{"paper_id":"4829c9d4-3f9a-49d3-a714-3f77e533cc3d","body_text":"Mitochondrial GPATs protect plants from water stress by impacting cuticle and secondary cell wall development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial GPATs protect plants from water stress by impacting cuticle and secondary cell wall development Qianru Jia, Yang Bai, Hongmei Zhang, Wei Zhang, Qiong Wang, Xiaoqing Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6445935/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Trichomes and cuticles are critical epidermal adaptations that serve protective roles in plants. The cuticle functions as a barrier, allowing for controlled interactions between the plant and its environment. Cutin synthesis is crucial for plants to withstand various external stresses. In this study, we report on the Arabidopsis mutant gpat1 gpat2 , which exhibits a highly permeable cuticle and defects in trichome development. Mutation of GPAT1 and GPAT2 resulted in a reduction of cutin monomer. In gpat1 gpat2 , the structure of the cuticular layer of the cell wall is notably altered. Additionally, GPAT1 and GPAT2 are found to negatively regulate the synthesis of lignin and cellulose, which are related to secondary cell wall (SCW) formation. The dysfunction of GPAT1 and GPAT2 disrupted the water balance of the plant. Our findings reveal a network where mitochondrial GPAT1 and GPAT2 play roles in maintaining water balance by participating in both Arabidopsis cutin synthesis and SCW formation. Mt-GPAT cutin trichome secondary cell wall water balance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message Mt-GPAT1/2 are involved in cutin biosynthesis and negatively regulate SCW formation by modulating the lignin and cellulose synthesis genes. Alterations in cuticle and SCW lead to plant water imbalance. Introduction The cuticle is composed of cutin, wax, and various polysaccharide molecules that penetrate the epidermal cell walls, forming a diffusion barrier covering almost all aerial surfaces of higher plants. This structure establishes a critical interface between the plant and its environment (Jung et al. 2006 ;Duo et al. 2024 ). The cuticle not only plays a pivotal role in plant processes, such as organogenesis and seed viability, but also provides protection against diverse stresses, including transpiration, elevated temperatures, ultraviolet radiation, and herbivory (Ingram and Nawrath, 2017 ༛Camoirano et al. 2020 ; González-Valenzuela et al. 2023 ; Wang et al. 2024a ). Trichomes, hair-like organs deriving from differentiation of the protoderm that extend from the surface of the epidermis (Donnelly et al. 1999 ). Trichomes fulfill diverse biological functions, including defense against herbivory, UV irradiation, and desiccation, and exhibit specific structural diversity in density and morphology to support these roles (Han et al., 2022 ). Furthermore, trichomes may undergo physiological adaptations to synthesize specialized metabolites or contribute to detoxification by sequestering toxic compounds (Traw and Bergelson, 2003 ; Matías-Hernández et al. 2016 ; Hülskamp et al. 2019; Zhan et al. 2024 ). In recent years, considerable research has been conducted to unravel the mechanisms of cutin biosynthesis, such as the ABCG family (Bird et al. 2007 ; Elejalde-Palmett et al. 2021 ; Fu et al. 2024 ) and cytochrome P450 enzymes (Wellesen et al. 2001 ; Li-Beisson et al. 2009 ; Huang et al. 2024 ). The Glycerol-3-Phosphate Acyltransferase (GPAT) family also plays a significant role in cutin biosynthesis. Based on subcellular localization, GPATs can be classified into mitochondrial (Mt), endoplasmic reticulum (ER), and chloroplast types (Jayawardhane et al. 2018 ). The Arabidopsis GPAT family consists of 10 members, among which GPAT1-3 belong to the mitochondrial type, GPAT4-8 are categorized as endoplasmic reticulum types, while GPAT9 and ATS1 are classified as plastid types (Jayawardhane et al. 2018 ). Several ER-GPATs from different plants are known to be involved in cutin synthesis across multiple organs, including AtGPAT4/6/8 (Li et al. 2007 ; Yang et al. 2012 ; Jayawardhane et al. 2018 ), BnGPAT4 (Chen et al. 2011 ), OsGPAT3 (Men et al. 2017 ), and SlGPAT6 (Petit et al. 2016 ; Jayawardhane et al. 2018 ). Furthermore, AtGPAT4/5/6/7/8 are involved in suberin synthesis, regulating plant salt tolerance and mechanical damage repair (Beisson et al. 2007 ; Li-Beisson et al. 2009 ; Gully et al. 2024 ). However, it has not yet been reported whether Mt-GPAT is involved in cutin synthesis. The secondary cell wall (SCW) is located between the primary cell wall and the plasma membrane and is composed of lignin, cellulose, and hemicelluloses (Wang et al. 2023 ). The formation network of the SCW is extremely complex and subject to multi-faceted regulation. Tier1 and Tier2 transcription factors (TFs) contain many MYB TFs, such as MYB46 and MYB83 in Tier2 TFs and MYB20, MYB42, MYB43, and MYB58 in Tier1 TFs (Ko et al., 2014 ; Geng et al., 2020 ) and NAC TFs, NST1, NST2, SND1, VND6, and VND7 in Tier3 TFs were characterized as master regulators of SCW biosynthesis (Kubo et al., 2005 ; Wang et al., 2011 ). Ubiquitination also plays a role in SCW formation (Wang et al. 2023 ). E3 Cullin Ring Ligase subunit DDB1a (Beris et al. 2016 ), cotton GhHUB2 (Feng et al. 2018 ), and OsFBK1 (Borah and Khurana, 2018 ), as components of an SCF E3 ligase complex, have all been reported to regulate SCW either directly or indirectly. To date, the role of GPAT in the regulation of SCW development remains unexamined. In this study, we report that the Arabidopsis double mutant gpat1 gpat2 exhibits higher permeability of water than the wild type (WT), suggesting that the cuticle layer in gpat1 gpat2 is disrupted. Our findings confirm that Mt-GPAT1 and Mt-GPAT2 are involved in plant cutin synthesis. The structure of the cutin layer is compromised, which hinders the development of trichomes. Further research has demonstrated that GPAT1 and GPAT2 regulate the formation of the SCW by negatively regulating genes associated with lignin and cellulose biosynthesis. Our findings underscore the importance of Mt-GPAT1 and Mt-GPAT2 in maintaining cellular water balance. Materials and Methods Plant materials and growth conditions The mutants gpat1 (Salk_052353) and gpat2 (Salk_064530C) were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. The double mutant gpat1 gpat2 , along with the complementary lines GPAT1:: gpat1 gpat2 and GPAT2:: gpat1 gpat2 , were previously characterized (Jia et al. 2022 ). Seeds were germinated on half-strength Murashige and Skoog (MS) medium and subsequently transferred to an illuminated incubator with a light intensity of 160 µmol m –2 s –1 , following a day/night cycle of 14/10 hours at 23°C, after a two-day vernalization period. Afterward, seedlings were transplanted into soil under the same conditions for continued cultivation. Toluidine Blue, Fluorol Yellow 088, phloroglucinol and zinc chloroiodide staining Six-day-old etiolated seedlings were stained with Toluidine Blue (TB) solution (0.05% (w/v) TB + 0.4% (v/v) Tween-20) for one minute, followed by rinsing under running tap water until no residual dye was visible in the rinse water. The samples were photographed using a stereomicroscope (Leica M165FC, Leica). Equal-weight samples were then immersed in 75% ethanol and extracted for two hours; the optical density was measured using a microplate reader (TECAN) at a wavelength of 626 nm. For Fluorol Yellow (FY) 088 (Macklin, China) staining, six-day-old seedlings were immersed in 0.01% (w/v, lactic acid) FY 088 for five minutes. The cuticle layer was then observed using a confocal laser scanning microscope with an excitation wavelength of 488 nm and emission wavelengths ranging from 500 to 550 nm (Zhang et al. 2024 ). For phloroglucinol and zinc chloroiodide staining, we utilized lignin staining solution (phloroglucinol method) (Macklin, China) and cellulose staining solution (Zinc chloroiodide method) (Yuanye, China). Six-day-old seedlings were immersed in the lignin acidification solution for ten minutes, after which an equal amount of phloroglucinol staining solution was added dropwise to the acidified material, and the mixture was allowed to sit for ten minutes. Alternatively, samples were immersed in the cellulose staining solution for five minutes. The samples were then observed using a stereomicroscope. Trichome observation Six-day-old seedlings, flowers and stems were imaged under an Ultra depth stereomicroscope (Leica DVM6, Leica). Electron microscopy techniques The leaf waxy crystal was observed using a freezing scanning electron microscope (SEM) (HITACHI S-3000, Japan). Twenty-day-old rosette leaves of WT and gpat1 gpat2 were adhered to conductive tape on the sample stage. The sample stages were then transferred to the SEM sample chamber, where they were cooled with liquid nitrogen for observation. The cold stage temperature was maintained at -140 ℃, and the acceleration voltage was set to 5 kV. The cotyledon cuticle layer was examined using a transmission electron microscope (TEM) (HITACHI HT7800, Japan). Specifically, cotyledons of WT and gpat1 gpat2 were fixed in a 2.5% (w/v) glutaraldehyde solution for more than two hours in 0.2 M PBS at 4°C. The samples were then washed three times with 0.1 M PBS and dehydrated through a graded series of ethanol. Following this, cotyledons were embedded in epoxy resin and subjected to ultra-thin slicing. Finally, the samples were placed on a copper mesh and stained with uranyl acetate and lead citrate before being observed under the TEM at 80.0 kV. Cutin monomer analysis Six-day-old seedlings were collected for the analysis of cutin monomers. The protocol used for extraction of cutin monomer followed a previous study with some modifications (Elejalde-Palmett et al. 2021 ). Samples were incubated in 10 mL preheated isopropanol /0.01% butylated hydroxytoluene (BHT) for 15 min at 85℃ and then washes twice in methanol:chloroform (V:V = 1:2) by 24 h. Samples were then dried under a gentle stream of N 2 and dried in a vacuum desiccator for one week. 25 µg ω-pentadecalactone and methyl heptadecanoate, 0.9 mL methyl acetate, 1.5 mL sodium methoxide and 3.6 mL methanol were added to the samples in turn and then incubated at 60℃ for 2 h. 3.5 mL dichloromethane, 0.7 mL glacial acetic acid and 1 mL 0.9% NaCl (w/v) were added to each sample and subsequently vortexed for 20 s. After centrifugation (1500 g for 2 min), the organic phase was collected, washed with 2 mL of 0.9% NaCl, and dried over sodium sulfate. The organic phase was then recovered and concentrated under a stream of N 2 . Monomers were derivatized by acetylation using 100 µL of anhydrous pyridine and acetic anhydride at 60℃ for 2h. A gas chromatograph coupled to a mass spectrometer (Agilent 7890A-5975CMS) with a DB-5 column (30 m×0.25 mm×0.25 µm, Agilent) was used to detect the cutin monomer. Column temperature program: 80℃, raised by 15℃/min to 200℃ and 2℃/min to 300℃ and held for 10 min. A total of five biological replicates were used for each sample. Wax analysis One month rosette leaves were collected for wax analysis. The protocol used for the extraction of wax monomer was based on a previous study with minor modifications (Elejalde-Palmett et al. 2021 ). Leaves were cut into small segments and then extracted twice using 10 mL of chloroform for 1 min. Samples were dried by N 2 and then derivatized with BFTSA/pyridine (1:1) at 100℃ for 30 min. Samples were resuspended with an appropriate volume of chloroform and detected by a gas chromatograph coupled to a mass spectrometer (Agilent 7890A-5975CMS) on a DB-5 column (30 m×0.25 mm×0.25 µm, Agilent). Column temperature program: 80℃ for 2 min, raised by 40℃/min to 200℃ and held for 2 min, raised by 10℃/min to 270℃ and held for 2 min, then raised by 2℃/min to 320℃ and held for 30 min. A total of five biological replicates were used for each sample. Lignin analysis Lignin was analyzed using the thioacidolysis method. Lignin content determination to cotyledons was carried out using a lignin content determination kit (Macklin, China), and the experimental procedures were conducted according to the manufacturer's instructions. Samples were dried at 80 ℃ to a constant weight, crushed and sieved through a 40 mesh sieve. Weigh about 2 mg into a 10 mL glass test tube. Add 500 µL Reagent I and 20 µL perchloric acid to the sample and blank control separately. Shake The above mixture was incubated in an 80 ℃ water bath for 40 min and shaken it gently every 10 minutes. After natural cooling, add 500 µL of Reagent II and mix thoroughly. Pipette 20 µL of the supernatant and add 980 µL of Reagent III. The optical density was measured using a microplate reader (TECAN) at a wavelength of 280 nm. Lignin content was calculated through a standard curve: y = 0.0347x + 0.0068, R 2 = 0.9889. Finally calculated by the formula: Lignin content (mg/g, DW)=(ΔA-0.0068) ÷ 0.0694×V×10 − 3 ÷ W×T. Where, V represents the total reaction volume; W represents DW of sample and T represents sample dilution ratios. Cellulose analysis Cellulose was stained using zinc chloroiodide solution. The quantification of cellulose content was performed using a cellulose content determination kit (Macklin, China), and the experimental procedures were conducted according to the manufacturer's instructions. Samples were dried at 80°C until a constant weight, then ground and sieved through a 40-mesh screen. 