Endo-β-1,4-glucanases in Arabidopsis thaliana: expression and putative roles in sexual reproduction

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However, their specific functions in plant sexual reproduction remain unclear. In this study, we systematically analyzed expression profiles of the GH9 gene family using in vivo observations of GFP-fusion proteins. Our results revealed that numerous GH9 genes were expressed across both vegetative and reproductive tissues: 5 in stomata, 12 in roots, 11 in mature pollen and 15 in mature ovules or seeds. Further analysis of reproductive tissues uncovered distinct expression specificities: GH9A4 as pollen-specific, GH9B6 as sperm cell-specific, GH9B12 as central cell- and endosperm cell-specific, GH9B11 as enriched in pollen and chalazal endosperm, and GH9B8 as vascular tissue-preferential. Additionally, GFP signals of up to 7 GH9 members were detected in the filiform apparatus, while 12 GH9 members showed signals in integuments and seed coats—hinting at diverse GH9 functions in reproduction. Furthermore, we conducted gene editing and phenotypic analysis on two subsets of GH9 genes highly expressed in pollen. While in vivo pollen germination and growth were unaffected, in vitro germination rates decreased significantly when GH9B5, GH9B7, and GH9A4 functionally deficient. Taken together, this study highlights the potential roles of the GH9 family in pollen germination and establishes a foundation for future functional analyses aimed at elucidating the putative roles of the GH9 family in plant reproduction. Endo-β-1 4-glucanases expression profile sexual reproduction pollen Arabidopsis thaliana Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message The in vivo expression patterns of the GH9 family and their involvement in pollen germination, collectively elucidate a putative regulatory function of this family in mediating plant sexual reproduction. Introduction The plant cell wall serves multiple functions, including defining cell shape, protecting the protoplast, mediating cell adhesion, maintaining normal water balance and turgor, and regulating the diffusion of macromolecules (Cosgrove 2024 ; Zhang, et al. 2021 ; Zhang, et al. 2025 ). Cellulose, as the most prominent constituent of the plant cell wall, is a complex carbohydrate chain consisting of up to 14,000 glucose molecules linked by β-1,4-glycosidic bonds (Somerville, et al. 2004 ). Endo-β-1,4-glucanase, also known as cellulases, are a widespread group of enzymes that cleave β-1,4-glucosidic bonds—such as those in cellulose and xyloglucan (Lopez-Casado, et al. 2008 ). These enzymes are encoded by genes expressed in diverse organisms, including bacteria, fungi, slime molds, gastropods, tunicates, termites, and plants (Libertini, et al. 2004 ). As a key class of enzymes, plant endoglucanases are assigned to glycosyl hydrolase family 9 (GH9), which has 25 members in Arabidopsis thaliana (Libertini, et al. 2004 ). These GH9 genes can be subdivided into three distinct structural subclasses: secreted members with a carbohydrate-binding module (CBM) (designated as GH9C), secreted members without a CBM (GH9B), and non-secreted members that have a membrane-anchoring structure (GH9A) (Urbanowicz, et al. 2007 ). The general roles of GH9 are known, which are involved in the synthesis, remodeling and turnover of cell wall components during multiple physiological processes (Libertini, et al. 2004 ). CBM49-containing GH9C enzymes are thought to participate in the degradation of crystalline cellulose, a process linked to irreversible cell wall disassembly, as exemplified by root hair emergence and the breakdown of the endosperm wall during germination (Perrot, et al. 2022 ). In Arabidopsis thaliana , knock-down mutants of AtGH9C1 exhibit defects in root hair formation and delayed germination (del Campillo, et al. 2012 ). Downregulation of AtGH9C2 expression reduces cellulose crystallinity, accompanied by increases in both plant height and rosette diameter (Glass, et al. 2015 ). While the CBM49 domain of the tomato β-1,4-glucanase Cel9C binds crystalline cellulose substrates in vitro, CBM49 is thought to contribute to substrate selection or modification rather than the direct hydrolysis of crystalline cellulose (Kundu 2019 ; Trainotti, et al. 1999 ; Urbanowicz, et al. 2007 ). In a separate study, the CBM domain of a rice GH9C endoglucanase was found to undergo post-translational cleavage after the protein is targeted to the cell wall; this cleavage may activate the enzyme’s hydrolytic activity or alter its substrate specificity (Yoshida and Komae 2006 ). Membrane-associated GH9A β-1,4-glucanases that do not possess a CBM49 domain, are likely primarily involved in the assembly, repair or editing of cellulose microfibrils during plant cellulose biosynthesis. Among GH9A β-1,4-glucanases, KORRIGAN1 (KOR1) is the most well-studied (Molhoj, et al. 2001 ; Nicol, et al. 1998 ). The β-1,4-glucanase activity of KOR1 is essential for the effective operation of the cellulose synthase complex (CSC); mutations in KOR1 disrupt the intracellular trafficking of CSC, reduce the cellulose polymerization rate, and cause abnormal deposition of crystalline cellulose (Mansoori, et al. 2014 ; Nicol, et al. 1998 ; Takahashi, et al. 2009 ; Vain, et al. 2014 ; Worden, et al. 2015 ; Zhang, et al. 2016 ). GH9B β-1,4-glucanases are believed to be secreted into the apoplast, where they can hydrolyze the amorphous regions of cellulose. These regions interact with other cell wall polymers, and their hydrolysis alters the mechanical properties of the cell wall, further promoting wall loosening or disassembly (Brummell, et al. 1999 ; Cosgrove 2005 ; del Campillo 1999 ; del Campillo, et al. 2004 ; Jara, et al. 2019 ; Karve, et al. 2016 ; Lewis, et al. 2013 ; Rose and Bennett 1999 ). Notably, a new function has recently been proposed for a specific clade of secreted GH9B β-1,4-glucanases: they may participate in cell-to-cell adhesion during grafting or in the interactions between parasitic plants and their hosts (Kurotani, et al. 2020 ; Notaguchi, et al. 2020 ; Wang, et al. 2017 ). GH9Bs have also been shown to be involved in plant–pathogen or plant–nematode interactions (Finiti, et al. 2013 ; Flors, et al. 2007 ; Goellner, et al. 2001 ; Tucker, et al. 2007 ; Wieczorek, et al. 2008 ; Woo, et al. 2014 ). To date, all known functions of the GH9 enzymes discussed above are associated with vegetative development. However, their involvement in reproductive development remains poorly understood, despite the fact that the cell wall undergoes substantial changes during both vegetative and reproductive growth. While promoter activity assays and transcriptome analyses have provided evidence that GH9 genes are transcribed in pollen, ovules and seeds, critical details of GH9 protein expression—including their presence, spatial localization, and abundance in these reproductive tissues—remain to be elucidated (del Campillo, et al. 2012 ; He, et al. 2018 ; Xie, et al. 2011 ; Yung, et al. 1999 ). In this study, we conducted a detailed tissue-specific analysis of GH9 family gene expression by visualizing the in vivo localization of their genomic green fluorescent protein (GFP)-fusion proteins. Using this approach, we identified candidate GH9 genes with potential involvement in reproductive processes, including pollen development, pollen tube growth and reception, embryo and endosperm development, and seed coat maturation. Additionally, we investigate the roles of two subsets of GH9 genes (highly expressed in pollen) in male reproductive development, and found that functional deficiency of GH9B5 , GH9B7 , and GH9A4 led to a significant reduction in germination rate in in vitro assays. Thus, our results suggest that GH9 β-1,4-glucanases may play essential roles in plant reproductive development, which warrants further investigation in future studies. Materials and methods Plant materials and growth conditions Arabidopsis thaliana ecotype Col-0 (wild-type), mutants, and transgenic lines were cultivated in a greenhouse under a photoperiod of 16 h light and 8 h dark at 22°C. Phylogenetic and collinearity analysis All Arabidopsis gene sequences were retrieved from TAIR (the Arabidopsis Information Resource; https://www.arabidopsis.org/ ). Phylogenetic analysis of the Arabidopsis endo-β-1,4-glucanase gene family was performed using MEGA 11 software, employing the neighbor-joining (NJ) method, with 1000 bootstrap replicates (Tamura, et al. 2021 ). Transcriptome data were obtained from the Arabidopsis RNA-seq Database ( https://plantrnadb.com/athrdb/ ) (Zhang, et al. 2020 ) and visualized with the ChiPlot tool (Xie, et al. 2023 ). To determine the replication patterns of each GH9 gene within the Arabidopsis genome, MCScanX was employed to assess genomic collinearity, utilizing the default parameters (Wang, et al. 2012 ). Constructs for plant transformation For the GH9s pro : GH9s-GFP construct, the GH9s promoter and the full-length GH9s coding sequence (without the stop codon) were amplified from Arabidopsis genomic DNA using the primer pairs GH9s-094-S/A , and then ligated into P094 (Wu, et al. 2012 ) upstream of GFP after XbalI/ EcoRI digestion. All primers used for cloning are listed in Supplementary Table S1 . For CRISPR/Cas9-mediated genome editing, four single guide RNAs (sgRNAs) were designed to target GH9B5/7/A1 and GH9B9/10/11/12 , respectively. Following established protocols, the four sgRNAs were first integrated into the entry vector pENTR-MSR (Xing, et al. 2014 ). The resulting expression cassettes were then digested with restriction enzymes KpnⅠ/XbaⅠ and ligated into the binary vector pCAMBIA1300-pYAO-cas9, which had been pre-digested with KpnⅠ/SpeⅠ (Yan, et al. 2015 ). All recombinant constructs were verified by Sanger sequencing and subsequently used to transform Arabidopsis thaliana via the floral dip method (Clough and Bent 1998 ). Phenotypic Analysis For in vitro pollen germination assays, mature pollen grains were evenly spread on solid pollen germination medium and cultured at 22°C for 5 h. The medium was composed of the following components: 0.05% boric acid, 5 mM CaCl 2 , 5 mM KCl, 1 mM MgSO4, 10% sucrose and 1% agarose (PH was adjusted to 7.5). Pollen grain viability was evaluated using Alexander’s staining. The Alexander’s staining solution (100mL total volume) contained 10 mL 95% ethanol, 1 mL malachite green (1% solution in 95% ethanol), 25 mL glycerol, 5 mL acid fuchsin (1% solution in water), 0.5 mL orange G (1% solution in water), 4 mL glacial acetic acid, and 54.5 mL distilled water. For DAPI (4,6-diamidino-2-phenylindole) staining, pollen tubes were incubated in liquid germination medium supplemented with 2.5 µg/mL DAPI and 0.01% (vol/vol) Triton X-100. Incubation was performed in the dark for 5 minutes prior to visualization using fluorescence microscopy. To analyze pollen-tube guidance, hand-pollinated pistils were fixed in Carnoy’s Fixative (ethanol:acetic acid = 3:1, v/v) for 12 h. After fixation, the pistils were rinsed three times with 0.1 M phosphate-buffered saline (PBS), and transferred into 5 M NaOH for overnight incubation to induce softening. Prior to staining, the pistils were rinsed three times with 0.1 M PBS, and then stained with 0.1% (wt/vol) aniline blue solution in the dark. To examine expression patterns of GH family genes, different tissues were observed under a confocal microscope (SP8 CLSM, Leica), using an excitation wavelength of 488 nm for green fluorescent protein. Results Phylogenetic analysis and expression profiling of GH9 family genes in Arabidopsis In Arabidopsis , there are currently 25 genes encoding β-1,4-glucanases, which are classified into three subgroups: 4 GH9A , 18 GH9B , and 3 GH9C genes (Urbanowicz, et al. 2007 ). Phylogenetic analysis confirmed that the protein sequences of these genes cluster into three distinct subfamilies, consistent with previous reports (Fig. 1 ) (Libertini, et al. 2004 ). To characterize the expression patterns of GH9 family genes, we extracted and summarized expression data from the Arabidopsis RNA-seq Database—encompassing various tissues across multiple stages