome 1. Subsequently, we refined this region to a 96-kb physical interval positioned between markers \u003cem\u003eM-29\u003c/em\u003e and \u003cem\u003eM-30\u003c/em\u003e. This interval encompasses 17 putative open reading frames (ORFs), as annotated by the Rice Genome Annotation Project Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Upon conducting sequencing and comparative analyses, a 30-base pair (bp) deletion is identified in the first exon of \u003cem\u003eORF16\u003c/em\u003e (\u003cem\u003eLOC_Os01g42210\u003c/em\u003e) in the \u003cem\u003eOspad1\u003c/em\u003e mutant. This deletion affects 10 amino acids within a region of \u003cem\u003eLOC_Os01g42210\u003c/em\u003e that is conserved across multiple species (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). To identify its homologs in other species, we performed BLASTP searches using amino acid sequences and subsequently constructed a phylogenetic tree (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). The results revealed the existence of OsPAD1 homologs in numerous plant species, many of which remain unstudied. Interestingly, \u003cem\u003eLOC_Os01g42210\u003c/em\u003e is the \u003cem\u003eOsLTP47\u003c/em\u003e gene (Chen et al. 2022). Notably, while a mutation in this gene led to male sterility, no report was provided regarding the phenotype of pollen aperture defection. Consequently, \u003cem\u003eLOC_Os01g42210\u003c/em\u003e was the most promising candidate gene. To identify the causal mutation, we firstly conducted a functional complementation experiment. Transgenic plants were generated by introducing a WT genomic fragment into \u003cem\u003eOspad1\u003c/em\u003e mutant callus. This approach aimed to confirm that the observed pollen aperture defect in the \u003cem\u003eOspad1\u003c/em\u003e mutant was indeed attributed to the disruption of \u003cem\u003eLOC_Os01g42210\u003c/em\u003e. Positive transgenic plants exhibited normal pollen aperture development, characterized by yellow anthers and restored pollen fertility (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, F, D, H; Supplementary Fig. S3A, C, E). Furthermore, we employed CRISPR/Cas9-targeted mutagenesis to generate a homozygous mutant, known as \u003cem\u003eCri-Ospad1\u003c/em\u003e. These \u003cem\u003eCri-Ospad1\u003c/em\u003e mutant plants exhibited an 848-bp deletion, situated 78-bp upstream of the ATG start codon, and displayed white anthers with non-fertile pollen, mirroring the phenotype of the \u003cem\u003eOspad1\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E; Supplementary Fig. S3B-E). TEM analysis revealed that pollen grains from the knockout plants lacked an operculum, similar to those observed in the \u003cem\u003eOspad1\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, I). Seed setting of the transgenic plants were sterile as expected (Supplementary Fig. S3F). In summary, our findings conclusively establish that \u003cem\u003eLOC_Os01g42210\u003c/em\u003e corresponds to \u003cem\u003eOsPAD1\u003c/em\u003e, confirming its pivotal role in pollen aperture development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular localization of\u003c/b\u003e \u003cb\u003eOsPAD1\u003c/b\u003e \u003cb\u003eand expression pattern\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the subcellular localization of the OsPAD1 protein, a transient expression experiment was conducted in rice protoplasts. We utilized a green fluorescent protein (GFP)-tagged OsPAD1 fusion protein, driven by the cauliflower mosaic virus (CaMV) 35S promoter. Our observations revealed that GFP signals were detected within two distinct subcellular compartments: the plasma membrane (PM) and the nucleus. Importantly, these signals colocalized with the PM marker SCAMP-mCherry and the nuclear marker D53-mCherry, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) (Cai et al. 2011; Zhou et al. 2013). A closer examination of the OsPAD1 amino acid sequence revealed the presence of a transmembrane domain spanning amino acids 52nd to 74th (Supplementary Fig. S4A). This domain effectively divided the protein into two segments: an N-terminal region (residues 1\u0026ndash;51) and a C-terminal region (residues 75\u0026ndash;268). Intriguingly, when the N-terminal region (OsPAD1\u003csup\u003e1\u0026ndash;51\u003c/sup\u003e-GFP) and the C-terminal region (OsPAD1\u003csup\u003e75\u0026ndash;268\u003c/sup\u003e-GFP) were individually controlled by the CaMV-35S promoter, their subcellular locations within the protoplast were not entirely identical. OsPAD1\u003csup\u003e1\u0026ndash;51\u003c/sup\u003e-GFP exhibited a presence in both the PM and the nucleus, mirroring the behavior of the full-length OsPAD1-GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). In