0.01 g of the sample is weighed, and 1 mL of 80% ethanol is added, followed by rapid homogenization at room temperature. The mixture is incubated in a 90°C water bath for 20 minutes. After cooling to room temperature, centrifugation is performed at 6000 × g and 25°C for 10 minutes, and the supernatant is discarded. The pellet is washed once with 1.5 mL of 80% ethanol and once with acetone (vortex mixing is performed for approximately 2 minutes, followed by centrifugation at 6000 × g and 25°C for 10 minutes, and the supernatant is discarded). The resulting pellet is designated as the crude cell wall. Add 1 mL of Reagent I and thoroughly mixed. The mixture is incubated in a 90°C water bath for 30 min. After cooling, centrifuged at 8000 × g for 10 min, and the supernatant is discarded. The pellets were washed three times with distilled water and then mixed with 1 mL of acetone, centrifuged at 8000 × g for 10 min, and the supernatant is discarded. Add 0.5 mL distilled water to the dried pellet and placed in an ice-water bath, and add 0.75 mL of concentrated sulfuric acid slowly. Then kept in the ice-water bath for 30 min. Centrifuged at 8000 × g and 4°C for 10 min, and the supernatant is collected. The supernatant was diluted 20-fold with distilled water for analysis. Add 150 µL sample (distilled water is used for the blank control), 35 µL working solution (add 4 mL Regent III to Regent II), and 315 µL concentrated sulfuric acid are sequentially added in a new tube and mixed thoroughly. The mixture is incubated in a 95°C water bath for 10 minutes. The optical density was measured using a microplate reader (TECAN) at a wavelength of 620 nm. Celluose content (mg/g, DW) was calculated by employing the dollowing formula: Celluose content (mg/g, DW)= [(△A + 0.0043) ÷ 5.25×V1]÷(W×V1 ÷ V2)×T, where ΔA = A determation -A blank ; V1, volume of added sample; V2, volume of added extraction solution; W, DW of sample; T, sample dilution ratios. RNA extraction and transcript analysis Total RNA was extracted from the cotyledons of WT and gpat1 gpat2 using the RNA-easy Isolation Reagent (Vazyme, China). First-strand cDNA was synthesized with the Hifair® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, China). Quantitative Real-time PCR (RT-qPCR) was performed using the Hieff® qPCR SYBR Green Master Mix (No Rox) (Yeasen, China). Relative quantitative results were normalized to the internal control ACTIN2 and presented as the mean normalized transcript levels derived from the comparative cycle threshold method (2 −ΔΔCt ). Assays were conducted using a Gentier 96E/96R system (TIANLONG, China). The primer sequences used for RT-qPCR and the accession numbers of the genes are listed in Supplemental Table S1 . Water loss analysis Plants were grown at 23 ℃ for 2 weeks before the water loss assay was conducted. The experiment was conducted in an artificial climate incubator with controlled environmental parameters. Temperature was maintained at 23°C (± 0.5°C) and relative humidity at 45% (± 3%), a vapor pressure deficit was approximately 1 kPa. To minimize the confounding effects of stomatal water loss on experimental measurements, parallel trials were performed under both light (photosynthetically active radiation 160 µmol m⁻² s⁻¹) and dark conditions. To measure the rate of water loss, we employed the following formula: Water loss rate = (Initial weight - Weight at a certain time point) / Initial weight × 100%. Statistical analysis The IBM SPSS Statistics 25 software was utilized for statistical analyses. Means were compared using one-way analysis of variance (ANOVA). Quantification of cuticle and cell wall thickness was conducted using ImageJ software. Results GPAT1 and GPAT2 involve in plant cuticle layer development In our previous study, we discovered that the Arabidopsis double mutant gpat1 gpat2 displayed significant developmental defects, including abnormalities in both embryonic and postembryonic stages (Jia et al. 2022 ). In addition to the previously documented phenotypes, we identified another noteworthy phenomenon. When placed on the surface of water, most cotyledons of wild-type (WT) plants float, while most cotyledons of gpat1 gpat2 sink to the bottom within seconds (Fig. 1 A). The gpat1 , gpat2 , and complement lines GPAT1::gpat1 gpat2 and GPAT2::gpat1 gpat2 exhibited a phenotype similar to that of WT (Supplemental Fig. S1 ). This observation suggests that the cuticle layer of gpat1 gpat2 is compromised or that the density of gpat1 gpat2 cotyledons has increased, making them denser than water. It was reported that gpat4 gpat8 double mutant strongly reduced in cutin and its cuticle showed high permeability to TB solution (Li et al., 2007 ). To verify this hypothesis, we set the abnormal cuticle mutant gpat4 gpat8 as a control. We found that gpat4 gpat8 also floated on the surface of water like the WT. This result suggested that abnormal cuticle can’t make cotyledons sank. So the sinking of gpat1 gpat2 to the bottom is most likely caused by an increase in its density.To investigate the reasons behind this phenomenon, we performed TB staining on etiolated seedlings of WT, gpat1 , gpat2 , gpat1 gpat2 , GPAT1::gpat1 gpat2 , and GPAT2::gpat1 gpat2 grown for six days on 1/2 MS medium to evaluate cuticle layer integrity. As shown in the results, the cotyledons of WT, gpat1 , gpat2 , GPAT1::gpat1 gpat2 , and GPAT2::gpat1 gpat2 remained unstained, while TB penetrated gpat1 gpat2 , causing the entire cotyledons and hypocotyls to appear blue (Fig. 1 C), indicating a compromised cuticle establishment in gpat1 gpat2 . Stained cotyledons of equal weight were collected, and the dye was extracted in 75% ethanol for two hours, followed by absorbance measurement at A626 using a spectrophotometer. The absorbance value of gpat1 gpat2 was significantly higher than those of WT, the single mutants gpat1 and gpat2 , as well as the complement lines GPAT1::gpat1 gpat2 and GPAT2::gpat1 gpat2 (Fig. 1 B). These results suggest that GPAT1 and GPAT2 are involved in cuticle development and show functional redundancy. Moreover, we found that trichome development in gpat1 gpat2 is defective across multiple organs (Fig. 2 ). Trichomes are specialized structures covered with the cuticular layer Using a stereomicroscope, we examined the trichomes on the seedlings, flowers, and stems of WT and gpat1 gpat2 . As shown in the figure, WT exhibited prominent trichomes on the leaves of seedlings, flowers, and stems, whereas some gpat1 gpat2 plants lacked trichomes altogether (Fig. 2 ). The proportions of abnormal phenotypes in leaves, flowers, and stems were 95.6%, 74.2%, and 18.3%, respectively (Fig. 2 ). gpat1 gpat2 has structural alterations in the cuticular layer Suberin is a plant polymer with water-repelling and protective properties, and its chemical composition is similar to that of cutin (Pollard et al. 2018). FY 088 specifically binds to fatty acids and used for cuticle layer staining. We stained the cotyledons of WT, gpat1 gpat2 and gpat4 gpat8 , using FY088 and observed the cuticle layer under CLSM (Fig. 3 A). The fluorescence intensity of WT and gpat1 gpat2 showed no significant differences, but the cuticle layer of gpat1 gpat2 presents irregular characteristic (Fig. 3 A). To observe the epidermal structure more clearly, we performed SEM on 20-day-old leaves and TEM on the cotyledons of WT and gpat1 gpat2 . We found that the cutin layer of gpat1 gpat2 cotyledons was compromised. Specifically, the cutin layer of WT exhibited a smooth surface, whereas gpat1 gpat2 displayed an eroded surface and was thinner compared to WT (Fig. 3 B and C). Interestingly, we also observed that the cell wall of gpat1 gpat2 was thicker than that of WT (Fig. 3 B and D). These observations suggests that the mutations in GPAT1 and GPAT2 may affect secondary cell wall (SCW) synthesis. gpat1 gpat2 shows a decrease in the amount of cutin monomers To elucidate the roles of GPAT1 and GPAT2 in cuticle permeability, we employed freeze SEM to observe the wax in one-month-old rosette leaves of WT and gpat1 gpat2 . The total wax content of rosette leaves in gpat1 gpat2 showed no significant difference compared to WT (Supplemental Fig. S2 and S3). We then measured the cutin monomers in seedlings of WT and gpat1 gpat2 . The results revealed that all the cutin monomer C16:0, C18:1, C18:2 ω-OH acid and C16:0, C18:0, C18:1, C18:2 dioic acid of gpat1 gpat2 were reduced significantly compared to WT (Fig. 4 A). The total cutin content in gpat1 gpat2 decreased by approximately roughly 33% (Fig. 4 B). This reduction corresponded closely to the cutin layer observations from TEM (Fig. 3 ). GPAT1 and GPAT2 regulate the formation of SCW During the experiment, we also found that the cotyledons and hypocotyls of gpat1 gpat2 were firmer than those of WT, prompting speculation that this phenomenon may be due to increased lignification or cellularization. To verify this hypothesis, we stained WT and gpat1 gpat2 seedlings with phloroglucinol, a dye specific for lignin. After staining, the degree of lignification was indicated by varying shades of red or purple-red, with darker colors indicating a greater degree of lignification. The results showed that the cotyledon and hypocotyl coloration of gpat1 gpat2 were more intense than those of WT, whereas the root coloration showed no significant differences between the two (Fig. 5 A and B). Furthermore, measurements of total lignin content revealed significantly higher levels in gpat1 gpat2 compared to WT (Fig. 5 C; Supplemental Fig. S3A). In addition, we assessed the cellulose content of WT and gpat1 gpat2 seedlings using the zinc chloroiodide method. The cotyledon and hypocotyl coloration of gpat1 gpat2 was significantly more intense than in the WT, indicating that the cellulose content in gpat1 gpat2 was higher than that of WT (Fig. 6 A). To further substantiate this observation, we quantified the cellulose content in WT and gpat1 gpat2 seedlings, confirming that gpat1 gpat2 contained higher cellulose levels compared to WT (Fig. 6 B). To investigate how GPAT1 and GPAT2 regulate lignin and cellulose content, we conducted RT-qPCR to detect the expression of lignin and cellulose synthesis genes in the cotyledons of WT and gpat1 gpat2 . Genes involved in lignin synthesis including PAL, C4H, 4CL, CCR, CAD, F5H, COM , and members of the LAC family were selected to assess transcript levels in WT and gpat1 gpat2 . The results showed that, except for LAC15 , almost all genes were upregulated, with LAC12 exhibiting the highest level of upregulation, followed closely by PAL3 . For cellulose synthesis, nine genes from the CESA family were analyzed (Kumar and Turner, 2015 ). Among these, CESA4 , CESA8 , and CESA9 were significantly upregulated, while CESA1 and CESA6 showed a slight downregulation in gpat1 gpat2 . These results suggest that GPAT1 and GPAT2 act as negative regulators of lignin and cellulose synthesis. In the absence of GPAT1 and GPAT2 , the expression of several lignin and cellulose synthesis genes was elevated, leading to an accumulation of lignin and cellulose in the cell wall of gpat1 gpat2 . Consequently, the cell wall becomes thicker and harder, resulting in a firmer texture of the cotyledons. This finding explains the observed thickening of the cell wall shown in Fig. 2 B. GPAT1 and GPAT2 maintain water balance in plants To investigate whether the absence of GPAT1 and GPAT2 affects the plants response to water stress, we conducted a water loss assay on the seedlings. In order to avoid the interference of stomatal water loss on the results, we conducted this assay under light and dark conditions respectively. The results indicated that when under light conditions, the water loss rates of all plant materials were faster than those under dark conditions. Notably, regardless of light or dark conditions, the water loss rate in gpat1 gpat2 was lower compared to that in the WT and complementary lines. (Fig. 7 A). Based on the findings presented in this article, we propose a model. In WT, with the involvement of GPAT1/2 , the biosynthesis of cutin monomers in plant cells proceeds normally, enabling proper development of secondary walls and trichomes, while maintaining balanced water flux in and out of the cells. But in gpat1 gpat2 , in the absence of GPAT1/2 , cutin synthesis is impaired, resulting in a thinner and irregular cuticle, which enhances cellular permeability, along with reduced trichome density. Concurrently, the levels of lignin and cellulose (major components of the cell wall) increase, leading to thickened cell walls and suppressed water efflux. Water deficit assay under light and dark conditions. Data are presented as means ± SD from three biologically independent experiments. Asterisks indicate significant differences compared with WT. Statistical analysis was performed using one-way ANOVA. *** indicates p < 0.001.