of plant development—to construct a comprehensive expression profile of these 25 genes (Zhang, et al. 2020 ). Among all GH9 genes, five ( GH9A1 , GH9B1 , GH9B7 , GH9B13 , and GH9C2 ) exhibited a ubiquitous expression pattern, with high transcript abundance in nearly all tissues and organs (Fig. 1 ). Another five genes ( GH9A4 , GH9B3 , GH9B5 , GH9B6 , and GH9B11 ) shared a similar expression pattern, being predominantly expressed in pollen. Additionally, GH9B8 and GH9C1 showed relatively high expression levels in the endosperm (Fig. 1 ). These results collectively demonstrate that Arabidopsis GH9 family genes display diverse expression profiles, implying their involvement in a broad range of plant developmental processes. Expression patterns of GH9-GFP fusion proteins in leaves and roots To validate the reliability of publicly available expression data, we generated transgenic Arabidopsis plants expressing GH9-GFP fusion proteins under the control of their respective native promoters. Among these transgenic lines, GH9B7–GFP signals were exclusively detectable in leaf epidermal cells (Fig. 2 ). Notably, to date, no direct evidence supports the involvement of GH9 proteins in the structure or function of leaf stomata. However, in transgenic leaves harboring pGH9B1::GH9B1–GFP , pGH9B7::GH9B7–GFP , pGH9B12::GH9B12–GFP , pGH9B14::GH9B14–GFP , pGH9B15::GH9B15–GFP constructs, green fluorescent signals localized to the plasma membrane or cell wall of stomatal cells, but not their cytoplasm (Fig. 2 ; Supplementary Table S2 ). This observation suggests that these GH9 proteins may indirectly affect stomatal function by contributing to the hydrolysis and modification of cell wall polysaccharides. In a previous study, promoter–reporter (GUS) assays and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that GH9B4 (also referred to as Cel5 ) was exclusively expressed in the root cap cells of both primary and lateral roots; this gene was further shown to mediate the sloughing of border cells from the root tip (del Campillo, et al. 2004 ; Karve, et al. 2016 ). Consistent with this expression pattern, in transgenic plants harboring the pGH9B4::GH9B4–GFP construct, GFP signals were exclusively detectable within the root cap (Fig. 3 ; Supplementary Table S2 ). Notably, GHB3 —the duplicate of GH9B4 , which is associated with lateral root induction—shares a similar root expression pattern. Besides GH9B3 and GH9B4, GH9B6 was also identified as a novel root cap marker, as evidenced by green fluorescent signals specifically observed in root cap cells (Fig. 3 ; Supplementary Table S2 ). Collectively, these three genes may exhibit functional redundancy; thus, simultaneous inhibition of all three will be necessary to investigate the mechanism governing border cell release from the root cap. The promoter of GH9B1 (also referred to as cel1 ) has been reported to be active in root elongation zones, and our observations of GH9B1-GFP expression in roots are consistent with this finding (Shani, et al. 2006 ). Among GH9B subfamily members, four additional members have also been shown to be expressed in roots. What is particularly interesting is the specific localization of GH9B8-GFP to vascular bundles (Fig. 3 ). Additionally, GH9C1 and GH9C2 , both of which contain a CBM49 domain, exhibited distinct root expression patterns: GH9C2 was constitutively expressed, whereas GH9C1 showed exclusive localization to root hairs (Fig. 3 ). Notably, the expression domain of GH9C1-GFP was narrower than that previously observed in GUS staining assays, yet this restricted expression was sufficient to support the phenotype of markedly reduced root hair density in gh9c1 mutants (del Campillo, et al. 2012 ). While the GH9A1 gene was expressed throughout the root, GH9A2-GFP, driven by its native promoter, was exclusively detected in the root differentiation zone, including root hairs (Molhoj, et al. 2001 ). In contrast to the subcellular localization of GH9B and GH9C subfamily members, the GFP signals of GH9A1 and GH9A2 predominantly accumulated in intracellular compartments, mirroring intracellular punctate pattern previously observed for GH9A1 via both immunofluorescence assays and GFP–GH9A1 fusion protein analyses (Fig. 3 ) (Robert, et al. 2005 ). Expression patterns of GH9-GFP fusion proteins in pollen To identify the potential roles of GH9 family genes in plant reproductive processes, we first analyzed the expression patterns and subcellular localization of GH9-GFP fusion proteins during male reproductive development. Based on transcriptomic data, the endoglucanases potentially involved in the cell wall remodeling during pollen development and pollen tube growth are GH9A4, GH9B3, GH9B5, GH9B6, and GH9B11 (Fig. 1 ). Among these genes, only the GH9A4 promoter has been demonstrated to drive pollen specific GUS activity (Xie, et al. 2011 ). In our study, we identified up to 10 GH9 gens expressed in mature pollen, including 2 from the GH9A subfamily, 7 from the GH9B subfamily, and 1 from the GH9C subfamily (Fig. 4 A; Supplementary Table S2 ). Notably, these GH9 genes exhibited differences in both expression levels and subcellular localization. In mature pollen, GH9A1-GFP or GH9A4-GFP exhibited strong intracellular signals with a filamentous pattern (Fig. 4 A). Among duplicated gene pairs, GH9B3/GH9B4 showed weak, punctate fluorescence, whereas GH9B5/GH9B7 displayed relatively strong, continuous fluorescence (Fig. 4 A; Supplementary Fig. S1 ). Weak plaque-like fluorescence was detected in pollen expressing GH9B9-GFP or GH9C2-GFP, while filamentous fluorescence was observed in pollen expressing GH9B11-GFP or GH9B18-GFP. GH9B and GH9C subfamily members do not appear to be secreted in mature pollen—despite the general consensus that they are secreted into the apoplast and prior findings indicating their likely cell wall localization in vegetative tissues (Fig. 4 A). Thus, the mechanisms governing the subcellular dynamics of GH9 β-1,4-glucanases across different tissues and developmental stages are complex yet intriguing, and warrant further investigation. The pollen tube, a fast tip-growing cell tasked with delivering two sperm cells to the ovule, depends on extensive cell wall deposition to drive its rapid elongation and on tight regulation of cell wall remodeling to modify mechanical properties (Chebli, et al. 2012 ; Cheung and Wu 2008 ; Dresselhaus and Franklin-Tong 2013 ; Hafidh and Honys 2021 ; Hepler, et al. 2013 ). To investigate the subcellular location of GH9 proteins in pollen tubes, we performed in vitro pollen germination assays to observe their distribution. Five GH9 genes highly expressed in pollen were selected for analysis. In pollen tubes expressing GH9A1-GFP, GFP signals were concentrated in the vacuole, although punctate fluorescence was also detected in the cytoplasm at the pollen tube tip (Fig. 4 C). In plasmolyzed pollen tubes, GH9A4-GFP, GH9B5-GFP and GH9B7-GFP signals were clearly visible in the pollen tube cell wall, whereas GH9B11-GFP signals were not, indicating that GH9A4, GH9B5 and GH9B7, but not GH9B11 can be secreted extracellularly (Fig. 4 D). Beyond the aforementioned GH9 genes expressed in pollen grains, GH9B6-GFP signals were specifically detected in sperm cells, suggesting a potential role for GH9B6 in either male gametophyte development or the subsequent double fertilization process (Fig. 4 B). Expression patterns of GH9-GFP fusion proteins in ovules and seeds Next, we analyzed the expression patterns of the GH9 family genes in female reproductive tissues at various developmental stages: mature ovules, seeds at 1–2 days after pollination (DAP), and seeds at 5–7 DAP (Fig. 5 ; Supplementary Table S2 ). Early reports have shown that GH9B2 transcripts were predominantly expressed in the developing septum and ovule primordia of young carpels (Yung, et al. 1999 ). In ovules harboring the GH9B2-GFP construct, we also observed strong fluorescence signals in the integuments. In addition to GH9B2 , GH9A1 , GH9B1 , GH9B5 , GH9B7 , GH9B13 , GH9B18 , and GH9C2 shared a similar expression pattern, with signals consistently detectable throughout the seed coat development (Fig. 5 ). GH9B8-GFP signals were visible in the integuments both before fertilization and after fertilization but disappeared in the seed coat by 5–7 DAP. GH9A2 , GH9B9 and GH9B10 exhibited a narrow expression pattern during seed coat development, as GFP signals were only observed in the seed coat at 5–7 DAP. Among these three genes, the expression of GH9B9 and GH9B10 was restricted to the inner integument (Fig. 5 ). The filiform apparatus (FA)—located at the micropylar end of synergid cells—is an active communication hub defined by intricate plasma membrane invaginations and thick cell walls. It is presumed to play pivotal roles in secreting pollen-attractant peptides and mediating male–female interactions during sexual reproduction (Johnson, et al. 2019 ; Susaki, et al. 2023 ). We found that green-fluorescent signals of seven GH9B-GFP fusion proteins (GH9B1, GH9B5, GH9B7, GH9B8, GH9B13, GH9B16 and GH9B18) localized to FA of the synergid cells but not their cytoplasm, suggesting that these GH9 proteins are continuously released into the micropylar region by these gland-like synergid cells prior to fertilization (Fig. 5 ; Supplementary Table S2 ). For female gametes, the duplicated gene pair GH9B5 / GH9B7 was expressed in both egg cells and central cells, but not in zygotes or the primary endosperm, suggesting their potential roles in cell wall formation of egg cells and central cells. After fertilization, only four GH9 genes were found to be expressed during embryonic development: GH9A2 in the pro-embryo, GH9B7 and GH9B8 in the globular embryo, and GH9B1 in the torpedo embryo (Fig. 5 ; Supplementary Table S2 ). While both GH9B8 and GH9B12 are expressed in central cells and endosperm, they exhibit distinct secretory characteristics (Fig. 5 ). GH9B8-GFP signals is mainly localized to the cytoplasm, whereas GH9B12-GFP showed secretion from central cells, as evidenced by the green-fluorescent signals in the cell walls of the inner integuments, particularly near the micropyle or at the chalazal end. GH9B5 , GH9B7 and GH9A4 are involved in pollen germination in vitro To further validate the involvement of β-1,4-glucanases in sexual reproduction, we focused on two groups of GH9 genes highly expressed in pollen to investigate their functions in male gametophyte development. The first group includes GH9B5 , GH9B7 , and GH9A4 , all highly expressed in mature pollen and capable of being secreted to the pollen tube cell wall. The second group consists of GH9B9 , GH9B10 , GH9B11 , and GH9B12 , which are tandemly linked on the chromosome; among these, GH9B9 and GH9B11 are expressed in pollen (Fig. 4 ). Using the CRISPR/Cas9 system, we generated genome-edited gh9b5/7/a4-1 , gh9b5/7/a4-2 , gh9b9/10/11/12 − 1 and gh9b9/10/11/12 − 2 mutants, in which all alleles harbor frameshift mutations accompanied by premature stop codons (Supplementary Fig. S2 ). Although pollen development and viability were normal in all mutants, gh9b5/7/a4 mutants showed a significantly reduced in vitro pollen germination rate, whereas WT and gh9b9/10/11/12 mutants not (Fig. 6 A-F). Next, we examined whether pollen tube growth was normal in gh9b5/7/a4 and gh9b9/10/11/12 mutants in vivo. At 3 hours after pollination (HAP), all pollen tubes reached the upper part of the pistil, with no differences in length among the genotypes (Fig. 6 G and I). By 8 HAP, the pollen tubes of both mutants also reached the bottom of the pistil (Fig. 6 H and J). This observation is further supported by the finding that neither gh9b5/7/a4 nor gh9b9/10/11/12 mutants affect plant fertility (Fig. 6 K and L). Thus, these results demonstrate that GH9B5 , GH9B7 , and GH9A4 are critical for pollen germination, though additional research is needed to determine which of these three genes exerts a dominant role. Discussion The plant cell wall is a dynamic composite structure with diverse functions, including providing a framework to support the cell structure, compartmentalizing specialized cells, protecting against infection by pathogens