contrast, OsPAD1\u003csup\u003e75\u0026ndash;268\u003c/sup\u003e-GFP was exclusively localized to the PM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). These findings collectively indicate that the N-terminal region is necessary for nuclear localization. In its absence, the protein still tends to localize to the PM, suggesting that the N-terminal region is not a prerequisite for PM localization. In addition, we also conducted transgenic experiments to investigate the protein localization of OsPAD1 on pollen grains at various developmental stages in OsPAD1-YFP transgenic plants. To begin with, we generated OsPAD1-YFP transgenic plants in the \u003cem\u003eOspad1\u003c/em\u003e mutant background. It is worth noting that pollen fertility was successfully restored in all positive transgenic plants, thus confirming the functionality of the transgene (Supplementary Fig. S4B-I). Subsequently, we meticulously examined the localization pattern of the OsPAD1 protein within pollen grains at different developmental stages. Our results revealed that starting from stage 9, the fluorescence signal of the OsPAD1 protein began to emerge at the pollen aperture site. As the developmental stages progressed to 10 and 11, the fluorescence signals exhibited more pronounced characteristics in proximity to the pollen aperture site. Consequently, during stages 12 and 13, the fluorescence signals gradually diminished in the vicinity of the pollen aperture site (Supplementary Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the expression patterns of \u003cem\u003eOsPAD1\u003c/em\u003e in both anthers and vegetative tissues of WT plants, we employed a combination of reverse-transcription quantitative PCR (RT-qPCR) and RNA in situ hybridization. Our results revealed that \u003cem\u003eOsPAD1\u003c/em\u003e expression levels were notably higher in anthers compared to roots, leaves, leaf sheaths, and stems. Furthermore, upon closer examination of anthers at different developmental stages, we observed a positive correlation between the maturation of anthers and the expression of \u003cem\u003eOsPAD1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). We also conducted spatial and temporal analysis of \u003cem\u003eOsPAD1\u003c/em\u003e expression within the anther using RNA in situ hybridization on sections of WT anthers. Initially, OsPAD1 exhibited very low levels of expression in the tapetum and microspores during stages 7 and 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I). By stage 9, \u003cem\u003eOsPAD1\u003c/em\u003e expression was significantly upregulated in the tapetum, with a comparatively lower expression in the microspores (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Notably, during stage 10, when the tapetum had undergone substantial degradation, \u003cem\u003eOsPAD1\u003c/em\u003e expression was absent in the microspores (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). However, at stage 11, \u003cem\u003eOsPAD1\u003c/em\u003e was once again expressed in the microspores (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). These dynamic expression patterns strongly support the role of \u003cem\u003eOsPAD1\u003c/em\u003e in tapetum development and the formation of pollen apertures.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDysfunction of\u003c/b\u003e \u003cb\u003eOsPAD1\u003c/b\u003e \u003cb\u003eleads to alterations in the lipid content of pollen grains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLTPs are typically characterized as small, 9-kDa proteins found in abundance within higher plants (Kader, 1996). However, it is noteworthy that the molecular weight of OsPAD1 is significantly larger, at 27 kDa, compared to the conventional LTP proteins. Despite this difference, OsPAD1 exhibits key structural features consistent with LTPs, including the presence of four conserved disulfide bridges and an eight-Cys motif, which serves as the hallmark signature of LTP proteins (Supplementary Fig. S6A). This structural arrangement forms the molecular basis through which LTPs transport lipids. Given that \u003cem\u003eOsC6\u003c/em\u003e, another gene in rice, encodes an LTP and has been shown to bind lipid molecules in vitro (Zhang et al. 2010), we hypothesized that OsPAD1 may similarly exhibit lipid-binding activity. Since we did not have access to purified full-length OsPAD1, to investigate this possibility, we isolated and purified the AAI domain within OsPAD1, a region predicted to interact with lipids, and assessed its ability to bind various phospholipids (Supplementary Fig. S6B). The results demonstrated that His-SUMO-AAI exhibited binding affinity to a variety of phospholipids, with a predominant binding to phosphatidylinositol (PI) and its mono/bisphosphates (PI(3)P, PI(4)P, PI(5)P, PI(3,5)P\u003csub\u003e2\u003c/sub\u003e, and PI(4,5)P\u003csub\u003e2\u003c/sub\u003e). In addition, there was notable binding to phosphatidic acid (PA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate whether the loss of lipid transport function in the \u003cem\u003eOspad1\u003c/em\u003e mutant contributes to the male sterility phenotype, we conducted measurements of lipid content in pollen grains at maturity for both mutant and WT plants (Supplementary Fig. S7). At maturity, significant reductions in the levels of various lipids were observed in the \u003cem\u003eOspad1\u003c/em\u003e mutant pollen grains compared to the WT, including PI, PA, lysophosphatidic acids, cardiolipins, phosphatidylglycerols, diacylglycerols, phosphatidylethanolamines, digalactosyl diacylglycerols, phosphatidylcholines, and monogalactosyl diacylglycerols (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Fig. S8, 9). Remarkably, the decreases in the levels of PI and PA, these lipids that OsPAD1 can bind to, have caught our attention. Therefore, we speculate that in the \u003cem\u003eOspad1\u003c/em\u003e mutant hinders the normal transport of these lipids, ultimately resulting pollen aperture defection.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOsPAD1 interacts with OsINP1 to affect the pollen aperture formation\u003c/h2\u003e \u003cp\u003eAt present, two genes, \u003cem\u003eOsINP1\u003c/em\u003e and \u003cem\u003eOsDAF1\u003c/em\u003e, associated with the aperture, were both found to be localized at the aperture (Zhang et al. 2020). An experiment using a yeast two-hybrid system confirmed the interaction between OsPAD1 and OsINP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Further bimolecular fluorescence complementation (BiFC) and pull-down assays showed direct the interaction between OsPAD1 and OsINP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). To delve deeper into the significance of this interaction, we generated the \u003cem\u003eOsinp1\u003c/em\u003e mutant using CRISPR/Cas9 technology on the WT background. In additon, a double mutant (\u003cem\u003eOspad1/Osinp1\u003c/em\u003e) was obtained by knocking out the \u003cem\u003eOsINP1\u003c/em\u003e allele with CRISPR/Cas9 technology in the \u003cem\u003eOspad1\u003c/em\u003e mutant. Under natural growing conditions, the phenotypes of \u003cem\u003eOspad1/Osinp1\u003c/em\u003e double mutant plants showed no apparent differences from the WT, \u003cem\u003eOspad1\u003c/em\u003e, and \u003cem\u003eOsinp1\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). Of interest, SEM revealed that, in comparison to the WT, the \u003cem\u003eOsinp1\u003c/em\u003e mutant exhibited no pollen aperture phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G), consistent with findings from previous studies (Zhang et al. 2020). However, the double mutant displayed heightened pollen sterility, characterized by complete shrinkage of pollen grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Further TEM analysis indicated that the \u003cem\u003eOsinp1\u003c/em\u003e mutant pollen grains solely lacked the aperture phenotype but showed an accumulation of starch contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI-K). In contrast, the double mutant pollen grains not only lacked the aperture phenotype but also exhibited no accumulation of starch contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL). These results suggest that OsPAD1 may play a role in the final stages of pollen development, contributing to the completion of pollen starch and nutrient filling, as well as participating in pollen aperture formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLipid transport plays a pivotal role in the development of plant reproductive tissues, particularly in the anther cuticle and pollen wall (Shi et al. 2015; Wan et al. 2020). Disruptions in lipid transport during anther and pollen development frequently result in male sterility. For instance, under the positive regulation of TDR, OsC6 facilitates lipid transport from tapetum cells to microspores, thereby promoting pollen wall formation (Zhang et al. 2010). Type III nsLTPs not only transport lipids from tapetum cells to microspores but also actively participate in pollen wall formation as essential components (Huang et al. 2013). EAT1, through its binding to the promoter of \u003cem\u003eOsLTPL94\u003c/em\u003e, activates this lipid transporter, enabling its secretion from microspores and tapetum to the pollen wall, further enhancing pollen wall formation (Tao et al. 2021). Notably, \u003cem\u003eOsLTP47\u003c/em\u003e encodes a grass-specific, membrane-localized nsLTP, and its dysfunction results in disrupted lipid metabolism in the anther and defective pollen walls (Chen et al. 2022). While nsLTPs have been studied in pollen grain development, research on pollen aperture development remains limited. In this study, we observed that the \u003cem\u003eOspad1\u003c/em\u003e mutant lacks the fibrillar-granular layer, while the rest of the pollen wall remains intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The absence of the fibrillar-granular layer in the pollen wall prevents the operculum from attaching, resulting in the rupture of the internal plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-L). Our results introduce novel insights into the mechanisms of lipid transporter proteins participating in the formation of the rice pollen aperture.