(B) A proposed model illustrating the mechanisms by which GPAT1 and GPAT2 regulate cuticle and SCW development. In WT, with the involvement of GPAT1/2 , the biosynthesis of cutin monomers in plant cells proceeds normally, enabling proper development of secondary walls and trichomes, while maintaining balanced water flux in and out of the cells. But in gpat1 gpat2 , in the absence of GPAT1/2 , cutin synthesis is impaired, resulting in a thinner and irregular cuticle, which enhances cellular permeability, along with reduced trichome density. Concurrently, the levels of lignin and cellulose—major components of the cell wall—increase, leading to thickened cell walls and suppressed water efflux. Discussion The cuticle plays a key role in biotic and abiotic defense, such as regulating water, gas, and ion fluxes, as well as providing protection against pathogens. Cutin is an extracellular lipid that, along with waxes and some polysaccharides, forms the cuticle layer (Ranathunge et al. 2011; Yang et al. 2012 ). Trichomes are epidermal appendages extending from plant surfaces, aiding in resistance to external stresses by preventing physical damage, deterring pests and diseases, and reducing water evaporation through slowed air movement, thereby aiding in maintaining plant water balance. The SCW provides strong mechanical support for plant organs such as stems and roots, while its hard texture and hydrophobic nature effectively resist external invasions. The cuticle, trichomes, and SCW serve as the first line of defense when plants interact with their external environment, coordinating with one another to support plants adaptation to their surroundings. Understanding the composition and changes in these structures is of great significance for plant stress tolerance. GPAT1 and GPAT2 influence cuticle and trichome development The cuticle layer envelops the trichomes, forming an integrated protective system for the plant surface. Cutin and wax are the primary components of the cuticle. In this study, we discovered that the deficiency of GPAT1 and GPAT2 led to defects in cuticle and trichome development (Fig. 1 and Fig. 2 ). Numerous studies have indicated that there is a genetic interplay between cuticle formation and trichomes development (Chalvin et al. 2020 ; Berhin et al. 2022 ). Several Arabidopsis cutin/wax-related mutants, such as glassy hair6/cer10 , yre/cer3 , and abcg11 were reported to exhibit trichome defects (Suo et al. 2013 ; Berhin et al. 2022 ). In wheat, AaABCG20 , which is responsible for cutin and wax transport, overexpression of AaABCG20 resulted in an increase in glandular trichome density (Fu et al. 2024 ). Additionally, modifications to the cell wall can affect proper cuticle deposition and, consequently, impact trichome integrity (Philippe et al. 2020 ). Studies on the reduced wall acetylation2 mutant, which displays collapsed trichomes, have confirmed a reduction in pectin and hemicellulose acetylation and highlighted the deposition of a thicker yet more permeable cuticle at the base of trichomes (Nafisi et al. 2015 ). These cuticle abnormalities suggest that cell wall acetylation is necessary for maintaining appropriate rigidity and the integrity of epidermal structures such as trichomes (Nafisi et al. 2015 ). Overexpression of SHN1/2/3 increases cuticle components while reducing trichome density. (Aharoni et al. 2004 ; Broun et al. 2004 ). AtMYB106 and AtMYB16 regulate cutin and wax biosynthesis by activating AtSHN1 , and induction of cuticle formation by AtMYB106 negatively impacts trichome branching (Oshima et al. 2013 ). Class I TCPs members such as TCP14 and TCP15, coordinate with AtMYB106 and cutin/wax-related genes to involve in trichome and cuticle development. Solanum lycopersicum SlMIXTA -like negatively regulates trichome development while positively influencing cutin deposition (Lashbrooke et al. 2015 ; Galdon-Armero et al. 2020). Although these studies suggest that there is an interaction between the cuticle, cell wall, and trichomes, the specific mechanisms underlying this interaction are still not well understood. The roles of GPAT1 and GPAT2 in regulating the relationship between the cuticle and trichomes remain particularly unclear. GPAT1 and GPAT2 are involved in cutin synthesis Indeed, many ER-GPATs have been reported to be involved in epidermal lipid synthesis across various plants, including Arabidopsis, rice, tomato, and rapeseed (Jayawardhane et al. 2018 ). However, no current evidence suggests that Mt-GPAT is involved in the synthesis of cuticular lipids in vivo. Previous reviews noted that GPAT1 and GPAT2 are involved in cutin synthesis, although this was merely summarized in tabular form without elaboration (Jayawardhane et al. 2018 ). In the current study, we found that the two Mt-GPATs, GPAT1 and GPAT2, redundantly contribute to cutin synthesis (Fig. 3 and Fig. 4 ). GPAT is recognized as the first rate-limiting enzyme in the Kennedy pathway, catalyzing the conversion of glycerol-3-phosphate (G-3-P) to lysophosphatidic acid (LPA) (Jayawardhane et al. 2018 ). GPAT1 exhibits specific sn -2 acyltransferase activity and utilizes ω-oxidized fatty acids and unmodified acyl-CoA as substrates, while GPAT2 uses DCA-CoA as its substrate (Yang et al. 2012 ; Jayawardhane et al. 2018 ). Yang et al. examined the polymeric lipids in gpat1 but did not observe a clear phenotype associated with cutin in the mutant (Yang et al. 2012 ). Our findings address this inquiry, indicating that GPAT1 and GPAT2 function redundantly and require co-expression to exhibit a phenotype. GPAT1 and GPAT2 regulate the formation of the SCW A certain thickness of the cell wall is essential for providing mechanical support and protection to plants, but excessively thick walls can be detrimental as they may impede water and nutrient transport, hinder light energy utilization, and delay defense mechanism activation (Barros et al. 2015 ; Liu et al. 2021 ). In the absence of GPAT1 and GPAT2 , transcriptional levels of genes associated with lignin and cellulose synthesis are upregulated (Figs. 5 and 6 ), causing excessive lignin and cellulose accumulation, which thickens the SCW (Fig. 3 , Fig. 5 D, and Fig. 6 C). When plants encounter external environmental stresses, the absence of GPAT1 and GPAT2 leads to a thinner cuticle, exposing them to osmotic stress (Fig. 1 C). To cope with this stress, plants may respond by thickening their cell walls to resist excessive water entry. Osmotic stress is known to enhance lignin accumulation by increasing G and S units (Yang et al. 2023 ). Phytohormones play a crucial role in regulating SCW deposition (Liu et al. 2021 ). In our previous study, we found that lysophosphatidic acid (LPA), another product of GPAT1 and GPAT2, can mediate auxin polar transport and distribution, regulating plant embryonic and postembryonic development (Jia et al. 2022 ). Recently, research demonstrated that auxin deficiency in vascular cells may lead to persistent lignin accumulation at wound sites (Xu et al. 2024 ). Therefore, GPAT1 and GPAT2 may also mediate auxin signaling to regulate SCW formation. Several studies reported that abscisic acid (ABA) and jasmonic acid (JA) signaling are involved in lignin synthesis. The ABA signaling regulator SnRK2 phosphorylates the master transcription factor AtNST1, enhancing SCW formation and lignin production (Liu et al. 2021 ). Exogenous ABA has been shown to induce phenylpropanoid metabolism, increasing the synthesis of phenolic acids and lignin monomers in muskmelon wounds during healing. (Wang et al. 2024b ). Whether GPAT1 and GPAT2 directly regulate genes related to SCW synthesis or influence them through signaling molecules like auxin and ABA is a topic worthy of further exploration. GPAT1 and GPAT2 help plants maintaining water balance The cutin layer of gpat1 gpat2 is relatively thinner compared to that of WT (Fig. 3 ), but its dehydration rate is slower than that of WT, indicating that the thickening of the SCW affects the outward loss of intracellular water (Fig. 7 ). Cutin possesses strong hydrophobic properties and effectively prevents excessive water loss within plants. In summary, trichomes, cutin, and the SCW likely interact with one another to maintain plant structure and function. We provided evidence that the loss of function of GPAT1 and GPAT2 results affects the structure of the epidermis in Arabidopsis cotyledons. Further research indicated that these two Mt-GPATs are involved in cutin synthesis. Our findings demonstrate that GPAT1 and GPAT2 negatively regulate SCW formation. The mutation of GPAT1 and GPAT2 leads to a thicker SCW, which affects the water balance within the cells. Author contribution statement All authors contributed to the study conception and design. Q.J. designed research; Q.J., Y.B., H.Z. and X.L. performed research; W.Z. and Q.W. analyzed data; Q.Z. and X.C. provided useful scientific advice; and Q.J. and H.C. wrote the manuscript. All authors commented on draft versions of the manuscript. All authors read and approved the final version of the manuscript. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by grants provided by the China Postdoctoral Science Foundation (2023M731401) and the Jiangsu Funding Program for Excellent Postdoctoral Talent (2023ZB647). Data availability The data and materials supporting the findings of this study are available from the corresponding author upon request. References Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16(9):2463–2480 Barros J, Serk H, Granlund I, Pesquet E (2015) The cell biology of lignification in higher plants. Ann Bot 115(7):1053–1074 Beisson F, Li Y, Bonaventure G, Pollard M, Ohlrogge JB (2007) The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19(1):351–368 Beris DG, Kapolas P, Livanos A, Roussis D, Milioni K, Haralampidis (2016) RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility. Plant Sci 245:71–83 Berhin A, Nawrath C, Hachez C (2022) Subtle interplay between trichome development and cuticle formation in plants. New Phytol 233(5):2036–2046 Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X, Yephremov A, Samuels L (2007) Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J 52(3):485–498 Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci U S A 101(13):4706–4711 Borah P, Khurana JP (2018) The OsFBK1 E3 ligase subunit affects anther and root secondary cell wall thickenings by mediating turnover of a Cinnamoyl-CoA reductase. Plant Physiol 176:2148–2165 Camoirano A, Arce AL, Ariel FD, Alem AL, Gonzalez DH, Viola IL (2020) Class I TCP transcription factors regulate trichome branching and cuticle development in Arabidopsis. J Exp Bot 71(18):5438–5453 Chalvin C, Drevensek S, Dron M, Bendahmane A, Boualem A (2020) Genetic control of glandular trichome development. Trends Plant Sci 25(5):477–487 Geng P, Zhang S, Liu J, Zhao C, Wu J, Cao Y, Fu C, Han X, He H, Zhao Q (2020) MYB20, MYB42, MYB43, and MYB85 regulate phenylalanine and lignin biosynthesis during secondary cell wall formation. Plant Physiol 182(3):1272–1283 González-Valenzuela L, Renard J, Depège-Fargeix N, Ingram G (2023) The plant cuticle. Curr Biol 33(6):R210–R214 Chen X, Truksa M, Snyder CL, El-Mezawy A, Shah S, Weselake RJ (2011) sn-Glycerol-3-phosphate acyltransferases in plants. Plant Signal Behav 6(11):1695–1699 Feng H, Li X, Chen H, Deng J, Zhang C, Liu J, Wang T, Zhang X, Dong J (2018) GhHUB2, a ubiquitin ligase, is involved in cotton fiber development via the ubiquitin-26S proteasome pathway. J Exp Bot 69(21):5059–5075 Fu X, Zheng H, Wang Y, Liu H, Liu P, Li L, Zhao J, Sun X, Tang K (2024) AaABCG20 transporter involved in cutin and wax secretion affects the initiation and development of glandular trichomes in Artemisia annua. Plant Sci 339:111959 Donnelly PM, Bonetta D, Tsukaya H, Dengler RE, Dengler NG (1999) Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215(2):407–419 Duo H, Yin M, Wang R (2024) Molecular mechanisms of resistance and future perspectives in plant breeding strategies against Sclerotinia sclerotiorum. New Crops 2:100046 Elejalde-Palmett C, Martinez San Segundo I, Garroum I, Charrier L, De Bellis D, Mucciolo A, Guerault A, Liu J, Zeisler-Diehl V, Aharoni A et al (2021) ABCG transporters export cutin precursors for the formation of the plant cuticle. Curr Biol 31(10):2111–2123 Gully K, Berhin A, De Bellis D, Herrfurth C, Feussner I, Nawrath C (2024) The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae. Proc Natl Acad Sci U S A 121(21):e2314570121 Han G, Li Y, Yang Z, Wang C, Zhang Y, Wang B (2022) Molecular mechanisms of plant trichome development. Front Plant Sci 13:910228 Huang H, Wang Y, Yang P, Zhao H, Jenks MA, Lü S, Yang X (2024) The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers. Plant J 118(5):1619–1634 Hülskamp M (2019) Trichomes Curr Biol 29(8):R273–R274 Ingram G, Nawrath C (2017) The roles of the cuticle in plant development: organ adhesions and beyond. J Exp Bot 68(19):5307–5321 Jayawardhane KN, Singer SD, Weselake RJ, Chen G (2018) Plant sn-Glycerol-3-Phosphate Acyltransferases: biocatalysts involved in the biosynthesis of intracellular and extracellular lipids. Lipids 53(5):469–480 Jia Q, Bai Y, Xu H, Liu Q, Li W, Li T, Lin F, Shen L, Xuan W, Zhang W et al (2022) Mitochondrial GPAT-derived LPA controls auxin-dependent embryonic and postembryonic development. Proc Natl Acad Sci U S A 119(49):e2212881119 Jung KH, Han MJ, Lee D, Lee YS, Schreiber L, Franke R, Faust A, Yephremov A, Saedler H, Kim YW et al (2006) Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development. Plant Cell 18(11):3015–3032 Ko JH, Jeon HW, Kim WC, Kim JY, Han KH (2014) The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Ann Bot 114(6):1099–1107 Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19(16):1855–1860 Kumar M, Turner S (2015) Plant cellulose synthesis: CESA proteins crossing kingdoms. Phytochemistry 112:91–99 Kumar M, Campbell L, Turner S (2016) Secondary cell walls: biosynthesis and manipulation. J Exp Bot 67(2):515–531 Lashbrooke JG, Adato A, Lotan O, Alkan N, Tsimbalist T, Rechav K, Fernandez Moreno J-P, Widemann E, Grausem B, Pinot F et al (2015) The tomato MIXTA-like transcription factor coordinates fruit epidermis conical cell development and cuticular lipid biosynthesis and assembly. Plant Physiol 169(4):2553–2571 Li-Beisson Y, Pollard M, Sauveplane V, Pinot F, Ohlrogge J, Beisson F (2009) Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester. Proc Natl Acad Sci U S A 106(51):22008–22013 Li Y, Beisson F, Koo AJK, Molina I, Pollard M, Ohlrogge J (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci U S A 104(46):18339–18344 Liu C, Yu H, Rao X, Li L, Dixon RA (2021) Abscisic acid regulates secondary cell-wall formation and lignin deposition in Arabidopsis thaliana through phosphorylation of NST1. Proc Natl Acad Sci U S A 118(5):e2010911118 Matías-Hernández L, Aguilar-Jaramillo AE, Cigliano RA, Sanseverino W, Pelaz S (2016) Flowering and trichome development share hormonal and transcription factor regulation. J Exp Bot 67(5):1209–1219 Men X, Shi J, Liang W, Zhang Q, Lian G, Quan S, Zhu L, Luo Z, Chen M, Zhang D (2017) Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3) is required for anther development and male fertility in rice. J Exp Bot 68(3):513–526 Nafisi M, Stranne M, Fimognari L, Atwell S, Martens HJ, Pedas PR, Hansen SF, Nawrath C, Scheller HV, Kliebenstein DJ et al (2015) Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses. Front Plant Sci 6:550 Oshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M (2013) MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. Plant Cell 25(5):1609–1624 Ohtani M, Demura T (2019) The quest for transcriptional hubs of lignin biosynthesis: beyond the NAC-MYB-gene regulatory network model. Curr Opin Biotechnol 56:82–87 Petit J, Bres C, Mauxion J-P, Wong Jun Tai F, Martin LBB, Fich EA, Joubès J, Rose JKC, Domergue F, Rothan C (2016) The glycerol-3-phosphate acyltransferase GPAT6 from tomato plays a central role in fruit cutin biosynthesis. Plant