and mediating the communication between cells to regulate plant development (Cosgrove 2024 ; Zhang, et al. 2021 ; Zhang, et al. 2025 ). Given these essential roles, plants have evolved β-1,4-glucanases to degrade cellulose and other polysaccharides containing 1,4-glycosidic bonds to remodel and disassemble the wall during cell growth (Cantarel, et al. 2009 ; del Campillo 1999 ; Lopez-Casado, et al. 2008 ). Numerous studies have reported that GH9 proteins are implicated in diverse physiological processes in higher plants, such as root hair emergence, endosperm breakdown, fruit ripening, grafting, plant parasitism, and nematode defense (del Campillo, et al. 2004 ; del Campillo, et al. 2012 ; Goellner, et al. 2001 ; Jara, et al. 2019 ; Kurotani, et al. 2020 ; Nicol, et al. 1998 ; Notaguchi, et al. 2020 ; Trainotti, et al. 1999 ; Tucker, et al. 2007 ; Wang, et al. 2017 ; Wieczorek, et al. 2008 ; Woo, et al. 2014 ). In Arabidopsis , the GH9 family comprises 25 members; however, to date, only a few of them have been well characterized regarding their in vivo functions. In the present study, we established detailed expression profiles of GH9 family genes by conducting in vivo observations of their GFP-fusion proteins. These observations reveal that GH9 genes are expressed not only in vegetative organs but also enriched in reproductive tissues. This work provides a valuable reference map for understanding the spatial distribution of GH9 β-1,4-glucanases and their dynamic behavior during plant development, thereby offering clear cues for further investigating their functional roles in reproductive processes. Potential roles of GH9 family genes in vegetable development Vegetative development, which involves the growth and maturation of non-reproductive organs such as roots, stems and leaves, relies strongly on dynamic cell wall remodeling. As key regulators of cell wall metabolism, GH9 β-1,4-glucanases contribute to multiple aspects of vegetative organ biology, including shaping their structure, optimizing their function, and enhancing their stress resilience (Perrot, et al. 2022 ). Roots are critical for water and nutrient uptake, plant anchorage and symbiotic interactions. Previous studies have indicated that GH9 genes are involved in nearly all stages of root development—for instance, GH9B1 in primary root elongation, GH9C1 in root hair formation, GH9B3 in lateral root emergence, and GH9B3 / GH9B4 in the sloughing of border cells from the root tip (del Campillo, et al. 2004 ; del Campillo, et al. 2012 ; Lewis, et al. 2013 ; Karve, et al. 2016 ; Tsabary, et al. 2003 ). Beyond the genes discussed above, our study identified eight additional GH9 genes, with expression localized to the root cap, elongation zone, vascular bundles, or root hairs (Fig. 3 ). Future studies will be needed to clarify whether these genes act redundantly with known GH9s or carry out novel functions in root development. Another interesting observation is that all five GH9B-GFP fusion proteins expressed in leaves localize to the stomatal cell wall (Fig. 2 ). Stomata are small pores on the leaf surface that play a critical role in regulating gas exchange and water loss, and their shape and function are physically constrained by the stomatal cell wall (Lawson and Matthews 2020 ). Cellulose microfibrils, long been recognized as the load-bearing components of cell walls, are central to maintaining this structural constraint (Cosgrove 2022 ; Gibson 2012 ; Markov, et al. 2017 ). Notably, the cellulose-deficient mutants cesa3 je5 exhibits aberrant cellulose microfibril reorientation, alongside abnormalities in stomatal apertures and guard cell lengths during stomatal movement (Rui and Anderson 2016 ). Whether cellulose remodeling is associated with stomatal development and function remains to be tested. Here, these five GH9B genes—with their potential roles in cell wall remodeling—represent an excellent entry point to address this unresolved question. Potential roles of GH9 family genes in plant reproduction from the male side To accommodate diverse cell shapes, plant cell walls exhibit varied forms, structures, and compositions, and are highly dynamic throughout the entire plant life cycle, whether during vegetative or reproductive development (Cosgrove 2024 ; Zhang, et al. 2021 ). How the molecular mechanisms controlling cell wall properties are regulated during plant reproduction is a fundamental question in plant biology. Studies on the expression and localization of GH9 family genes not only clarify the spatiotemporal patterns of these genes but also provide insights into the functions of β-1,4-glucanases in plant reproduction, from both male and female perspectives. In mature pollen, ten GH9 genes were found to be expressed, with distinct expression levels and subcellular localizations (Fig. 4 A). Furthermore, functional deficiency of GH9B5 , GH9B7 , and GH9A4 led to a significantly reduced germination rate in in vitro pollen germination assays, suggesting their potential roles in cell wall degradation or remodeling during the initiation of pollen germination (Fig. 6 E and F). Regarding whether other GH family members exhibit redundant functions in pollen germination, this possibility cannot be ruled out, which might also explain why no obvious phenotypes were observed in vivo. Pollen tube growth is a critical step toward fertilization in flowering plants. During pollen tube growth, cell wall dynamics balance cell expansion and turgor resistance, with biosynthesis and modification of the cell wall being crucial for maintaining pollen tube integrity (Chebli, et al. 2012 ; Cheung and Wu 2008 ; Dresselhaus and Franklin-Tong 2013 ; Hafidh and Honys 2021 ; Hepler, et al. 2013 ). Despite the low cellulose content in the pollen tube wall, crosslinking between cellulose, pectin, and callose endows the pollen tube with distinct mechanical properties necessary for its growth (Chebli, et al. 2012 ). Plant cellulose biosynthesis occurs at the plasma membrane via large cellulose synthase (CESA) complexes. However, in the growing tips of pollen tubes, cellulose synthase-like D (CSLD) proteins appear to localize to the apical plasma membrane, while CESAs are mostly confined to internal compartments—opposite to their distribution in somatic cells (Park, et al. 2011 ; Wang, et al. 2011 ). Pollen tubes lacking CSLD1 and/or CSLD4 exhibit reduced cellulose deposition, compromising the genetic transmission of the male gametophyte (Bernal, et al. 2008 ; Wang, et al. 2011 ). Additionally, loss of function of two pollen-specific glycerophosphodiester phosphodiesterase-like genes in Arabidopsis causes a severe reduction in cellulose deposition at the mutant pollen tube tip walls, ultimately resulting in pollen tube bursting (Wang, et al. 2023 ). However, to date, no direct evidence has been reported for cell wall remodeling mediated by GH9 β-1,4-glucanases during pollen tube growth. The enzymes potentially involved may include not only the ten pollen-expressed GH9 genes identified in this study, but also those up-regulated in pollen tubes or enriched in female tissues that the pollen tube encounters during its journey. Potential roles of GH9 family genes in plant reproduction from the female side In addition to the roles mentioned above, GH9 family members also exhibit potential functions in the female reproductive development. In most flowering plants, including Arabidopsis thaliana , the female gametophyte—also known as the embryo sac—comprises highly differentiated cells: two female gametes (the haploid egg cell and homodiploid central cell), two synergid cells (which function in pollen tube attraction and reception), and three antipodal cells (with poorly characterized functions) (Hater, et al. 2020 ; Wang, et al. 2025 ; Yadegari and Drews 2004 ; Yang, et al. 2010 ). These cells not only have distinct morphologies but also exhibit ultrastructural differences in their cell walls. Moreover, the chalazal region of the egg cell, the target site for sperm entry and fusion during fertilization, exhibits thin furrows between thick cell wall structures (Tekleyohans, et al. 2017 ). Future work will be necessary to elucidate two key questions: first, whether these indentations (i.e., the thin furrows) correspond to the actual sites of sperm entry; and second, whether the cell wall apposition pattern is regulated by GH9 proteins expressed in egg cells or central cells, that were identified in our study. Although the synergid cell itself is not fertilized, it mediates extensive communication between female and male gametophytes prior to double fertilization. The filiform apparatus, located at the micropylar end of synergid cells, is enriched in both pollen tube attractant peptides and various pollen tube reception factors (Capron, et al. 2008 ; Escobar-Restrepo, et al. 2007 ; Galindo-Trigo, et al. 2020 ; Márton, et al. 2005 ; Meng, et al. 2019 ; Okuda, et al. 2009 ; Takeuchi and Higashiyama 2012 ; Tsukamoto, et al. 2010 ; Zhong, et al. 2019 ). A recent study has revealed that the FA exhibits a well characterized sponge-like structure, consisting of intricate plasma membrane invaginations and thick cell walls; notably, the surface area of the FA is 3.7-fold larger than the estimated cell wall area excluding invaginations (Susaki, et al. 2023 ). Thus, two key questions remain to be addressed in future studies: first, how is this widely conserved synergid cell wall structure formed? Second, are the seven GH9Bs expressed in the FA involved in this formation process? Potential roles of GH9 family genes in plant reproduction during seed development Seed development is a complex, highly regulated biological process in plants that transforms a fertilized ovule into a mature seed, which typically consists of three major structures: the embryo, the endosperm, and the seed coat (Figueiredo and Kohler 2014 ; Kesavan, et al. 2013 ; Lafon-Placette and Kohler 2014 ; Orozco-Arroyo, et al. 2015 ; Wang, et al. 2022 ). Seed development relies on dynamic changes in cell wall composition, structure, and remodeling to support key developmental processes, including embryo growth and differentiation, endosperm cellularization, as well as seed coat formation. The cell wall is not merely a rigid "support structure", but also acts as a regulatory interface that coordinates growth, nutrient transport, and stress resistance across these three core seed components (Huang, et al. 2023 ). Although several GH9 genes were identified in the embryo and endosperm in this study, 12 out of 25 GH9 family members were found to be expressed in the seed coat (Fig. 5 ). These seed coat-expressed GH9 genes may remodel and disassemble the wall during cell growth, by degrading cellulose and other polysaccharides, to help the seed coat adapt to the turgor pressure generated by internal embryo and endosperm growth. However, this functional hypothesis requires extensive experimental validation in future studies. The integuments connect to the vascular tissues of the maternal funiculus at the chalazal region, a connection that ensures the nutritional supply required for seed development. A recent study has reported a fertilization-dependent phloem end gate at the chalazal end of the ovule, which regulates nutrient transport into the developing seed (Liu, et al. 2025 ). Before fertilization, this gate is blocked by callose deposition; after fertilization, specifically following fertilization of the central cell, this callose block is removed, a process controlled by genes encoding β-1,3-glucanases (callose-degrading enzymes) (Liu, et al. 2025 ). In the present study, GH9B8-GFP was detected in the vascular bundles of the funicle. Of particular note is the localization of GH9A2-GFP in integument cells at the funicle attachment site, as well as the localization of GH9B11-GFP in the chalazal zone of the endosperm (CZE) (Fig. 5 ). A key question for future investigation is whether these GH9 genes could also form a "cellulase gate" at the chalazal end of the ovule, analogous to the previously reported callose-based phloem end gate. If this hypothesis is validated, it could potentially offer a widely applicable strategy in angiosperms to increase seed size in the future. Conclusions This study delved into expression patterns and putative roles of the GH9 family gene in sexual reproduction. By visualizing the in vivo localization of GH9-GFP fusion proteins, comprehensive expression profile analysis reveals that GH9 family genes are expressed not only in vegetative organs but also enriched in reproductive tissues, including mature pollen, unfertilized ovules and seeds at different developmental stages. Moreover, we found that functional deficiency of GH9B5 , GH9B7 , and GH9A4 led to a significant reduction in pollen germination rate in in vitro assays, implying their critical roles in pollen germination. Taken together, this study constructs a comprehensive expression profile of GH9 family genes and highlights their potential roles in plant reproduction. Declarations Conflict of interests The authors have no competing interests to declare that are relevant to the content of this article. Author contributions Study design, project management: WW, HXX. Experimental Design: WW, HXX. Data Collection: HXX, XDG. Sample processing: HXX, XDG. Data Analysis: WW, HXX, XDG. Draft manuscript: WW, HXX. All Authors read, edited, and approved the manuscript. Acknowledgments This work was supported by National Natural Science Foundation of China grants (32200702 to HXX.; 31800264 to WW) and by the High-level Talent Introduction Project of Central China Normal University to WW. Data availability All data is available in the main text or the supplementary information. 