\u003c/p\u003e \u003cp\u003eAn intriguing finding from our research is the phylogenetic relationship analysis, which reveals that OsPAD1 and some of its homologous proteins are classified within the monocotyledonous branch, while homologous proteins found in other species, such as willow, rose, pomegranate, and Arabidopsis, are categorized within the dicotyledonous branch. Notably, most of the species closely related to OsPAD1 belong to the Poaceae family, which typically possess only one aperture in their pollen. This suggests a conserved role for OsPAD1 related to aperture development. Additionally, OsPAD1 homologs are exclusively found in both monocotyledonous and dicotyledonous plants, indicating that OsPAD1 likely emerged during the later stages of plant evolution and might play a crucial role in the evolution of angiosperms (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). These unique phenotypes not only distinguish themselves from those previously reported in male sterile mutants (Zhang et al. 2010; Wan et al. 2020; Tao et al. 2021; Chen et al. 2022), but also open up new avenues for investigating pollen aperture formation, particularly in relation to the formation of the fibrillar-granular layer in grasses.\u003c/p\u003e \u003cp\u003eIn Arabidopsis, INP1 plays a critical role in regulating aperture formation by preventing sporopollenin deposition at future aperture sites (Dobritsa and Coerper 2012). Another key Arabidopsis protein, D6PKL3, also involved in aperture regulation, does not directly interact with INP1. Nevertheless, these two proteins mutually influence each other's localization (Lee et al. 2018). Importantly, both INP1 and D6PKL3 interact with various PI, including PI(4)P and PI(4,5)P\u003csub\u003e2\u003c/sub\u003e, known to accumulate in the future aperture region of Arabidopsis. This accumulation suggests their potential role in recruiting proteins related to aperture development to these specific regions (Lee et al. 2018). Similarly, our lipid-binding experiments have demonstrated that OsPAD1 can interact with PI and its mono/bisphosphates, including PI(4)P and PI(4,5)P\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This suggests that OsPAD1 might be recruited by PI(4)P and PI(4,5)P\u003csub\u003e2\u003c/sub\u003e to carry out its function. OsINP1, a homolog of INP1 in rice, serves a similar role to its Arabidopsis counterpart, accumulating at the future aperture position during the late tetrad stage. OsDAF1, another protein, interacts with OsINP1, and both proteins localize to the aperture. However, the polarized accumulation of OsDAF1 at the aperture depends on its interaction with OsINP1, whereas the polarization of OsINP1 occurs independently of OsDAF1 (Zhang et al. 2020). In our current study, the pollen aperture of the \u003cem\u003eOspad1\u003c/em\u003e mutant was found to lack an operculum. TEM revealed that the absence of an operculum in the mutant was due to the absence of the fibrillar-granular layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Since OsINP1 aggregates at the future aperture position during the late stage 8 and converges to the fibrillar-granular layer during pollen wall formation, it is plausible that during the development of the pollen outer wall at stage 9, OsPAD1 accumulates unevenly on the pollen surface under the guidance of OsINP1. This accumulation likely involves the utilization of lipids from the tapetum to form the fibrillar-granular layer. These findings shed light on the specific molecular function of OsINP1 in aperture formation and provide new insights for the study of aperture formation mechanisms. Notably, the double mutant's pollen displayed a typical abortion phenotype, even in the absence of an aperture (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, L). The result suggests that OsPAD1 plays a crucial role not only in lipid transport for the fibrillar-granular layer formation but also potentially contributes to starch accumulation in pollen during late flower development.