Physiol 171(2):894–913 Philippe G, Geneix N, Petit J, Guillon F, Sandt C, Rothan C, Lahaye M, Marion D, Bakan B (2020) Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides. New Phytol 226(3):809–822 Pollard M, Beisson F, Li Y, Ohlrogge JB (2008) Building lipid barriers: biosynthesis of cutin and suberin. Trends Plant Sci 13(5):236–246 Suo B, Seifert S, Kirik V (2013) Arabidopsis GLASSY HAIR genes promote trichome papillae development. J Exp Bot 64(16):4981–4991 Traw MB, Bergelson J (2003) Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Plant Physiol 133(3):1367–1375 Wang Q, Lei S, Yan J, Song Y, Qian J, Zheng M, Hsu YF (2023) UBC6, a ubiquitin-conjugating enzyme, participates in secondary cell wall thickening in the inflorescence stem of Arabidopsis. Plant Physiol Biochem 205:108152 Wang H, Zhao Q, Chen F, Wang M, Dixon RA (2011) NAC domain function and transcriptional control of a secondary cell wall master switch. Plant J 68(6):1104–1114 Wang Q, Liu N, Yang R, Zhang X, Wang Y, Li Y, Prusky D, Bi Y, Han Y (2024a) Essential role of ABA signaling and related transcription factors in phenolic acid and lignin synthesis during muskmelon wound healing. Front Plant Sci 15:1404477 Wang Y, Li P, Sun W, Zhang T (2024b) Plant cell walls: Emerging targets of stomata engineering to improve photosynthesis and water use efficiency. New Crops 1:100021 Waszczak C, Carmody M, Kangasjärvi J (2018) Reactive oxygen species in plant signaling. Annu Rev Plant Biol 69:209–236 Wellesen K, Durst F, Pinot F, Benveniste I, Nettesheim K, Wisman E, Steiner-Lange S, Saedler H, Yephremov A (2001) Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development. Proc Natl Acad Sci U S A 98(17):9694–9699 Xu H, Dong C, Wu Y, Fu S, Tauqeer A, Gu X, Li Q, Niu X, Liu P, Zhang X et al (2024) The JA-to-ABA signaling relay promotes lignin deposition for wound healing in Arabidopsis. Mol Plant 17(10):1594–1605 Yang J, Song J, Feng Y, Cao Y, Fu B, Zhang Z, Ma N, Li Q, Hu T, Wang Y et al (2023) Osmotic stress-induced lignin synthesis is regulated at multiple levels in alfalfa (Medicago sativa L). Int J Biol Macromol 246:125501 Yang W, Simpson JP, Li-Beisson Y, Beisson F, Pollard M, Ohlrogge JB (2012) A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution. Plant Physiol 160(2):638–652 Zhan J, Di T, Chen X, Zheng T, Sun W, Yang M, Zhou M, Shen Z, Chen H, Su N (2024) CbMYB108 integrates the regulation of diterpene biosynthesis and trichome development in Conyza blinii against UV-B. Plant Cell Environ 47(4):1300–1318 Zhang X, Gao H, Liu Y, Zhao H, Lü S (2024) Function identification of Arabidopsis GPAT4 and GPAT8 in the biosynthesis of suberin and cuticular wax. Plant Sci 339:111933 Zhong R, Ye ZH (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav 4(11):1028–1034 Supplementary Files SupplementalFigures.docx SupplementalTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6445935\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":450922643,\"identity\":\"164c856e-4517-4af8-9222-8684e7b3e24b\",\"order_by\":0,\"name\":\"Qianru Jia\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBAC9mYgwfjvvxwbe/sB4rQwgrQwsDEb8/GcSSBSSwNES+I8CQcDIrW0Mz+8XcHDlt4mwZDA8KNiGzEOYzO2PCPBk9sm3XiAsefMbWK0MJhJNhhI5LbJHEhgZmwjSgv7N8mGBIN0NokEA+K0CDbzAG05kJBAvBZpZp5iy8aGA4ZtwEA+SJRf+PiPb7wJ1CIv395+8MGPCiK0gIAEjHGAOPXIWkbBKBgFo2AUYAUAn+g1/dPd7VYAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0009-0009-9858-1619\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Qianru\",\"middleName\":\"\",\"lastName\":\"Jia\",\"suffix\":\"\"},{\"id\":450922644,\"identity\":\"40d370b6-afad-4994-b28e-b5330d01b5f7\",\"order_by\":1,\"name\":\"Yang Bai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Botanical Garden Mem Sun Yat-Sen: Institute of Botany Jiangsu Province and Chinese Academy of Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yang\",\"middleName\":\"\",\"lastName\":\"Bai\",\"suffix\":\"\"},{\"id\":450922645,\"identity\":\"18737d9a-09ca-4c5a-86d2-dc2d4c5edf50\",\"order_by\":2,\"name\":\"Hongmei Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hongmei\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":450922646,\"identity\":\"a435eaee-bdcf-45cf-92c4-60b15c8e015e\",\"order_by\":3,\"name\":\"Wei Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wei\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":450922647,\"identity\":\"61ba7f5e-4ed5-4438-832d-affff1d3c146\",\"order_by\":4,\"name\":\"Qiong Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qiong\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":450922648,\"identity\":\"54e0e178-0f01-4d46-a475-f84b82d0df5b\",\"order_by\":5,\"name\":\"Xiaoqing Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaoqing\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":450922649,\"identity\":\"ea0b3c2e-289d-44b9-a46f-be4ac0bccc93\",\"order_by\":6,\"name\":\"Xin Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xin\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":450922650,\"identity\":\"5c79bf93-1e5b-4181-9eb7-f908d060127b\",\"order_by\":7,\"name\":\"Qun Zhang\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-4477-1478\",\"institution\":\"Nanjing Agricultural University College of Life Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qun\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":450922651,\"identity\":\"38eac72c-e08e-4167-8206-4c5396e51db6\",\"order_by\":8,\"name\":\"Huatao Chen\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-5468-9999\",\"institution\":\"Jiangsu Academy of Agricultural Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Huatao\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-04-14 12:22:42\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6445935/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6445935/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":82209991,\"identity\":\"21d60d2b-5a10-454d-a0ea-965f9ea6d4fe\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 18:52:11\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":320183,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe loss of function of GPAT1 and GPAT2 led to an increase in permeability of the plant cuticle.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Six-day-old cotyledons of WT, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003egpat4 gpat8 \\u003c/em\\u003ewere placed on the surface of water. Most cotyledons from WT and \\u003cem\\u003egpat4 gpat8 \\u003c/em\\u003efloated, whereas most cotyledons from \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e sank. \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e was\\u003cem\\u003e \\u003c/em\\u003eset as a control.\\u003cem\\u003e \\u003c/em\\u003eWhite arrows indicate cotyledons sinking to the bottom.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Cuticle permeability to TB solution in etiolated seedlings of WT, \\u003cem\\u003egpat1\\u003c/em\\u003e, \\u003cem\\u003egpat2\\u003c/em\\u003e, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e, \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e. The two images on the right of the first row are two repetitions of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e was\\u003cem\\u003e \\u003c/em\\u003eset as a positive control. Scale bar = 1 mm.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) \\u0026nbsp;Absorbance readings from cotyledons of WT, \\u003cem\\u003egpat1\\u003c/em\\u003e, \\u003cem\\u003egpat2\\u003c/em\\u003e, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e, and \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e at the same weight, measured at a wavelength of 626 nm. All data are shown as means ± standard deviation (SD) from three biologically independent replicates. Asterisks indicate significant differences from WT (***p \\u0026lt; 0.001; one-way ANOVA). n.s., no significance.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/e61fd570a4275b959a707118.png\"},{\"id\":82210371,\"identity\":\"58fa662c-7939-499b-a71a-6d5c43fd88ec\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 19:00:11\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":342069,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003egpat1 gpat2 \\u003c/em\\u003emutant is affected in trichome development.\\u003c/p\\u003e\\n\\u003cp\\u003e(A-C) Six-day-old seedlings of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. (B) and (C) are two replicates of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. n represents total number of seedlings. The percentage indicates the proportion of abnormal phenotypes within the total. All images were captured using an ultra-depth stereomicroscope. Images are representative phenotypes. White arrows indicate the trichomes on the leaves. Scale bar = 500 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(D and E) Flowers of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. n represents the total number of flowers. The percentage indicates the proportion of abnormal phenotypes within the total. White arrows indicate the trichomes on the flowers. Scale bar = 500 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(F and G) Stems of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, respectively. Scale bars represent 1 mm. n indicates the total number of stems. The percentage indicates the proportion of abnormal phenotypes within the total. Scale bar = 500 μm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/6bb3821cc8004d13c84e05ff.png\"},{\"id\":82210372,\"identity\":\"cc05761c-2ef9-400b-aa37-300682cb2434\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 19:00:11\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":386515,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe loss of function of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e led to the reduction of cutin layer of the cotyledon.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Cuticle layer of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e cotyledons. Suberin of WT, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003eand \\u003cem\\u003egpat4 gpat8 \\u003c/em\\u003ecotyledons were observed by CLSM. \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e was set as a control. Scale bars=100 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Transmission electron microscopy (TEM) images of the surface of cotyledons from WT and \\u003cem\\u003egpat1\\u003c/em\\u003e \\u003cem\\u003egpat2\\u003c/em\\u003e. CW indicates the cell wall. Black arrows highlight the cutin layer. Scale bar = 200 nm.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Quantification of cuticle thickness from panel A. Asterisks indicate significant differences compared with WT. *** indicates p \\u0026lt; 0.001, one-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Quantification of cell wall thickness from panel A. Asterisks indicate significant differences compared with WT. *** indicates p \\u0026lt; 0.001, one-way ANOVA.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/1fa857054f4664acd9fa5172.png\"},{\"id\":82209992,\"identity\":\"7324a5e5-1d89-4aa1-8cc0-05bc26d7c5fc\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 18:52:11\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":155248,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCutin compositions of WT and \\u003cem\\u003egpat1 gpat2 \\u003c/em\\u003eseedlings.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Comparation of cutin monomer of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e seedlings. All data are shown as means ± SD from five biologically independent. Asterisks indicate significant differences compared with WT. *** indicates p \\u0026lt; 0.001, one-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Comparation of total cutin of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e seedlings. All data are shown as means ± SD from five biologically independent. Asterisks indicate significant differences compared with WT. *** represents p \\u0026lt; 0.001, one-way ANOVA.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/de2a55ce2cd668bd8d6a7f63.png\"},{\"id\":82209998,\"identity\":\"7bdfdb52-3295-48e6-8281-643eb6f850ea\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 18:52:11\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":311127,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e regulate the synthesis of lignin.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e cotyledons stained with phloroglucinol. Scale bars = 500 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e roots stained with phloroglucinol. Scale bars = 100 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Quantification of lignin content. *** indicates \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt;0.001.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) RT-qPCR analysis of lignin biosynthesis genes. Data are presented as means ± SD from three biologically independent experiments, with three independent trials yielding similar results. Statistical analysis was performed using one-way ANOVA. * indicates p \\u0026lt; 0.05, ** indicates p \\u0026lt; 0.01, and *** indicates p \\u0026lt; 0.001. n.s., no significance.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/54f35c74b8a75d8e7547b988.png\"},{\"id\":82210373,\"identity\":\"b7007c09-df42-4a31-bb7a-a28c45eb642d\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 19:00:11\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":256115,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2 \\u003c/em\\u003eregulate the synthesis of cellulose.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003eseedlings stained with phloroglucinol. Scale bar = 500 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Analysis of cellulose content conducted using a cellulose content determination kit. \\u0026nbsp;All data are shown as means ± SD from three biologically independent. Asterisks indicate significant differences compared with WT. ***indicates p\\u0026lt;0.001.