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19:30:56","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211421,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/efea36b8c0347305cfb3c4ac.html"},{"id":95412342,"identity":"91852d0c-2194-4a9c-85ae-6367b6da1da9","added_by":"auto","created_at":"2025-11-07 19:30:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":431091,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis and expression patterns of the GH9 family genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA11, based on GH9 protein sequences. Three subfamilies are distinguished by different colors. Expression profiles across various tissues were retrieved from the \u003cem\u003eArabidopsis\u003c/em\u003e RNA-seq Database. Both the heatmap and phylogenetic tree were visualized using the ChiPlot tool.\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/d86441b965596e4ec216c9e8.jpg"},{"id":95412341,"identity":"a007a18c-a21c-479b-952d-28a824b093c8","added_by":"auto","created_at":"2025-11-07 19:30:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":310008,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization of GH9-GFP fusion proteins in \u003cem\u003eArabidopsis\u003c/em\u003eleaves. \u003cem\u003eGH9\u003c/em\u003e coding sequences were fused in-frame with the green fluorescent protein (GFP) and stably expressed in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants under their native promoters. Scale bars, 25μm.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/6f6e40078600fe9b7ad7d6c7.jpg"},{"id":95526903,"identity":"9ee7459b-99d6-4eab-a55d-24a7683a1b10","added_by":"auto","created_at":"2025-11-10 10:08:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529060,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of GH9-GFP fusion proteins expressed in roots under the control of their native promoters. Scale bars, 50μm.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/0e7b7c5c63d4eeb6858f9d1a.jpg"},{"id":95412349,"identity":"4b93efe6-9b03-46e8-90a6-b646338b38e2","added_by":"auto","created_at":"2025-11-07 19:30:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":531081,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of different \u003cem\u003eGH9\u003c/em\u003e genes in mature pollen and pollen tubes.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGFP signals in mature pollen expressing various GH9-GFP fusion proteins (driven by their native promoters). (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGH9A1-GFP signals in the pollen tube. (\u003cstrong\u003eD\u003c/strong\u003e) GFP signals in plasmolyzed pollen tubes expressing GH9A4-GFP, GH9B5-GFP, GH9B7-GFP and GH9B11-GFP. Scale bars, 5 μm.\u003c/p\u003e","description":"","filename":"F4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/f8035a008bee88334019e071.jpg"},{"id":95527541,"identity":"7d4e959f-c087-4abf-a482-c6ebf98541a6","added_by":"auto","created_at":"2025-11-10 10:14:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":765484,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of diverse \u003cem\u003eGH9\u003c/em\u003e genes in unfertilized and fertilized ovules. Arrowheads indicate GFP signals in the filiform apparatus. Abbreviations: EC, egg cell; EM, embryo; CC, central cell; CZE, chalazal endosperm; DAP, days after pollination. Scale bars, 25 μm.\u003c/p\u003e","description":"","filename":"F5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/4329d11652a6347df393965b.jpg"},{"id":95527065,"identity":"0c75237c-92b1-4c01-9371-d06e00d01941","added_by":"auto","created_at":"2025-11-10 10:09:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":949351,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic observations of the \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e CRISPR mutants. (\u003cstrong\u003eA) \u003c/strong\u003eDAPI staining of pollen from wild type (Col-0), \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutant plants. VN, vegetative cell nucleus; SN, sperm nuclei. (\u003cstrong\u003eB)\u003c/strong\u003e Percentages of tricellular pollen corresponding to (\u003cstrong\u003eA\u003c/strong\u003e). From left to right, n=364, 336, 363, 396 and 339 pollen, respectively. (\u003cstrong\u003eC) \u003c/strong\u003eAlexander staining to assess pollen viability in wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e plants. (\u003cstrong\u003eD)\u003c/strong\u003ePercentages of viable pollen analyzed in (\u003cstrong\u003eC\u003c/strong\u003e). From left to right, n=362, 371, 347, 370 and 365 pollen, respectively. (\u003cstrong\u003eE) \u003c/strong\u003eRepresentative images of wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e pollen at 5 hours after germination. (\u003cstrong\u003eF)\u003c/strong\u003e Analysis of pollen germination rates in wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutants. Note that \u003cem\u003egh9b5/7/a4 \u003c/em\u003epollen exhibits a significantly reduced germination rate. From left to right, n=564, 446, 756, 350 and 382 pollen, respectively. (\u003cstrong\u003eG \u003c/strong\u003eand\u003cstrong\u003e H) \u003c/strong\u003eAniline blue staining of wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e pollen tubes in wild type pistils at 3 (\u003cstrong\u003eG\u003c/strong\u003e) or 8 (\u003cstrong\u003eH\u003c/strong\u003e) hours after pollination (HAP). Arrow heads indicate the region where the pollen tubes contacted the pistil tissue. (\u003cstrong\u003eI \u003c/strong\u003eand\u003cstrong\u003e J)\u003c/strong\u003e Statistical analysis of wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e pollen tube length grown in pistils at 3HAP (\u003cstrong\u003eI\u003c/strong\u003e) or 8HAP (\u003cstrong\u003eJ\u003c/strong\u003e). n=10 pistils for each. (\u003cstrong\u003eK) \u003c/strong\u003eSeed morphology in self-pollinated siliques of wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutant plants. (\u003cstrong\u003eL) \u003c/strong\u003eSeed set rates of wild type, \u003cem\u003egh9b5/7/a4 \u003c/em\u003eand \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutants (10 siliques for each). Data are presented as mean ± SD. Means with no common letter differ significantly (P \u0026lt; 0.01) based on multiple comparisons using one-way ANOVA and Tukey’s range test. Scale bars, 5 µm in (\u003cstrong\u003eA\u003c/strong\u003e), 25 µm in (\u003cstrong\u003eC\u003c/strong\u003e), 50 µm in (\u003cstrong\u003eE\u003c/strong\u003e), 75 µm in (\u003cstrong\u003eG \u003c/strong\u003eand\u003cstrong\u003e H\u003c/strong\u003e) and 0.5 mm in (\u003cstrong\u003eK\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"F6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/f256a6c5e1b6627ba2d8496e.jpg"},{"id":98425678,"identity":"490b63c0-d15c-4751-a1fb-7aa1c1d96a0f","added_by":"auto","created_at":"2025-12-17 16:35:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4591174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/bf30518f-1562-459f-bc4e-98188fa220ac.pdf"},{"id":95412367,"identity":"9fb14a9b-9adb-4833-aa3f-9bd1548999d0","added_by":"auto","created_at":"2025-11-07 19:30:56","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":2267593,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/f4cb9fc391da9ebdfa21a4e0.docx"},{"id":95412350,"identity":"cccc1f12-e26e-4c94-b292-3031c5d263ac","added_by":"auto","created_at":"2025-11-07 19:30:56","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":16486,"visible":true,"origin":"","legend":"","description":"","filename":"TableS12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7805288/v1/69e74223a1f667addc1c2820.xlsx"}],"financialInterests":"","formattedTitle":"Endo-β-1,4-glucanases in Arabidopsis thaliana: expression and putative roles in sexual reproduction","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cstrong\u003eThe in vivo expression patterns of the GH9 family and their involvement in pollen germination, collectively elucidate a putative regulatory function of this family in mediating plant sexual reproduction.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe plant cell wall serves multiple functions, including defining cell shape, protecting the protoplast, mediating cell adhesion, maintaining normal water balance and turgor, and regulating the diffusion of macromolecules (Cosgrove \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cellulose, as the most prominent constituent of the plant cell wall, is a complex carbohydrate chain consisting of up to 14,000 glucose molecules linked by β-1,4-glycosidic bonds (Somerville, et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Endo-β-1,4-glucanase, also known as cellulases, are a widespread group of enzymes that cleave β-1,4-glucosidic bonds\u0026mdash;such as those in cellulose and xyloglucan (Lopez-Casado, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These enzymes are encoded by genes expressed in diverse organisms, including bacteria, fungi, slime molds, gastropods, tunicates, termites, and plants (Libertini, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). As a key class of enzymes, plant endoglucanases are assigned to glycosyl hydrolase family 9 (GH9), which has 25 members in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Libertini, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These \u003cem\u003eGH9\u003c/em\u003e genes can be subdivided into three distinct structural subclasses: secreted members with a carbohydrate-binding module (CBM) (designated as GH9C), secreted members without a CBM (GH9B), and non-secreted members that have a membrane-anchoring structure (GH9A) (Urbanowicz, et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe general roles of \u003cem\u003eGH9\u003c/em\u003e are known, which are involved in the synthesis, remodeling and turnover of cell wall components during multiple physiological processes (Libertini, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). CBM49-containing GH9C enzymes are thought to participate in the degradation of crystalline cellulose, a process linked to irreversible cell wall disassembly, as exemplified by root hair emergence and the breakdown of the endosperm wall during germination (Perrot, et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, knock-down mutants of \u003cem\u003eAtGH9C1\u003c/em\u003e exhibit defects in root hair formation and delayed germination (del Campillo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Downregulation of \u003cem\u003eAtGH9C2\u003c/em\u003e expression reduces cellulose crystallinity, accompanied by increases in both plant height and rosette diameter (Glass, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). While