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Wenhua Zhang (Nanjing Agricultural University) for useful comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.M.W. and Z.G.Z. directed the project. J.M.W., Z.G.Z., and Q.M.W. designed the experiments and wrote the paper. Y.L.T. provided the Ospad1 mutant material. Q.M.W. and K.Y.C. performed most of the experiments. S.S.Z., Y.H.X., C.L.W, X.W.Y., W.T.B., H.Z., and S.M.Y. helped to analyze data. Y.H., D.K.L., A.Q.J., J.Y.L., and H.Y. helped to generate the transgenic plants. X.Z., Y.L.R., C.L.L., Z.J.C., Q.B.L., and L.J. provided technical support. All authors read the final manuscript and approved of its content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Key Research and Development Program of China (2022YFD1201504, 2022YFD1200801), the National Nature Science Foundation of China (U2002202, 31991224, 31971909), the Key Research and Development Program of Jiangsu Province (BE2021360), the Key Laboratory of Biology, Genetics and Breeding of Japonica Rice in the Mid-lower Yangtze River, and the Jiangsu Collaborative Innovation Center for Modern Crop Production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnquiries about data availability should be directed to the authors.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlackmore S, Wortley AH, Skvarla JJ, and Rowley JR (2007) Pollen wall development in flowering plants. 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Nature 504: 406-410.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sterile, Fibrillar-granular layer, Operculum, Lipid binding, Oriza sativa","lastPublishedDoi":"10.21203/rs.3.rs-3873582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3873582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant lipid transfer proteins (LTPs) are distinguished by their capacity to facilitate lipid transport in vitro between membranes. This includes the transportation of lipid constituents from the tapetum to the microspore, thereby playing a pivotal role in the synthesis and construction of the pollen wall, encompassing the formation of the pollen aperture. However, our understanding of LTPs and their role in pollen aperture formation in rice remains limited. In this study, we have isolated and characterized a male sterile rice mutant named as \u003cem\u003epollen aperture defect 1\u003c/em\u003e (\u003cem\u003eOspad1\u003c/em\u003e). When compared to the wild type, \u003cem\u003eOspad1\u003c/em\u003e mutant plants exhibit pollen grain abortion due to the absence of the fibrillar-granular layer, ultimately leading to the leakage of contents from the malformed aperture. \u003cem\u003eOsPAD1\u003c/em\u003e encodes a non-specific LTP and is specifically expressed in the tapetum and microspore during male development. Subsequently, \u003cem\u003ein vitro\u003c/em\u003e lipid binding assays reveal that the recombinant OsPAD1 protein has the capability to bind to a broad spectrum of lipids. The malfunction of OsPAD1 results in disrupted lipid metabolism and compromised pollen aperture, ultimately leading to male sterility. Furthermore, yeast two-hybrid, bimolecular fluorescent complementation and pull-down assays all demonstrate that OsPAD1 can directly interact with OsINP1, an orthologue of a crucial aperture factor in Arabidopsis, together regulating rice aperture development. These findings offer new insights into the molecular mechanisms that underlie the function of LTPs in rice pollen aperture formation. This research holds potential implications not only for rice but also for other cereal crops.\u003c/p\u003e","manuscriptTitle":"OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 20:52:18","doi":"10.21203/rs.3.rs-3873582/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-02-26T08:54:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-26T08:52:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Molecular Biology","date":"2024-01-28T03:16:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2024-01-24T20:25:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"58961017-a668-4d62-87eb-aa7d1732f670","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:05:01+00:00","versionOfRecord":{"articleIdentity":"rs-3873582","link":"https://doi.org/10.1007/s11103-024-01531-z","journal":{"identity":"plant-molecular-biology","isVorOnly":false,"title":"Plant Molecular Biology"},"publishedOn":"2024-12-22 15:58:12","publishedOnDateReadable":"December 22nd, 2024"},"versionCreatedAt":"2024-02-28 20:52:18","video":"","vorDoi":"10.1007/s11103-024-01531-z","vorDoiUrl":"https://doi.org/10.1007/s11103-024-01531-z","workflowStages":[]},"version":"v1","identity":"rs-3873582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3873582","identity":"rs-3873582","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}