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) RT-qPCR analysis of lignin biosynthesis genes. Data are presented as means ± SD from three biologically independent experiments. Three independent trials yielded similar results. Asterisks indicate significant differences compared with WT. Statistical analysis was performed using one-way ANOVA. ** indicates p \\u0026lt; 0.01, *** indicates p \\u0026lt; 0.001, and n.s. indicates no significance.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/f75c3218fed8b0fa3d496fa1.png\"},{\"id\":82211295,\"identity\":\"cfd8bb6c-b7dc-4b55-95cf-ab409cf28be8\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 19:24:11\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":255719,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWater deficit sensitivity of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/66da3777a702cce37152097b.png\"},{\"id\":83461072,\"identity\":\"28867a21-9eaa-4eef-bd6d-eaf8fb4c7820\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 16:22:17\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2992068,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/4b9e927f-0437-4e33-bddb-1d599601e390.pdf\"},{\"id\":82210007,\"identity\":\"5245def9-68c8-4a61-8799-205717db2fcb\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 18:52:11\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1318481,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalFigures.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/9001424382c45008e821d348.docx\"},{\"id\":82210018,\"identity\":\"371978f1-0150-4c7f-8b08-41b55926f0a7\",\"added_by\":\"auto\",\"created_at\":\"2025-05-07 18:52:11\",\"extension\":\"docx\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21398,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalTables.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6445935/v1/3b5fd6259879c6b0515c33f8.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Mitochondrial GPATs protect plants from water stress by impacting cuticle and secondary cell wall development\",\"fulltext\":[{\"header\":\"Key Message\",\"content\":\"\\u003cp\\u003eMt-GPAT1/2 are involved in cutin biosynthesis and negatively regulate SCW formation by modulating the lignin and cellulose synthesis genes. Alterations in cuticle and SCW lead to plant water imbalance.\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe cuticle is composed of cutin, wax, and various polysaccharide molecules that penetrate the epidermal cell walls, forming a diffusion barrier covering almost all aerial surfaces of higher plants. This structure establishes a critical interface between the plant and its environment (Jung et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e;Duo et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The cuticle not only plays a pivotal role in plant processes, such as organogenesis and seed viability, but also provides protection against diverse stresses, including transpiration, elevated temperatures, ultraviolet radiation, and herbivory (Ingram and Nawrath, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e༛Camoirano et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Gonz\\u0026aacute;lez-Valenzuela et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2024a\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTrichomes, hair-like organs deriving from differentiation of the protoderm that extend from the surface of the epidermis (Donnelly et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e). Trichomes fulfill diverse biological functions, including defense against herbivory, UV irradiation, and desiccation, and exhibit specific structural diversity in density and morphology to support these roles (Han et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Furthermore, trichomes may undergo physiological adaptations to synthesize specialized metabolites or contribute to detoxification by sequestering toxic compounds (Traw and Bergelson, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Mat\\u0026iacute;as-Hern\\u0026aacute;ndez et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; H\\u0026uuml;lskamp et al. 2019; Zhan et al. \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn recent years, considerable research has been conducted to unravel the mechanisms of cutin biosynthesis, such as the ABCG family (Bird et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Elejalde-Palmett et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Fu et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e) and cytochrome P450 enzymes (Wellesen et al. \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e; Li-Beisson et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Huang et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The Glycerol-3-Phosphate Acyltransferase (GPAT) family also plays a significant role in cutin biosynthesis. Based on subcellular localization, GPATs can be classified into mitochondrial (Mt), endoplasmic reticulum (ER), and chloroplast types (Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). The Arabidopsis GPAT family consists of 10 members, among which GPAT1-3 belong to the mitochondrial type, GPAT4-8 are categorized as endoplasmic reticulum types, while GPAT9 and ATS1 are classified as plastid types (Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Several ER-GPATs from different plants are known to be involved in cutin synthesis across multiple organs, including AtGPAT4/6/8 (Li et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Yang et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), BnGPAT4 (Chen et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e), OsGPAT3 (Men et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e), and SlGPAT6 (Petit et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Furthermore, AtGPAT4/5/6/7/8 are involved in suberin synthesis, regulating plant salt tolerance and mechanical damage repair (Beisson et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Li-Beisson et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Gully et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). However, it has not yet been reported whether Mt-GPAT is involved in cutin synthesis.\\u003c/p\\u003e \\u003cp\\u003eThe secondary cell wall (SCW) is located between the primary cell wall and the plasma membrane and is composed of lignin, cellulose, and hemicelluloses (Wang et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The formation network of the SCW is extremely complex and subject to multi-faceted regulation. Tier1 and Tier2 transcription factors (TFs) contain many MYB TFs, such as MYB46 and MYB83 in Tier2 TFs and MYB20, MYB42, MYB43, and MYB58 in Tier1 TFs (Ko et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Geng et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) and NAC TFs, NST1, NST2, SND1, VND6, and VND7 in Tier3 TFs were characterized as master regulators of SCW biosynthesis (Kubo et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Wang et al., \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). Ubiquitination also plays a role in SCW formation (Wang et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). E3 Cullin Ring Ligase subunit DDB1a (Beris et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), cotton GhHUB2 (Feng et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), and OsFBK1 (Borah and Khurana, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), as components of an SCF E3 ligase complex, have all been reported to regulate SCW either directly or indirectly. To date, the role of GPAT in the regulation of SCW development remains unexamined.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we report that the Arabidopsis double mutant \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e exhibits higher permeability of water than the wild type (WT), suggesting that the cuticle layer in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e is disrupted. Our findings confirm that Mt-GPAT1 and Mt-GPAT2 are involved in plant cutin synthesis. The structure of the cutin layer is compromised, which hinders the development of trichomes. Further research has demonstrated that GPAT1 and GPAT2 regulate the formation of the SCW by negatively regulating genes associated with lignin and cellulose biosynthesis. Our findings underscore the importance of Mt-GPAT1 and Mt-GPAT2 in maintaining cellular water balance.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePlant materials and growth conditions\\u003c/h2\\u003e \\u003cp\\u003eThe mutants \\u003cem\\u003egpat1\\u003c/em\\u003e (Salk_052353) and \\u003cem\\u003egpat2\\u003c/em\\u003e (Salk_064530C) were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. The double mutant \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, along with the complementary lines \\u003cem\\u003eGPAT1:: gpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003eGPAT2:: gpat1 gpat2\\u003c/em\\u003e, were previously characterized (Jia et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Seeds were germinated on half-strength Murashige and Skoog (MS) medium and subsequently transferred to an illuminated incubator with a light intensity of 160 \\u0026micro;mol m\\u003csup\\u003e\\u0026ndash;2\\u003c/sup\\u003e s\\u003csup\\u003e\\u0026ndash;1\\u003c/sup\\u003e, following a day/night cycle of 14/10 hours at 23\\u0026deg;C, after a two-day vernalization period. Afterward, seedlings were transplanted into soil under the same conditions for continued cultivation.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eToluidine Blue, Fluorol Yellow 088, phloroglucinol and zinc chloroiodide staining\\u003c/h3\\u003e\\n\\u003cp\\u003eSix-day-old etiolated seedlings were stained with Toluidine Blue (TB) solution (0.05% (w/v) TB\\u0026thinsp;+\\u0026thinsp;0.4% (v/v) Tween-20) for one minute, followed by rinsing under running tap water until no residual dye was visible in the rinse water. The samples were photographed using a stereomicroscope (Leica M165FC, Leica). Equal-weight samples were then immersed in 75% ethanol and extracted for two hours; the optical density was measured using a microplate reader (TECAN) at a wavelength of 626 nm.\\u003c/p\\u003e \\u003cp\\u003eFor Fluorol Yellow (FY) 088 (Macklin, China) staining, six-day-old seedlings were immersed in 0.01% (w/v, lactic acid) FY 088 for five minutes. The cuticle layer was then observed using a confocal laser scanning microscope with an excitation wavelength of 488 nm and emission wavelengths ranging from 500 to 550 nm (Zhang et al. \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFor phloroglucinol and zinc chloroiodide staining, we utilized lignin staining solution (phloroglucinol method) (Macklin, China) and cellulose staining solution (Zinc chloroiodide method) (Yuanye, China). Six-day-old seedlings were immersed in the lignin acidification solution for ten minutes, after which an equal amount of phloroglucinol staining solution was added dropwise to the acidified material, and the mixture was allowed to sit for ten minutes. Alternatively, samples were immersed in the cellulose staining solution for five minutes. The samples were then observed using a stereomicroscope.\\u003c/p\\u003e\\n\\u003ch3\\u003eTrichome observation\\u003c/h3\\u003e\\n\\u003cp\\u003eSix-day-old seedlings, flowers and stems were imaged under an Ultra depth stereomicroscope (Leica DVM6, Leica).\\u003c/p\\u003e\\n\\u003ch3\\u003eElectron microscopy techniques\\u003c/h3\\u003e\\n\\u003cp\\u003eThe leaf waxy crystal was observed using a freezing scanning electron microscope (SEM) (HITACHI S-3000, Japan). Twenty-day-old rosette leaves of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e were adhered to conductive tape on the sample stage. The sample stages were then transferred to the SEM sample chamber, where they were cooled with liquid nitrogen for observation. The cold stage temperature was maintained at -140 ℃, and the acceleration voltage was set to 5 kV.\\u003c/p\\u003e \\u003cp\\u003eThe cotyledon cuticle layer was examined using a transmission electron microscope (TEM) (HITACHI HT7800, Japan). Specifically, cotyledons of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e were fixed in a 2.5% (w/v) glutaraldehyde solution for more than two hours in 0.2 M PBS at 4\\u0026deg;C. The samples were then washed three times with 0.1 M PBS and dehydrated through a graded series of ethanol. Following this, cotyledons were embedded in epoxy resin and subjected to ultra-thin slicing. Finally, the samples were placed on a copper mesh and stained with uranyl acetate and lead citrate before being observed under the TEM at 80.0 kV.\\u003c/p\\u003e\\n\\u003ch3\\u003eCutin monomer analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eSix-day-old seedlings were collected for the analysis of cutin monomers. The protocol used for extraction of cutin monomer followed a previous study with some modifications (Elejalde-Palmett et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Samples were incubated in 10 mL preheated isopropanol /0.01% butylated hydroxytoluene (BHT) for 15 min at 85℃ and then washes twice in methanol:chloroform (V:V\\u0026thinsp;=\\u0026thinsp;1:2) by 24 h. Samples were then dried under a gentle stream of N\\u003csub\\u003e2\\u003c/sub\\u003e and dried in a vacuum desiccator for one week. 25 \\u0026micro;g ω-pentadecalactone and methyl heptadecanoate, 0.9 mL methyl acetate, 1.5 mL sodium methoxide and 3.6 mL methanol were added to the samples in turn and then incubated at 60℃ for 2 h. 3.5 mL dichloromethane, 0.7 mL glacial acetic acid and 1 mL 0.9% NaCl (w/v) were added to each sample and subsequently vortexed for 20 s. After centrifugation (1500 g for 2 min), the organic phase was collected, washed with 2 mL of 0.9% NaCl, and dried over sodium sulfate. The organic phase was then recovered and concentrated under a stream of N\\u003csub\\u003e2\\u003c/sub\\u003e. Monomers were derivatized by acetylation using 100 \\u0026micro;L of anhydrous pyridine and acetic anhydride at 60℃ for 2h. A gas chromatograph coupled to a mass spectrometer (Agilent 7890A-5975CMS) with a DB-5 column (30 m\\u0026times;0.25 mm\\u0026times;0.25 \\u0026micro;m, Agilent) was used to detect the cutin monomer. Column temperature program: 80℃, raised by 15℃/min to 200℃ and 2℃/min to 300℃ and held for 10 min. A total of five biological replicates were used for each sample.