the CBM49 domain of the tomato β-1,4-glucanase Cel9C binds crystalline cellulose substrates in vitro, CBM49 is thought to contribute to substrate selection or modification rather than the direct hydrolysis of crystalline cellulose (Kundu \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trainotti, et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Urbanowicz, et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In a separate study, the CBM domain of a rice GH9C endoglucanase was found to undergo post-translational cleavage after the protein is targeted to the cell wall; this cleavage may activate the enzyme\u0026rsquo;s hydrolytic activity or alter its substrate specificity (Yoshida and Komae \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Membrane-associated GH9A β-1,4-glucanases that do not possess a CBM49 domain, are likely primarily involved in the assembly, repair or editing of cellulose microfibrils during plant cellulose biosynthesis. Among GH9A β-1,4-glucanases, KORRIGAN1 (KOR1) is the most well-studied (Molhoj, et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Nicol, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The β-1,4-glucanase activity of KOR1 is essential for the effective operation of the cellulose synthase complex (CSC); mutations in \u003cem\u003eKOR1\u003c/em\u003e disrupt the intracellular trafficking of CSC, reduce the cellulose polymerization rate, and cause abnormal deposition of crystalline cellulose (Mansoori, et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nicol, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Takahashi, et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Vain, et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Worden, et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). GH9B β-1,4-glucanases are believed to be secreted into the apoplast, where they can hydrolyze the amorphous regions of cellulose. These regions interact with other cell wall polymers, and their hydrolysis alters the mechanical properties of the cell wall, further promoting wall loosening or disassembly (Brummell, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Cosgrove \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; del Campillo \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; del Campillo, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Jara, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Karve, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lewis, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rose and Bennett \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Notably, a new function has recently been proposed for a specific clade of secreted GH9B β-1,4-glucanases: they may participate in cell-to-cell adhesion during grafting or in the interactions between parasitic plants and their hosts (Kurotani, et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Notaguchi, et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eGH9Bs\u003c/em\u003e have also been shown to be involved in plant\u0026ndash;pathogen or plant\u0026ndash;nematode interactions (Finiti, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Flors, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Goellner, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Tucker, et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wieczorek, et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Woo, et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo date, all known functions of the GH9 enzymes discussed above are associated with vegetative development. However, their involvement in reproductive development remains poorly understood, despite the fact that the cell wall undergoes substantial changes during both vegetative and reproductive growth. While promoter activity assays and transcriptome analyses have provided evidence that \u003cem\u003eGH9\u003c/em\u003e genes are transcribed in pollen, ovules and seeds, critical details of GH9 protein expression\u0026mdash;including their presence, spatial localization, and abundance in these reproductive tissues\u0026mdash;remain to be elucidated (del Campillo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; He, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xie, et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yung, et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In this study, we conducted a detailed tissue-specific analysis of GH9 family gene expression by visualizing the in vivo localization of their genomic green fluorescent protein (GFP)-fusion proteins. Using this approach, we identified candidate \u003cem\u003eGH9\u003c/em\u003e genes with potential involvement in reproductive processes, including pollen development, pollen tube growth and reception, embryo and endosperm development, and seed coat maturation. Additionally, we investigate the roles of two subsets of \u003cem\u003eGH9\u003c/em\u003e genes (highly expressed in pollen) in male reproductive development, and found that functional deficiency of \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, and \u003cem\u003eGH9A4\u003c/em\u003e led to a significant reduction in germination rate in in vitro assays. Thus, our results suggest that GH9 β-1,4-glucanases may play essential roles in plant reproductive development, which warrants further investigation in future studies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Col-0 (wild-type), mutants, and transgenic lines were cultivated in a greenhouse under a photoperiod of 16 h light and 8 h dark at 22\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhylogenetic and collinearity analysis\u003c/h3\u003e\n\u003cp\u003eAll \u003cem\u003eArabidopsis\u003c/em\u003e gene sequences were retrieved from TAIR (the \u003cem\u003eArabidopsis\u003c/em\u003e Information Resource; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenetic analysis of the \u003cem\u003eArabidopsis\u003c/em\u003e endo-β-1,4-glucanase gene family was performed using MEGA 11 software, employing the neighbor-joining (NJ) method, with 1000 bootstrap replicates (Tamura, et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Transcriptome data were obtained from the \u003cem\u003eArabidopsis\u003c/em\u003e RNA-seq Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plantrnadb.com/athrdb/\u003c/span\u003e\u003cspan address=\"https://plantrnadb.com/athrdb/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Zhang, et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and visualized with the ChiPlot tool (Xie, et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To determine the replication patterns of each GH9 gene within the \u003cem\u003eArabidopsis\u003c/em\u003e genome, MCScanX was employed to assess genomic collinearity, utilizing the default parameters (Wang, et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eConstructs for plant transformation\u003c/h3\u003e\n\u003cp\u003eFor the \u003cem\u003eGH9s\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eGH9s-GFP\u003c/em\u003e construct, the \u003cem\u003eGH9s\u003c/em\u003e promoter and the full-length \u003cem\u003eGH9s\u003c/em\u003e coding sequence (without the stop codon) were amplified from \u003cem\u003eArabidopsis\u003c/em\u003e genomic DNA using the primer pairs \u003cem\u003eGH9s-094-S/A\u003c/em\u003e, and then ligated into P094 (Wu, et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) upstream of GFP after XbalI/ EcoRI digestion. All primers used for cloning are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFor CRISPR/Cas9-mediated genome editing, four single guide RNAs (sgRNAs) were designed to target \u003cem\u003eGH9B5/7/A1\u003c/em\u003e and \u003cem\u003eGH9B9/10/11/12\u003c/em\u003e, respectively. Following established protocols, the four sgRNAs were first integrated into the entry vector pENTR-MSR (Xing, et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The resulting expression cassettes were then digested with restriction enzymes KpnⅠ/XbaⅠ and ligated into the binary vector pCAMBIA1300-pYAO-cas9, which had been pre-digested with KpnⅠ/SpeⅠ (Yan, et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). All recombinant constructs were verified by Sanger sequencing and subsequently used to transform \u003cem\u003eArabidopsis thaliana\u003c/em\u003e via the floral dip method (Clough and Bent \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhenotypic Analysis\u003c/h3\u003e\n\u003cp\u003eFor in vitro pollen germination assays, mature pollen grains were evenly spread on solid pollen germination medium and cultured at 22\u0026deg;C for 5 h. The medium was composed of the following components: 0.05% boric acid, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 mM KCl, 1 mM MgSO4, 10% sucrose and 1% agarose (PH was adjusted to 7.5). Pollen grain viability was evaluated using Alexander\u0026rsquo;s staining. The Alexander\u0026rsquo;s staining solution (100mL total volume) contained 10 mL 95% ethanol, 1 mL malachite green (1% solution in 95% ethanol), 25 mL glycerol, 5 mL acid fuchsin (1% solution in water), 0.5 mL orange G (1% solution in water), 4 mL glacial acetic acid, and 54.5 mL distilled water. For DAPI (4,6-diamidino-2-phenylindole) staining, pollen tubes were incubated in liquid germination medium supplemented with 2.5 \u0026micro;g/mL DAPI and 0.01% (vol/vol) Triton X-100. Incubation was performed in the dark for 5 minutes prior to visualization using fluorescence microscopy.\u003c/p\u003e\u003cp\u003eTo analyze pollen-tube guidance, hand-pollinated pistils were fixed in Carnoy\u0026rsquo;s Fixative (ethanol:acetic acid\u0026thinsp;=\u0026thinsp;3:1, v/v) for 12 h. After fixation, the pistils were rinsed three times with 0.1 M phosphate-buffered saline (PBS), and transferred into 5 M NaOH for overnight incubation to induce softening. Prior to staining, the pistils were rinsed three times with 0.1 M PBS, and then stained with 0.1% (wt/vol) aniline blue solution in the dark. To examine expression patterns of GH family genes, different tissues were observed under a confocal microscope (SP8 CLSM, Leica), using an excitation wavelength of 488 nm for green fluorescent protein.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePhylogenetic analysis and expression profiling of GH9 family genes in\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, there are currently 25 genes encoding β-1,4-glucanases, which are classified into three subgroups: 4 \u003cem\u003eGH9A\u003c/em\u003e, 18 \u003cem\u003eGH9B\u003c/em\u003e, and 3 \u003cem\u003eGH9C\u003c/em\u003e genes (Urbanowicz, et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Phylogenetic analysis confirmed that the protein sequences of these genes cluster into three distinct subfamilies, consistent with previous reports (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Libertini, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). To characterize the expression patterns of GH9 family genes, we extracted and summarized expression data from the \u003cem\u003eArabidopsis\u003c/em\u003e RNA-seq Database\u0026mdash;encompassing various tissues across multiple stages of plant development\u0026mdash;to construct a comprehensive expression profile of these 25 genes (Zhang, et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among all \u003cem\u003eGH9\u003c/em\u003e genes, five (\u003cem\u003eGH9A1\u003c/em\u003e, \u003cem\u003eGH9B1\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, \u003cem\u003eGH9B13\u003c/em\u003e, and \u003cem\u003eGH9C2\u003c/em\u003e) exhibited a ubiquitous expression pattern, with high transcript abundance in nearly all tissues and organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Another five genes (\u003cem\u003eGH9A4\u003c/em\u003e, \u003cem\u003eGH9B3\u003c/em\u003e, \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B6\u003c/em\u003e, and \u003cem\u003eGH9B11\u003c/em\u003e) shared a similar expression pattern, being predominantly expressed in pollen. Additionally, \u003cem\u003eGH9B8\u003c/em\u003e and \u003cem\u003eGH9C1\u003c/em\u003e showed relatively high expression levels in the endosperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results collectively demonstrate that \u003cem\u003eArabidopsis\u003c/em\u003e GH9 family genes display diverse expression profiles, implying their involvement in a broad range of plant developmental processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eExpression patterns of GH9-GFP fusion proteins in leaves and roots\u003c/h2\u003e\u003cp\u003eTo validate the reliability of publicly available expression data, we generated transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants expressing GH9-GFP fusion proteins under the control of their respective native promoters. Among these transgenic lines, GH9B7\u0026ndash;GFP signals were exclusively detectable in leaf epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, to date, no direct evidence supports the involvement of GH9 proteins in the structure or function of leaf stomata. However, in transgenic leaves harboring \u003cem\u003epGH9B1::GH9B1\u0026ndash;GFP\u003c/em\u003e, \u003cem\u003epGH9B7::GH9B7\u0026ndash;GFP\u003c/em\u003e, \u003cem\u003epGH9B12::GH9B12\u0026ndash;GFP\u003c/em\u003e, \u003cem\u003epGH9B14::GH9B14\u0026ndash;GFP\u003c/em\u003e, \u003cem\u003epGH9B15::GH9B15\u0026ndash;GFP\u003c/em\u003e constructs, green fluorescent signals localized to the plasma membrane or cell wall of stomatal cells, but not their cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This observation suggests that these GH9 proteins may indirectly affect stomatal function by contributing to the hydrolysis and modification of cell wall polysaccharides.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn a previous study, promoter\u0026ndash;reporter (GUS) assays and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that \u003cem\u003eGH9B4\u003c/em\u003e (also referred to as \u003cem\u003eCel5\u003c/em\u003e) was exclusively expressed in the root cap cells of both primary and lateral roots; this gene was further shown to mediate the sloughing of border cells from the root tip (del Campillo, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Karve, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consistent with this expression pattern, in transgenic plants harboring the \u003cem\u003epGH9B4::GH9B4\u0026ndash;GFP\u003c/em\u003e construct, GFP signals were exclusively detectable within the root cap (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, \u003cem\u003eGHB3\u003c/em\u003e\u0026mdash;the duplicate of \u003cem\u003eGH9B4\u003c/em\u003e, which is associated with lateral root induction\u0026mdash;shares a similar root expression pattern. Besides \u003cem\u003eGH9B3\u003c/em\u003e and \u003cem\u003eGH9B4, GH9B6\u003c/em\u003e was also identified as a novel root cap marker, as evidenced by green fluorescent signals specifically observed in root cap cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Collectively, these three genes may exhibit functional redundancy; thus, simultaneous inhibition of all three will be necessary to investigate the mechanism governing border cell release from the root cap.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe promoter of \u003cem\u003eGH9B1\u003c/em\u003e (also referred to as \u003cem\u003ecel1\u003c/em\u003e) has been reported to be active in root elongation zones, and our observations of GH9B1-GFP expression in roots are consistent with this finding (Shani, et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Among GH9B subfamily members, four additional members have also been shown to be expressed in roots. What is particularly interesting is the specific localization of GH9B8-GFP to vascular bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Additionally, \u003cem\u003eGH9C1\u003c/em\u003e and \u003cem\u003eGH9C2\u003c/em\u003e, both of which contain a CBM49 domain, exhibited distinct root expression patterns: \u003cem\u003eGH9C2\u003c/em\u003e was constitutively expressed, whereas \u003cem\u003eGH9C1\u003c/em\u003e showed exclusive localization to root hairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, the expression domain of GH9C1-GFP was narrower than that previously observed in GUS staining assays, yet this restricted expression was sufficient to support the phenotype of markedly reduced root hair density in \u003cem\u003egh9c1\u003c/em\u003e mutants (del Campillo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile the \u003cem\u003eGH9A1\u003c/em\u003e gene was expressed throughout the root, GH9A2-GFP, driven by its native promoter, was exclusively detected in the root differentiation zone, including root hairs (Molhoj, et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In contrast to the subcellular localization of GH9B and GH9C subfamily members, the GFP signals of GH9A1 and GH9A2 predominantly accumulated in intracellular compartments, mirroring intracellular punctate pattern previously observed for GH9A1 via both immunofluorescence assays and GFP\u0026ndash;GH9A1 fusion protein analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (Robert, et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExpression patterns of GH9-GFP fusion proteins in pollen\u003c/h3\u003e\n\u003cp\u003eTo identify the potential roles of GH9 family genes in plant reproductive processes, we first analyzed the expression patterns and subcellular localization of GH9-GFP fusion proteins during male reproductive development. Based on transcriptomic data, the endoglucanases potentially involved in the cell wall remodeling during pollen development and pollen tube growth are GH9A4, GH9B3, GH9B5, GH9B6, and GH9B11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these genes, only the \u003cem\u003eGH9A4\u003c/em\u003e promoter has been demonstrated to drive pollen specific GUS activity (Xie, et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In our study, we identified up to 10 \u003cem\u003eGH9\u003c/em\u003e gens expressed in mature pollen, including 2 from the GH9A subfamily, 7 from the GH9B subfamily, and 1 from the GH9C subfamily (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, these \u003cem\u003eGH9\u003c/em\u003e genes exhibited differences in both expression levels and subcellular localization. In mature pollen, GH9A1-GFP or GH9A4-GFP exhibited strong intracellular signals with a filamentous pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among duplicated gene pairs, GH9B3/GH9B4 showed weak, punctate fluorescence, whereas GH9B5/GH9B7 displayed relatively strong, continuous fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Weak plaque-like fluorescence was detected in pollen expressing GH9B9-GFP or GH9C2-GFP, while filamentous fluorescence was observed in pollen expressing GH9B11-GFP or GH9B18-GFP. GH9B and GH9C subfamily members do not appear to be secreted in mature pollen\u0026mdash;despite the general consensus that they are secreted into the apoplast and prior findings indicating their likely cell wall localization in vegetative tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Thus, the mechanisms governing the subcellular dynamics of GH9 β-1,4-glucanases across different tissues and developmental stages are complex yet intriguing, and warrant further investigation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe pollen tube, a fast tip-growing cell tasked with delivering two sperm cells to the ovule, depends on extensive cell wall deposition to drive its rapid elongation and on tight regulation of cell wall remodeling to modify mechanical properties (Chebli, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cheung and Wu \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dresselhaus and Franklin-Tong \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hafidh and Honys \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hepler, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To investigate the subcellular location of GH9 proteins in pollen tubes, we performed in vitro pollen germination assays to observe their distribution. Five \u003cem\u003eGH9\u003c/em\u003e genes highly expressed in pollen were selected for analysis. In pollen tubes expressing GH9A1-GFP, GFP signals were concentrated in the vacuole, although punctate fluorescence was also detected in the cytoplasm at the pollen tube tip (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In plasmolyzed pollen tubes, GH9A4-GFP, GH9B5-GFP and GH9B7-GFP signals were clearly visible in the pollen tube cell wall, whereas GH9B11-GFP signals were not, indicating that GH9A4, GH9B5 and GH9B7, but not GH9B11 can be secreted extracellularly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Beyond the aforementioned \u003cem\u003eGH9\u003c/em\u003e genes expressed in pollen grains, GH9B6-GFP signals were specifically detected in sperm cells, suggesting a potential role for \u003cem\u003eGH9B6\u003c/em\u003e in either male gametophyte development or the subsequent double fertilization process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003eExpression patterns of GH9-GFP fusion proteins in ovules and seeds\u003c/h3\u003e\n\u003cp\u003eNext, we analyzed the expression patterns of the GH9 family genes in female reproductive tissues at various developmental stages: mature ovules, seeds at 1\u0026ndash;2 days after pollination (DAP), and seeds at 5\u0026ndash;7 DAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Early reports have shown that \u003cem\u003eGH9B2\u003c/em\u003e transcripts were predominantly expressed in the developing septum and ovule primordia of young carpels (Yung, et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In ovules harboring the GH9B2-GFP construct, we also observed strong fluorescence signals in the integuments. In addition to \u003cem\u003eGH9B2\u003c/em\u003e, \u003cem\u003eGH9A1\u003c/em\u003e, \u003cem\u003eGH9B1\u003c/em\u003e, \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, \u003cem\u003eGH9B13\u003c/em\u003e, \u003cem\u003eGH9B18\u003c/em\u003e, and \u003cem\u003eGH9C2\u003c/em\u003e shared a similar expression pattern, with signals consistently detectable throughout the seed coat development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). GH9B8-GFP signals were visible in the integuments both before fertilization and after fertilization but disappeared in the seed coat by 5\u0026ndash;7 DAP. \u003cem\u003eGH9A2\u003c/em\u003e, \u003cem\u003eGH9B9\u003c/em\u003e and \u003cem\u003eGH9B10\u003c/em\u003e exhibited a narrow expression pattern during seed coat development, as GFP signals were only observed in the seed coat at 5\u0026ndash;7 DAP. Among these three genes, the expression of \u003cem\u003eGH9B9\u003c/em\u003e and \u003cem\u003eGH9B10\u003c/em\u003e was restricted to the inner integument (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe filiform apparatus (FA)\u0026mdash;located at the micropylar end of synergid cells\u0026mdash;is an active communication hub defined by intricate plasma membrane invaginations and thick cell walls. It is presumed to play pivotal roles in secreting pollen-attractant peptides and mediating male\u0026ndash;female interactions during sexual reproduction (Johnson, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Susaki, et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We found that green-fluorescent signals of seven GH9B-GFP fusion proteins (GH9B1, GH9B5, GH9B7, GH9B8, GH9B13, GH9B16 and GH9B18) localized to FA of the synergid cells but not their cytoplasm, suggesting that these GH9 proteins are continuously released into the micropylar region by these gland-like synergid cells prior to fertilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor female gametes, the duplicated gene pair \u003cem\u003eGH9B5\u003c/em\u003e/\u003cem\u003eGH9B7\u003c/em\u003e was expressed in both egg cells and central cells, but not in zygotes or the primary endosperm, suggesting their potential roles in cell wall formation of egg cells and central cells. After fertilization, only four \u003cem\u003eGH9\u003c/em\u003e genes were found to be expressed during embryonic development: \u003cem\u003eGH9A2\u003c/em\u003e in the pro-embryo, \u003cem\u003eGH9B7\u003c/em\u003e and \u003cem\u003eGH9B8\u003c/em\u003e in the globular embryo, and \u003cem\u003eGH9B1\u003c/em\u003e in the torpedo embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). While both \u003cem\u003eGH9B8\u003c/em\u003e and \u003cem\u003eGH9B12\u003c/em\u003e are expressed in central cells and endosperm, they exhibit distinct secretory characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). GH9B8-GFP signals is mainly localized to the cytoplasm, whereas GH9B12-GFP showed secretion from central cells, as evidenced by the green-fluorescent signals in the cell walls of the inner integuments, particularly near the micropyle or at the chalazal end.