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWax analysis\\u003c/h2\\u003e \\u003cp\\u003eOne month rosette leaves were collected for wax analysis. The protocol used for the extraction of wax monomer was based on a previous study with minor modifications (Elejalde-Palmett et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Leaves were cut into small segments and then extracted twice using 10 mL of chloroform for 1 min. Samples were dried by N\\u003csub\\u003e2\\u003c/sub\\u003e and then derivatized with BFTSA/pyridine (1:1) at 100℃ for 30 min. Samples were resuspended with an appropriate volume of chloroform and detected by a gas chromatograph coupled to a mass spectrometer (Agilent 7890A-5975CMS) on a DB-5 column (30 m\\u0026times;0.25 mm\\u0026times;0.25 \\u0026micro;m, Agilent). Column temperature program: 80℃ for 2 min, raised by 40℃/min to 200℃ and held for 2 min, raised by 10℃/min to 270℃ and held for 2 min, then raised by 2℃/min to 320℃ and held for 30 min. A total of five biological replicates were used for each sample.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eLignin analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eLignin was analyzed using the thioacidolysis method. Lignin content determination to cotyledons was carried out using a lignin content determination kit (Macklin, China), and the experimental procedures were conducted according to the manufacturer's instructions. Samples were dried at 80 ℃ to a constant weight, crushed and sieved through a 40 mesh sieve. Weigh about 2 mg into a 10 mL glass test tube. Add 500 \\u0026micro;L Reagent I and 20 \\u0026micro;L perchloric acid to the sample and blank control separately. Shake The above mixture was incubated in an 80 ℃ water bath for 40 min and shaken it gently every 10 minutes. After natural cooling, add 500 \\u0026micro;L of Reagent II and mix thoroughly. Pipette 20 \\u0026micro;L of the supernatant and add 980 \\u0026micro;L of Reagent III. The optical density was measured using a microplate reader (TECAN) at a wavelength of 280 nm. Lignin content was calculated through a standard curve: y\\u0026thinsp;=\\u0026thinsp;0.0347x\\u0026thinsp;+\\u0026thinsp;0.0068, R\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;0.9889. Finally calculated by the formula: Lignin content (mg/g, DW)=(ΔA-0.0068)\\u0026thinsp;\\u0026divide;\\u0026thinsp;0.0694\\u0026times;V\\u0026times;10\\u0026thinsp;\\u0026minus;\\u0026thinsp;3\\u0026thinsp;\\u0026divide;\\u0026thinsp;W\\u0026times;T. Where, V represents the total reaction volume; W represents DW of sample and T represents sample dilution ratios.\\u003c/p\\u003e\\n\\u003ch3\\u003eCellulose analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eCellulose was stained using zinc chloroiodide solution. The quantification of cellulose content was performed using a cellulose content determination kit (Macklin, China), and the experimental procedures were conducted according to the manufacturer's instructions. Samples were dried at 80\\u0026deg;C until a constant weight, then ground and sieved through a 40-mesh screen. 0.01 g of the sample is weighed, and 1 mL of 80% ethanol is added, followed by rapid homogenization at room temperature. The mixture is incubated in a 90\\u0026deg;C water bath for 20 minutes. After cooling to room temperature, centrifugation is performed at 6000 \\u0026times; g and 25\\u0026deg;C for 10 minutes, and the supernatant is discarded. The pellet is washed once with 1.5 mL of 80% ethanol and once with acetone (vortex mixing is performed for approximately 2 minutes, followed by centrifugation at 6000 \\u0026times; g and 25\\u0026deg;C for 10 minutes, and the supernatant is discarded). The resulting pellet is designated as the crude cell wall. Add 1 mL of Reagent I and thoroughly mixed. The mixture is incubated in a 90\\u0026deg;C water bath for 30 min. After cooling, centrifuged at 8000 \\u0026times; g for 10 min, and the supernatant is discarded. The pellets were washed three times with distilled water and then mixed with 1 mL of acetone, centrifuged at 8000 \\u0026times; g for 10 min, and the supernatant is discarded. Add 0.5 mL distilled water to the dried pellet and placed in an ice-water bath, and add 0.75 mL of concentrated sulfuric acid slowly. Then kept in the ice-water bath for 30 min. Centrifuged at 8000 \\u0026times; g and 4\\u0026deg;C for 10 min, and the supernatant is collected. The supernatant was diluted 20-fold with distilled water for analysis. Add 150 \\u0026micro;L sample (distilled water is used for the blank control), 35 \\u0026micro;L working solution (add 4 mL Regent III to Regent II), and 315 \\u0026micro;L concentrated sulfuric acid are sequentially added in a new tube and mixed thoroughly. The mixture is incubated in a 95\\u0026deg;C water bath for 10 minutes. The optical density was measured using a microplate reader (TECAN) at a wavelength of 620 nm. Celluose content (mg/g, DW) was calculated by employing the dollowing formula: Celluose content (mg/g, DW)= [(△A\\u0026thinsp;+\\u0026thinsp;0.0043)\\u0026thinsp;\\u0026divide;\\u0026thinsp;5.25\\u0026times;V1]\\u0026divide;(W\\u0026times;V1\\u0026thinsp;\\u0026divide;\\u0026thinsp;V2)\\u0026times;T, where ΔA\\u0026thinsp;=\\u0026thinsp;A \\u003csub\\u003edetermation\\u003c/sub\\u003e-A\\u003csub\\u003eblank\\u003c/sub\\u003e; V1, volume of added sample; V2, volume of added extraction solution; W, DW of sample; T, sample dilution ratios.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRNA extraction and transcript analysis\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from the cotyledons of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e using the RNA-easy Isolation Reagent (Vazyme, China). First-strand cDNA was synthesized with the Hifair\\u0026reg; Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, China). Quantitative Real-time PCR (RT-qPCR) was performed using the Hieff\\u0026reg; qPCR SYBR Green Master Mix (No Rox) (Yeasen, China). Relative quantitative results were normalized to the internal control \\u003cem\\u003eACTIN2\\u003c/em\\u003e and presented as the mean normalized transcript levels derived from the comparative cycle threshold method (2\\u003csup\\u003e\\u0026minus;ΔΔCt\\u003c/sup\\u003e). Assays were conducted using a Gentier 96E/96R system (TIANLONG, China). The primer sequences used for RT-qPCR and the accession numbers of the genes are listed in \\u003cb\\u003eSupplemental Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWater loss analysis\\u003c/h2\\u003e \\u003cp\\u003ePlants were grown at 23 ℃ for 2 weeks before the water loss assay was conducted. The experiment was conducted in an artificial climate incubator with controlled environmental parameters. Temperature was maintained at 23\\u0026deg;C (\\u0026plusmn;\\u0026thinsp;0.5\\u0026deg;C) and relative humidity at 45% (\\u0026plusmn;\\u0026thinsp;3%), a vapor pressure deficit was approximately 1 kPa. To minimize the confounding effects of stomatal water loss on experimental measurements, parallel trials were performed under both light (photosynthetically active radiation 160 \\u0026micro;mol m⁻\\u0026sup2; s⁻\\u0026sup1;) and dark conditions. To measure the rate of water loss, we employed the following formula: Water loss rate = (Initial weight - Weight at a certain time point) / Initial weight \\u0026times; 100%.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe IBM SPSS Statistics 25 software was utilized for statistical analyses. Means were compared using one-way analysis of variance (ANOVA). Quantification of cuticle and cell wall thickness was conducted using ImageJ software.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eGPAT1\\u003c/strong\\u003e \\u003cstrong\\u003eand\\u003c/strong\\u003e \\u003cstrong\\u003eGPAT2\\u003c/strong\\u003e \\u003cstrong\\u003einvolve in plant cuticle layer development\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn our previous study, we discovered that the Arabidopsis double mutant \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e displayed significant developmental defects, including abnormalities in both embryonic and postembryonic stages (Jia et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In addition to the previously documented phenotypes, we identified another noteworthy phenomenon. When placed on the surface of water, most cotyledons of wild-type (WT) plants float, while most cotyledons of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e sink to the bottom within seconds (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The \\u003cem\\u003egpat1\\u003c/em\\u003e, \\u003cem\\u003egpat2\\u003c/em\\u003e, and complement lines \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e exhibited a phenotype similar to that of WT (Supplemental Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). This observation suggests that the cuticle layer of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e is compromised or that the density of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e cotyledons has increased, making them denser than water. It was reported that \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e double mutant strongly reduced in cutin and its cuticle showed high permeability to TB solution (Li et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). To verify this hypothesis, we set the abnormal cuticle mutant \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e as a control. We found that \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e also floated on the surface of water like the WT. This result suggested that abnormal cuticle can\\u0026rsquo;t make cotyledons sank. So the sinking of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e to the bottom is most likely caused by an increase in its density.To investigate the reasons behind this phenomenon, we performed TB staining on etiolated seedlings of WT, \\u003cem\\u003egpat1\\u003c/em\\u003e, \\u003cem\\u003egpat2\\u003c/em\\u003e, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e, and \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e grown for six days on 1/2 MS medium to evaluate cuticle layer integrity. As shown in the results, the cotyledons of WT, \\u003cem\\u003egpat1\\u003c/em\\u003e, \\u003cem\\u003egpat2\\u003c/em\\u003e, \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e, and \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e remained unstained, while TB penetrated \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, causing the entire cotyledons and hypocotyls to appear blue (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC), indicating a compromised cuticle establishment in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. Stained cotyledons of equal weight were collected, and the dye was extracted in 75% ethanol for two hours, followed by absorbance measurement at A626 using a spectrophotometer. The absorbance value of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e was significantly higher than those of WT, the single mutants \\u003cem\\u003egpat1\\u003c/em\\u003e and \\u003cem\\u003egpat2\\u003c/em\\u003e, as well as the complement lines \\u003cem\\u003eGPAT1::gpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003eGPAT2::gpat1 gpat2\\u003c/em\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). These results suggest that \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e are involved in cuticle development and show functional redundancy.\\u003c/p\\u003e\\n\\u003cp\\u003eMoreover, we found that trichome development in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e is defective across multiple organs (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Trichomes are specialized structures covered with the cuticular layer Using a stereomicroscope, we examined the trichomes on the seedlings, flowers, and stems of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. As shown in the figure, WT exhibited prominent trichomes on the leaves of seedlings, flowers, and stems, whereas some \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e plants lacked trichomes altogether (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The proportions of abnormal phenotypes in leaves, flowers, and stems were 95.6%, 74.2%, and 18.3%, respectively (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003egpat1 gpat2\\u003c/strong\\u003e \\u003cstrong\\u003ehas structural alterations in the cuticular layer\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSuberin is a plant polymer with water-repelling and protective properties, and its chemical composition is similar to that of cutin (Pollard et al. 2018).\\u003c/p\\u003e\\n\\u003cp\\u003eFY 088 specifically binds to fatty acids and used for cuticle layer staining. We stained the cotyledons of WT, \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e and \\u003cem\\u003egpat4 gpat8\\u003c/em\\u003e, using FY088 and observed the cuticle layer under CLSM (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). The fluorescence intensity of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e showed no significant differences, but the cuticle layer of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e presents irregular characteristic (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA).\\u003c/p\\u003e\\n\\u003cp\\u003eTo observe the epidermal structure more clearly, we performed SEM on 20-day-old leaves and TEM on the cotyledons of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. We found that the cutin layer of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e cotyledons was compromised. Specifically, the cutin layer of WT exhibited a smooth surface, whereas gpat1 gpat2 displayed an eroded surface and was thinner compared to WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and C). Interestingly, we also observed that the cell wall of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e was thicker than that of WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and D). These observations suggests that the mutations in \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e may affect secondary cell wall (SCW) synthesis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003egpat1 gpat2\\u003c/strong\\u003e \\u003cstrong\\u003eshows a decrease in the amount of cutin monomers\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo elucidate the roles of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e in cuticle permeability, we employed freeze SEM to observe the wax in one-month-old rosette leaves of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. The total wax content of rosette leaves in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e showed no significant difference compared to WT (Supplemental Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e and S3). We then measured the cutin monomers in seedlings of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. The results revealed that all the cutin monomer C16:0, C18:1, C18:2 \\u0026omega;-OH acid and C16:0, C18:0, C18:1, C18:2 dioic acid of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e were reduced significantly compared to WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The total cutin content in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e decreased by approximately roughly 33% (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). This reduction corresponded closely to the cutin layer observations from TEM (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGPAT1\\u003c/strong\\u003e \\u003cstrong\\u003eand\\u003c/strong\\u003e \\u003cstrong\\u003eGPAT2\\u003c/strong\\u003e \\u003cstrong\\u003eregulate the formation of SCW\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDuring the experiment, we also found that the cotyledons and hypocotyls of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e were firmer than those of WT, prompting speculation that this phenomenon may be due to increased lignification or cellularization. To verify this hypothesis, we stained WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e seedlings with phloroglucinol, a dye specific for lignin. After staining, the degree of lignification was indicated by varying shades of red or purple-red, with darker colors indicating a greater degree of lignification. The results showed that the cotyledon and hypocotyl coloration of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e were more intense than those of WT, whereas the root coloration showed no significant differences between the two (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and B). Furthermore, measurements of total lignin content revealed significantly higher levels in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e compared to WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC; Supplemental Fig. S3A).\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition, we assessed the cellulose content of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e seedlings using the zinc chloroiodide method. The cotyledon and hypocotyl coloration of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e was significantly more intense than in the WT, indicating that the cellulose content in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e was higher than that of WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). To further substantiate this observation, we quantified the cellulose content in WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e seedlings, confirming that \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e contained higher cellulose levels compared to WT (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB).\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate how \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e regulate lignin and cellulose content, we conducted RT-qPCR to detect the expression of lignin and cellulose synthesis genes in the cotyledons of WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. Genes involved in lignin synthesis including \\u003cem\\u003ePAL, C4H, 4CL, CCR, CAD, F5H, COM\\u003c/em\\u003e, and members of the \\u003cem\\u003eLAC\\u003c/em\\u003e family were selected to assess transcript levels in WT and \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. The results showed that, except for \\u003cem\\u003eLAC15\\u003c/em\\u003e, almost all genes were upregulated, with \\u003cem\\u003eLAC12\\u003c/em\\u003e exhibiting the highest level of upregulation, followed closely by \\u003cem\\u003ePAL3\\u003c/em\\u003e. For cellulose synthesis, nine genes from the \\u003cem\\u003eCESA\\u003c/em\\u003e family were analyzed (Kumar and Turner, \\u003cspan class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Among these, \\u003cem\\u003eCESA4\\u003c/em\\u003e, \\u003cem\\u003eCESA8\\u003c/em\\u003e, and \\u003cem\\u003eCESA9\\u003c/em\\u003e were significantly upregulated, while \\u003cem\\u003eCESA1\\u003c/em\\u003e and \\u003cem\\u003eCESA6\\u003c/em\\u003e showed a slight downregulation in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. These results suggest that \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e act as negative regulators of lignin and cellulose synthesis. In the absence of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e, the expression of several lignin and cellulose synthesis genes was elevated, leading to an accumulation of lignin and cellulose in the cell wall of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e. Consequently, the cell wall becomes thicker and harder, resulting in a firmer texture of the cotyledons. This finding explains the observed thickening of the cell wall shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGPAT1\\u003c/strong\\u003e \\u003cstrong\\u003eand\\u003c/strong\\u003e \\u003cstrong\\u003eGPAT2\\u003c/strong\\u003e \\u003cstrong\\u003emaintain water balance in plants\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate whether the absence of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e affects the plants response to water stress, we conducted a water loss assay on the seedlings. In order to avoid the interference of stomatal water loss on the results, we conducted this assay under light and dark conditions respectively. The results indicated that when under light conditions, the water loss rates of all plant materials were faster than those under dark conditions. Notably, regardless of light or dark conditions, the water loss rate in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e was lower compared to that in the WT and complementary lines. (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA). Based on the findings presented in this article, we propose a model. In WT, with the involvement of \\u003cem\\u003eGPAT1/2\\u003c/em\\u003e, the biosynthesis of cutin monomers in plant cells proceeds normally, enabling proper development of secondary walls and trichomes, while maintaining balanced water flux in and out of the cells. But in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, in the absence of \\u003cem\\u003eGPAT1/2\\u003c/em\\u003e, cutin synthesis is impaired, resulting in a thinner and irregular cuticle, which enhances cellular permeability, along with reduced trichome density. Concurrently, the levels of lignin and cellulose (major components of the cell wall) increase, leading to thickened cell walls and suppressed water efflux.\\u003c/p\\u003e\\n\\u003cp\\u003eWater deficit assay under light and dark conditions. Data are presented as means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD from three biologically independent experiments. Asterisks indicate significant differences compared with WT. Statistical analysis was performed using one-way ANOVA. *** indicates p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001.(B) A proposed model illustrating the mechanisms by which \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e regulate cuticle and SCW development. In WT, with the involvement of \\u003cem\\u003eGPAT1/2\\u003c/em\\u003e, the biosynthesis of cutin monomers in plant cells proceeds normally, enabling proper development of secondary walls and trichomes, while maintaining balanced water flux in and out of the cells. But in \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, in the absence of \\u003cem\\u003eGPAT1/2\\u003c/em\\u003e, cutin synthesis is impaired, resulting in a thinner and irregular cuticle, which enhances cellular permeability, along with reduced trichome density. Concurrently, the levels of lignin and cellulose\\u0026mdash;major components of the cell wall\\u0026mdash;increase, leading to thickened cell walls and suppressed water efflux.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe cuticle plays a key role in biotic and abiotic defense, such as regulating water, gas, and ion fluxes, as well as providing protection against pathogens. Cutin is an extracellular lipid that, along with waxes and some polysaccharides, forms the cuticle layer (Ranathunge et al. 2011; Yang et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Trichomes are epidermal appendages extending from plant surfaces, aiding in resistance to external stresses by preventing physical damage, deterring pests and diseases, and reducing water evaporation through slowed air movement, thereby aiding in maintaining plant water balance. The SCW provides strong mechanical support for plant organs such as stems and roots, while its hard texture and hydrophobic nature effectively resist external invasions. The cuticle, trichomes, and SCW serve as the first line of defense when plants interact with their external environment, coordinating with one another to support plants adaptation to their surroundings. Understanding the composition and changes in these structures is of great significance for plant stress tolerance.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGPAT1 and GPAT2 influence cuticle and trichome development\\u003c/b\\u003e The cuticle layer envelops the trichomes, forming an integrated protective system for the plant surface. Cutin and wax are the primary components of the cuticle. In this study, we discovered that the deficiency of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e led to defects in cuticle and trichome development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Numerous studies have indicated that there is a genetic interplay between cuticle formation and trichomes development (Chalvin et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Berhin et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Several Arabidopsis cutin/wax-related mutants, such as \\u003cem\\u003eglassy hair6/cer10\\u003c/em\\u003e, \\u003cem\\u003eyre/cer3\\u003c/em\\u003e, and \\u003cem\\u003eabcg11\\u003c/em\\u003ewere reported to exhibit trichome defects (Suo et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Berhin et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In wheat, \\u003cem\\u003eAaABCG20\\u003c/em\\u003e, which is responsible for cutin and wax transport, overexpression of \\u003cem\\u003eAaABCG20\\u003c/em\\u003e resulted in an increase in glandular trichome density (Fu et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Additionally, modifications to the cell wall can affect proper cuticle deposition and, consequently, impact trichome integrity (Philippe et al. \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Studies on the \\u003cem\\u003ereduced wall acetylation2\\u003c/em\\u003e mutant, which displays collapsed trichomes, have confirmed a reduction in pectin and hemicellulose acetylation and highlighted the deposition of a thicker yet more permeable cuticle at the base of trichomes (Nafisi et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). These cuticle abnormalities suggest that cell wall acetylation is necessary for maintaining appropriate rigidity and the integrity of epidermal structures such as trichomes (Nafisi et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Overexpression of \\u003cem\\u003eSHN1/2/3\\u003c/em\\u003e increases cuticle components while reducing trichome density. (Aharoni et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Broun et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). AtMYB106 and AtMYB16 regulate cutin and wax biosynthesis by activating \\u003cem\\u003eAtSHN1\\u003c/em\\u003e, and induction of cuticle formation by AtMYB106 negatively impacts trichome branching (Oshima et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Class I TCPs members such as TCP14 and TCP15, coordinate with \\u003cem\\u003eAtMYB106\\u003c/em\\u003e and cutin/wax-related genes to involve in trichome and cuticle development. \\u003cem\\u003eSolanum lycopersicum SlMIXTA\\u003c/em\\u003e-like negatively regulates trichome development while positively influencing cutin deposition (Lashbrooke et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Galdon-Armero et al. 2020). Although these studies suggest that there is an interaction between the cuticle, cell wall, and trichomes, the specific mechanisms underlying this interaction are still not well understood. The roles of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e in regulating the relationship between the cuticle and trichomes remain particularly unclear.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGPAT1\\u003c/b\\u003e \\u003cb\\u003eand\\u003c/b\\u003e \\u003cb\\u003eGPAT2\\u003c/b\\u003e \\u003cb\\u003eare involved in cutin synthesis\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eIndeed, many ER-GPATs have been reported to be involved in epidermal lipid synthesis across various plants, including Arabidopsis, rice, tomato, and rapeseed (Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). However, no current evidence suggests that Mt-GPAT is involved in the synthesis of cuticular lipids in vivo. Previous reviews noted that GPAT1 and GPAT2 are involved in cutin synthesis, although this was merely summarized in tabular form without elaboration (Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). In the current study, we found that the two Mt-GPATs, GPAT1 and GPAT2, redundantly contribute to cutin synthesis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eGPAT is recognized as the first rate-limiting enzyme in the Kennedy pathway, catalyzing the conversion of glycerol-3-phosphate (G-3-P) to lysophosphatidic acid (LPA) (Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). GPAT1 exhibits specific \\u003cem\\u003esn\\u003c/em\\u003e-2 acyltransferase activity and utilizes ω-oxidized fatty acids and unmodified acyl-CoA as substrates, while GPAT2 uses DCA-CoA as its substrate (Yang et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Jayawardhane et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Yang et al. examined the polymeric lipids in \\u003cem\\u003egpat1\\u003c/em\\u003e but did not observe a clear phenotype associated with cutin in the mutant (Yang et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Our findings address this inquiry, indicating that GPAT1 and GPAT2 function redundantly and require co-expression to exhibit a phenotype.