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGH9B5\u003c/b\u003e, \u003cb\u003eGH9B7\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eGH9A4\u003c/b\u003e \u003cb\u003eare involved in pollen germination in vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further validate the involvement of β-1,4-glucanases in sexual reproduction, we focused on two groups of \u003cem\u003eGH9\u003c/em\u003e genes highly expressed in pollen to investigate their functions in male gametophyte development. The first group includes \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, and \u003cem\u003eGH9A4\u003c/em\u003e, all highly expressed in mature pollen and capable of being secreted to the pollen tube cell wall. The second group consists of \u003cem\u003eGH9B9\u003c/em\u003e, \u003cem\u003eGH9B10\u003c/em\u003e, \u003cem\u003eGH9B11\u003c/em\u003e, and \u003cem\u003eGH9B12\u003c/em\u003e, which are tandemly linked on the chromosome; among these, \u003cem\u003eGH9B9\u003c/em\u003e and \u003cem\u003eGH9B11\u003c/em\u003e are expressed in pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Using the CRISPR/Cas9 system, we generated genome-edited \u003cem\u003egh9b5/7/a4-1\u003c/em\u003e, \u003cem\u003egh9b5/7/a4-2\u003c/em\u003e, \u003cem\u003egh9b9/10/11/12\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/em\u003e and \u003cem\u003egh9b9/10/11/12\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/em\u003e mutants, in which all alleles harbor frameshift mutations accompanied by premature stop codons (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Although pollen development and viability were normal in all mutants, \u003cem\u003egh9b5/7/a4\u003c/em\u003e mutants showed a significantly reduced in vitro pollen germination rate, whereas WT and \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutants not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we examined whether pollen tube growth was normal in \u003cem\u003egh9b5/7/a4\u003c/em\u003e and \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutants in vivo. At 3 hours after pollination (HAP), all pollen tubes reached the upper part of the pistil, with no differences in length among the genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and I). By 8 HAP, the pollen tubes of both mutants also reached the bottom of the pistil (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and J). This observation is further supported by the finding that neither \u003cem\u003egh9b5/7/a4\u003c/em\u003e nor \u003cem\u003egh9b9/10/11/12\u003c/em\u003e mutants affect plant fertility (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and L). Thus, these results demonstrate that \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, and \u003cem\u003eGH9A4\u003c/em\u003e are critical for pollen germination, though additional research is needed to determine which of these three genes exerts a dominant role.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe plant cell wall is a dynamic composite structure with diverse functions, including providing a framework to support the cell structure, compartmentalizing specialized cells, protecting against infection by pathogens and mediating the communication between cells to regulate plant development (Cosgrove \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Given these essential roles, plants have evolved β-1,4-glucanases to degrade cellulose and other polysaccharides containing 1,4-glycosidic bonds to remodel and disassemble the wall during cell growth (Cantarel, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; del Campillo \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Lopez-Casado, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Numerous studies have reported that GH9 proteins are implicated in diverse physiological processes in higher plants, such as root hair emergence, endosperm breakdown, fruit ripening, grafting, plant parasitism, and nematode defense (del Campillo, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; del Campillo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Goellner, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Jara, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kurotani, et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nicol, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Notaguchi, et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Trainotti, et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Tucker, et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wieczorek, et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Woo, et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, the GH9 family comprises 25 members; however, to date, only a few of them have been well characterized regarding their in vivo functions. In the present study, we established detailed expression profiles of GH9 family genes by conducting in vivo observations of their GFP-fusion proteins. These observations reveal that \u003cem\u003eGH9\u003c/em\u003e genes are expressed not only in vegetative organs but also enriched in reproductive tissues. This work provides a valuable reference map for understanding the spatial distribution of GH9 β-1,4-glucanases and their dynamic behavior during plant development, thereby offering clear cues for further investigating their functional roles in reproductive processes.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePotential roles of GH9 family genes in vegetable development\u003c/h2\u003e\u003cp\u003eVegetative development, which involves the growth and maturation of non-reproductive organs such as roots, stems and leaves, relies strongly on dynamic cell wall remodeling. As key regulators of cell wall metabolism, GH9 β-1,4-glucanases contribute to multiple aspects of vegetative organ biology, including shaping their structure, optimizing their function, and enhancing their stress resilience (Perrot, et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Roots are critical for water and nutrient uptake, plant anchorage and symbiotic interactions. Previous studies have indicated that \u003cem\u003eGH9\u003c/em\u003e genes are involved in nearly all stages of root development\u0026mdash;for instance, \u003cem\u003eGH9B1\u003c/em\u003e in primary root elongation, \u003cem\u003eGH9C1\u003c/em\u003e in root hair formation, \u003cem\u003eGH9B3\u003c/em\u003e in lateral root emergence, and \u003cem\u003eGH9B3\u003c/em\u003e/\u003cem\u003eGH9B4\u003c/em\u003e in the sloughing of border cells from the root tip (del Campillo, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; del Campillo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lewis, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Karve, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tsabary, et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Beyond the genes discussed above, our study identified eight additional \u003cem\u003eGH9\u003c/em\u003e genes, with expression localized to the root cap, elongation zone, vascular bundles, or root hairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Future studies will be needed to clarify whether these genes act redundantly with known \u003cem\u003eGH9s\u003c/em\u003e or carry out novel functions in root development.\u003c/p\u003e\u003cp\u003eAnother interesting observation is that all five GH9B-GFP fusion proteins expressed in leaves localize to the stomatal cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Stomata are small pores on the leaf surface that play a critical role in regulating gas exchange and water loss, and their shape and function are physically constrained by the stomatal cell wall (Lawson and Matthews \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cellulose microfibrils, long been recognized as the load-bearing components of cell walls, are central to maintaining this structural constraint (Cosgrove \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gibson \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Markov, et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Notably, the cellulose-deficient mutants \u003cem\u003ecesa3\u003c/em\u003e\u003csup\u003e\u003cem\u003eje5\u003c/em\u003e\u003c/sup\u003e exhibits aberrant cellulose microfibril reorientation, alongside abnormalities in stomatal apertures and guard cell lengths during stomatal movement (Rui and Anderson \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Whether cellulose remodeling is associated with stomatal development and function remains to be tested. Here, these five \u003cem\u003eGH9B\u003c/em\u003e genes\u0026mdash;with their potential roles in cell wall remodeling\u0026mdash;represent an excellent entry point to address this unresolved question.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePotential roles of GH9 family genes in plant reproduction from the male side\u003c/h2\u003e\u003cp\u003eTo accommodate diverse cell shapes, plant cell walls exhibit varied forms, structures, and compositions, and are highly dynamic throughout the entire plant life cycle, whether during vegetative or reproductive development (Cosgrove \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). How the molecular mechanisms controlling cell wall properties are regulated during plant reproduction is a fundamental question in plant biology. Studies on the expression and localization of GH9 family genes not only clarify the spatiotemporal patterns of these genes but also provide insights into the functions of β-1,4-glucanases in plant reproduction, from both male and female perspectives.\u003c/p\u003e\u003cp\u003eIn mature pollen, ten \u003cem\u003eGH9\u003c/em\u003e genes were found to be expressed, with distinct expression levels and subcellular localizations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, functional deficiency of \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, and \u003cem\u003eGH9A4\u003c/em\u003e led to a significantly reduced germination rate in in vitro pollen germination assays, suggesting their potential roles in cell wall degradation or remodeling during the initiation of pollen germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). Regarding whether other GH family members exhibit redundant functions in pollen germination, this possibility cannot be ruled out, which might also explain why no obvious phenotypes were observed in vivo.\u003c/p\u003e\u003cp\u003ePollen tube growth is a critical step toward fertilization in flowering plants. During pollen tube growth, cell wall dynamics balance cell expansion and turgor resistance, with biosynthesis and modification of the cell wall being crucial for maintaining pollen tube integrity (Chebli, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cheung and Wu \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dresselhaus and Franklin-Tong \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hafidh and Honys \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hepler, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Despite the low cellulose content in the pollen tube wall, crosslinking between cellulose, pectin, and callose endows the pollen tube with distinct mechanical properties necessary for its growth (Chebli, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Plant cellulose biosynthesis occurs at the plasma membrane via large cellulose synthase (CESA) complexes. However, in the growing tips of pollen tubes, cellulose synthase-like D (CSLD) proteins appear to localize to the apical plasma membrane, while CESAs are mostly confined to internal compartments\u0026mdash;opposite to their distribution in somatic cells (Park, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Pollen tubes lacking CSLD1 and/or CSLD4 exhibit reduced cellulose deposition, compromising the genetic transmission of the male gametophyte (Bernal, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, loss of function of two pollen-specific glycerophosphodiester phosphodiesterase-like genes in \u003cem\u003eArabidopsis\u003c/em\u003e causes a severe reduction in cellulose deposition at the mutant pollen tube tip walls, ultimately resulting in pollen tube bursting (Wang, et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, to date, no direct evidence has been reported for cell wall remodeling mediated by GH9 β-1,4-glucanases during pollen tube growth. The enzymes potentially involved may include not only the ten pollen-expressed \u003cem\u003eGH9\u003c/em\u003e genes identified in this study, but also those up-regulated in pollen tubes or enriched in female tissues that the pollen tube encounters during its journey.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePotential roles of GH9 family genes in plant reproduction from the female side\u003c/h2\u003e\u003cp\u003eIn addition to the roles mentioned above, GH9 family members also exhibit potential functions in the female reproductive development. In most flowering plants, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the female gametophyte\u0026mdash;also known as the embryo sac\u0026mdash;comprises highly differentiated cells: two female gametes (the haploid egg cell and homodiploid central cell), two synergid cells (which function in pollen tube attraction and reception), and three antipodal cells (with poorly characterized functions) (Hater, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yadegari and Drews \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yang, et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These cells not only have distinct morphologies but also exhibit ultrastructural differences in their cell walls. Moreover, the chalazal region of the egg cell, the target site for sperm entry and fusion during fertilization, exhibits thin furrows between thick cell wall structures (Tekleyohans, et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Future work will be necessary to elucidate two key questions: first, whether these indentations (i.e., the thin furrows) correspond to the actual sites of sperm entry; and second, whether the cell wall apposition pattern is regulated by GH9 proteins expressed in egg cells or central cells, that were identified in our study.\u003c/p\u003e\u003cp\u003eAlthough the synergid cell itself is not fertilized, it mediates extensive communication between female and male gametophytes prior to double fertilization. The filiform apparatus, located at the micropylar end of synergid cells, is enriched in both pollen tube attractant peptides and various pollen tube reception factors (Capron, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Escobar-Restrepo, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Galindo-Trigo, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; M\u0026aacute;rton, et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Meng, et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Okuda, et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Takeuchi and Higashiyama \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tsukamoto, et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A recent study has revealed that the FA exhibits a well characterized sponge-like structure, consisting of intricate plasma membrane invaginations and thick cell walls; notably, the surface area of the FA is 3.7-fold larger than the estimated cell wall area excluding invaginations (Susaki, et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, two key questions remain to be addressed in future studies: first, how is this widely conserved synergid cell wall structure formed? Second, are the seven \u003cem\u003eGH9Bs\u003c/em\u003e expressed in the FA involved in this formation process?\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePotential roles of GH9 family genes in plant reproduction during seed development\u003c/h2\u003e\u003cp\u003eSeed development is a complex, highly regulated biological process in plants that transforms a fertilized ovule into a mature seed, which typically consists of three major structures: the embryo, the endosperm, and the seed coat (Figueiredo and Kohler \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kesavan, et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lafon-Placette and Kohler \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Orozco-Arroyo, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Seed development relies on dynamic changes in cell wall composition, structure, and remodeling to support key developmental processes, including embryo growth and differentiation, endosperm cellularization, as well as seed coat formation. The cell wall is not merely a rigid \"support structure\", but also acts as a regulatory interface that coordinates growth, nutrient transport, and stress resistance across these three core seed components (Huang, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough several \u003cem\u003eGH9\u003c/em\u003e genes were identified in the embryo and endosperm in this study, 12 out of 25 GH9 family members were found to be expressed in the seed coat (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These seed coat-expressed \u003cem\u003eGH9\u003c/em\u003e genes may remodel and disassemble the wall during cell growth, by degrading cellulose and other polysaccharides, to help the seed coat adapt to the turgor pressure generated by internal embryo and endosperm growth. However, this functional hypothesis requires extensive experimental validation in future studies. The integuments connect to the vascular tissues of the maternal funiculus at the chalazal region, a connection that ensures the nutritional supply required for seed development. A recent study has reported a fertilization-dependent phloem end gate at the chalazal end of the ovule, which regulates nutrient transport into the developing seed (Liu, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Before fertilization, this gate is blocked by callose deposition; after fertilization, specifically following fertilization of the central cell, this callose block is removed, a process controlled by genes encoding β-1,3-glucanases (callose-degrading enzymes) (Liu, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present study, GH9B8-GFP was detected in the vascular bundles of the funicle. Of particular note is the localization of GH9A2-GFP in integument cells at the funicle attachment site, as well as the localization of GH9B11-GFP in the chalazal zone of the endosperm (CZE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A key question for future investigation is whether these \u003cem\u003eGH9\u003c/em\u003e genes could also form a \"cellulase gate\" at the chalazal end of the ovule, analogous to the previously reported callose-based phloem end gate. If this hypothesis is validated, it could potentially offer a widely applicable strategy in angiosperms to increase seed size in the future.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study delved into expression patterns and putative roles of the GH9 family gene in sexual reproduction. By visualizing the in vivo localization of GH9-GFP fusion proteins, comprehensive expression profile analysis reveals that GH9 family genes are expressed not only in vegetative organs but also enriched in reproductive tissues, including mature pollen, unfertilized ovules and seeds at different developmental stages. Moreover, we found that functional deficiency of \u003cem\u003eGH9B5\u003c/em\u003e, \u003cem\u003eGH9B7\u003c/em\u003e, and \u003cem\u003eGH9A4\u003c/em\u003e led to a significant reduction in pollen germination rate in in vitro assays, implying their critical roles in pollen germination. Taken together, this study constructs a comprehensive expression profile of GH9 family genes and highlights their potential roles in plant reproduction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eStudy design, project management: WW, HXX. Experimental Design: WW, HXX. Data Collection: HXX, XDG. Sample processing: HXX, XDG. Data Analysis: WW, HXX, XDG. Draft manuscript: WW, HXX. All Authors read, edited, and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was supported by National Natural Science Foundation of China grants (32200702 to HXX.; 31800264 to WW) and by the High-level Talent Introduction Project of Central China Normal University to WW.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll data is available in the main text or the supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBernal AJ, Yoo CM, Mutwil M, Jensen JK, Hou G, Blaukopf C, Sorensen I, Blancaflor EB, Scheller HV, Willats WGT (2008) Functional analysis of the cellulose synthase-like genes \u003cem\u003eCSLD1\u003c/em\u003e, \u003cem\u003eCSLD2\u003c/em\u003e, and \u003cem\u003eCSLD4\u003c/em\u003e in tip-growing \u003cem\u003eArabidopsis\u003c/em\u003e cells. 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Nat Commun 7:11656\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong S, Liu ML, Wang ZJ, Huang QP, Hou SY, Xu YC, Ge ZX, Song ZH, Huang JY, Qiu XY, Shi YH, Xiao JY, Liu P, Guo YL, Dong J, Dresselhaus T, Gu HY, Qu LJ (2019) Cysteine-rich peptides promote interspecific genetic isolation in \u003cem\u003eArabidopsis\u003c/em\u003e. Science 364:eaau9564\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endo-β-1,4-glucanases, expression profile, sexual reproduction, pollen, Arabidopsis thaliana","lastPublishedDoi":"10.21203/rs.3.rs-7805288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7805288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Plant endoglucanases of glycosyl hydrolase family 9 (GH9), which cleave 1,4-β-glucosidic bonds in glycan chains, have been shown to play pivotal roles in cellulose degradation, as well as in the relaxation or construction of cell walls during diverse plant growth and developmental processes. However, their specific functions in plant sexual reproduction remain unclear. In this study, we systematically analyzed expression profiles of the GH9 gene family using in vivo observations of GFP-fusion proteins. Our results revealed that numerous GH9 genes were expressed across both vegetative and reproductive tissues: 5 in stomata, 12 in roots, 11 in mature pollen and 15 in mature ovules or seeds. Further analysis of reproductive tissues uncovered distinct expression specificities: GH9A4 as pollen-specific, GH9B6 as sperm cell-specific, GH9B12 as central cell- and endosperm cell-specific, GH9B11 as enriched in pollen and chalazal endosperm, and GH9B8 as vascular tissue-preferential. Additionally, GFP signals of up to 7 GH9 members were detected in the filiform apparatus, while 12 GH9 members showed signals in integuments and seed coats—hinting at diverse GH9 functions in reproduction. Furthermore, we conducted gene editing and phenotypic analysis on two subsets of GH9 genes highly expressed in pollen. While in vivo pollen germination and growth were unaffected, in vitro germination rates decreased significantly when GH9B5, GH9B7, and GH9A4 functionally deficient. Taken together, this study highlights the potential roles of the GH9 family in pollen germination and establishes a foundation for future functional analyses aimed at elucidating the putative roles of the GH9 family in plant reproduction.","manuscriptTitle":"Endo-β-1,4-glucanases in Arabidopsis thaliana: expression and putative roles in sexual reproduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 19:30:51","doi":"10.21203/rs.3.rs-7805288/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f33d89bc-9c8a-4742-bfd1-acc6fb771859","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T16:32:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 19:30:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7805288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7805288","identity":"rs-7805288","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}