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGPAT1\\u003c/b\\u003e \\u003cb\\u003eand\\u003c/b\\u003e \\u003cb\\u003eGPAT2\\u003c/b\\u003e \\u003cb\\u003eregulate the formation of the SCW\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eA certain thickness of the cell wall is essential for providing mechanical support and protection to plants, but excessively thick walls can be detrimental as they may impede water and nutrient transport, hinder light energy utilization, and delay defense mechanism activation (Barros et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Liu et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). In the absence of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e, transcriptional levels of genes associated with lignin and cellulose synthesis are upregulated (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e and \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e), causing excessive lignin and cellulose accumulation, which thickens the SCW (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD, and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003eWhen plants encounter external environmental stresses, the absence of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e leads to a thinner cuticle, exposing them to osmotic stress (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). To cope with this stress, plants may respond by thickening their cell walls to resist excessive water entry. Osmotic stress is known to enhance lignin accumulation by increasing G and S units (Yang et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Phytohormones play a crucial role in regulating SCW deposition (Liu et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn our previous study, we found that lysophosphatidic acid (LPA), another product of GPAT1 and GPAT2, can mediate auxin polar transport and distribution, regulating plant embryonic and postembryonic development (Jia et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Recently, research demonstrated that auxin deficiency in vascular cells may lead to persistent lignin accumulation at wound sites (Xu et al. \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Therefore, GPAT1 and GPAT2 may also mediate auxin signaling to regulate SCW formation. Several studies reported that abscisic acid (ABA) and jasmonic acid (JA) signaling are involved in lignin synthesis. The ABA signaling regulator SnRK2 phosphorylates the master transcription factor AtNST1, enhancing SCW formation and lignin production (Liu et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Exogenous ABA has been shown to induce phenylpropanoid metabolism, increasing the synthesis of phenolic acids and lignin monomers in muskmelon wounds during healing. (Wang et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2024b\\u003c/span\\u003e). Whether \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e directly regulate genes related to SCW synthesis or influence them through signaling molecules like auxin and ABA is a topic worthy of further exploration.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGPAT1\\u003c/b\\u003e \\u003cb\\u003eand\\u003c/b\\u003e \\u003cb\\u003eGPAT2\\u003c/b\\u003e \\u003cb\\u003ehelp plants maintaining water balance\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe cutin layer of \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e is relatively thinner compared to that of WT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), but its dehydration rate is slower than that of WT, indicating that the thickening of the SCW affects the outward loss of intracellular water (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Cutin possesses strong hydrophobic properties and effectively prevents excessive water loss within plants.\\u003c/p\\u003e \\u003cp\\u003eIn summary, trichomes, cutin, and the SCW likely interact with one another to maintain plant structure and function. We provided evidence that the loss of function of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e results affects the structure of the epidermis in Arabidopsis cotyledons. Further research indicated that these two Mt-GPATs are involved in cutin synthesis. Our findings demonstrate that \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e negatively regulate SCW formation. The mutation of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e leads to a thicker SCW, which affects the water balance within the cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAuthor contribution statement\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eAll authors contributed to the study conception and design. Q.J. designed research; Q.J., Y.B., H.Z. and X.L. performed research; W.Z. and Q.W. analyzed data; Q.Z. and X.C. provided useful scientific advice; and Q.J. and H.C. wrote the manuscript. All authors commented on draft versions of the manuscript. All authors read and approved the final version of the manuscript.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eCompeting Interests\\u003c/h2\\u003e \\u003cp\\u003eThe authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis work was supported by grants provided by the China Postdoctoral Science Foundation (2023M731401) and the Jiangsu Funding Program for Excellent Postdoctoral Talent (2023ZB647).\\u003c/p\\u003e\\u003ch2\\u003eData availability\\u003c/h2\\u003e \\u003cp\\u003eThe data and materials supporting the findings of this study are available from the corresponding author upon request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16(9):2463\\u0026ndash;2480\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarros J, Serk H, Granlund I, Pesquet E (2015) The cell biology of lignification in higher plants. Ann Bot 115(7):1053\\u0026ndash;1074\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBeisson F, Li Y, Bonaventure G, Pollard M, Ohlrogge JB (2007) The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19(1):351\\u0026ndash;368\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBeris DG, Kapolas P, Livanos A, Roussis D, Milioni K, Haralampidis (2016) RNAi-mediated silencing of the Arabidopsis thaliana \\u003cem\\u003eULCS1\\u003c/em\\u003e gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility. Plant Sci 245:71\\u0026ndash;83\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBerhin A, Nawrath C, Hachez C (2022) Subtle interplay between trichome development and cuticle formation in plants. New Phytol 233(5):2036\\u0026ndash;2046\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X, Yephremov A, Samuels L (2007) Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J 52(3):485\\u0026ndash;498\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBroun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci U S A 101(13):4706\\u0026ndash;4711\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBorah P, Khurana JP (2018) The OsFBK1 E3 ligase subunit affects anther and root secondary cell wall thickenings by mediating turnover of a Cinnamoyl-CoA reductase. Plant Physiol 176:2148\\u0026ndash;2165\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCamoirano A, Arce AL, Ariel FD, Alem AL, Gonzalez DH, Viola IL (2020) Class I TCP transcription factors regulate trichome branching and cuticle development in Arabidopsis. J Exp Bot 71(18):5438\\u0026ndash;5453\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChalvin C, Drevensek S, Dron M, Bendahmane A, Boualem A (2020) Genetic control of glandular trichome development. Trends Plant Sci 25(5):477\\u0026ndash;487\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGeng P, Zhang S, Liu J, Zhao C, Wu J, Cao Y, Fu C, Han X, He H, Zhao Q (2020) MYB20, MYB42, MYB43, and MYB85 regulate phenylalanine and lignin biosynthesis during secondary cell wall formation. Plant Physiol 182(3):1272\\u0026ndash;1283\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGonz\\u0026aacute;lez-Valenzuela L, Renard J, Dep\\u0026egrave;ge-Fargeix N, Ingram G (2023) The plant cuticle. Curr Biol 33(6):R210\\u0026ndash;R214\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChen X, Truksa M, Snyder CL, El-Mezawy A, Shah S, Weselake RJ (2011) sn-Glycerol-3-phosphate acyltransferases in plants. Plant Signal Behav 6(11):1695\\u0026ndash;1699\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFeng H, Li X, Chen H, Deng J, Zhang C, Liu J, Wang T, Zhang X, Dong J (2018) GhHUB2, a ubiquitin ligase, is involved in cotton fiber development via the ubiquitin-26S proteasome pathway. J Exp Bot 69(21):5059\\u0026ndash;5075\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFu X, Zheng H, Wang Y, Liu H, Liu P, Li L, Zhao J, Sun X, Tang K (2024) AaABCG20 transporter involved in cutin and wax secretion affects the initiation and development of glandular trichomes in Artemisia annua. Plant Sci 339:111959\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDonnelly PM, Bonetta D, Tsukaya H, Dengler RE, Dengler NG (1999) Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215(2):407\\u0026ndash;419\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDuo H, Yin M, Wang R (2024) Molecular mechanisms of resistance and future perspectives in plant breeding strategies against Sclerotinia sclerotiorum. New Crops 2:100046\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eElejalde-Palmett C, Martinez San Segundo I, Garroum I, Charrier L, De Bellis D, Mucciolo A, Guerault A, Liu J, Zeisler-Diehl V, Aharoni A et al (2021) ABCG transporters export cutin precursors for the formation of the plant cuticle. Curr Biol 31(10):2111\\u0026ndash;2123\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGully K, Berhin A, De Bellis D, Herrfurth C, Feussner I, Nawrath C (2024) The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae. Proc Natl Acad Sci U S A 121(21):e2314570121\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHan G, Li Y, Yang Z, Wang C, Zhang Y, Wang B (2022) Molecular mechanisms of plant trichome development. Front Plant Sci 13:910228\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHuang H, Wang Y, Yang P, Zhao H, Jenks MA, L\\u0026uuml; S, Yang X (2024) The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers. Plant J 118(5):1619\\u0026ndash;1634\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eH\\u0026uuml;lskamp M (2019) Trichomes Curr Biol 29(8):R273\\u0026ndash;R274\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eIngram G, Nawrath C (2017) The roles of the cuticle in plant development: organ adhesions and beyond. J Exp Bot 68(19):5307\\u0026ndash;5321\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJayawardhane KN, Singer SD, Weselake RJ, Chen G (2018) Plant sn-Glycerol-3-Phosphate Acyltransferases: biocatalysts involved in the biosynthesis of intracellular and extracellular lipids. Lipids 53(5):469\\u0026ndash;480\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJia Q, Bai Y, Xu H, Liu Q, Li W, Li T, Lin F, Shen L, Xuan W, Zhang W et al (2022) Mitochondrial GPAT-derived LPA controls auxin-dependent embryonic and postembryonic development. Proc Natl Acad Sci U S A 119(49):e2212881119\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJung KH, Han MJ, Lee D, Lee YS, Schreiber L, Franke R, Faust A, Yephremov A, Saedler H, Kim YW et al (2006) Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development. Plant Cell 18(11):3015\\u0026ndash;3032\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKo JH, Jeon HW, Kim WC, Kim JY, Han KH (2014) The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Ann Bot 114(6):1099\\u0026ndash;1107\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19(16):1855\\u0026ndash;1860\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKumar M, Turner S (2015) Plant cellulose synthesis: CESA proteins crossing kingdoms. Phytochemistry 112:91\\u0026ndash;99\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKumar M, Campbell L, Turner S (2016) Secondary cell walls: biosynthesis and manipulation. J Exp Bot 67(2):515\\u0026ndash;531\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLashbrooke JG, Adato A, Lotan O, Alkan N, Tsimbalist T, Rechav K, Fernandez Moreno J-P, Widemann E, Grausem B, Pinot F et al (2015) The tomato MIXTA-like transcription factor coordinates fruit epidermis conical cell development and cuticular lipid biosynthesis and assembly. Plant Physiol 169(4):2553\\u0026ndash;2571\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi-Beisson Y, Pollard M, Sauveplane V, Pinot F, Ohlrogge J, Beisson F (2009) Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester. Proc Natl Acad Sci U S A 106(51):22008\\u0026ndash;22013\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi Y, Beisson F, Koo AJK, Molina I, Pollard M, Ohlrogge J (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci U S A 104(46):18339\\u0026ndash;18344\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiu C, Yu H, Rao X, Li L, Dixon RA (2021) Abscisic acid regulates secondary cell-wall formation and lignin deposition in Arabidopsis thaliana through phosphorylation of NST1. Proc Natl Acad Sci U S A 118(5):e2010911118\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMat\\u0026iacute;as-Hern\\u0026aacute;ndez L, Aguilar-Jaramillo AE, Cigliano RA, Sanseverino W, Pelaz S (2016) Flowering and trichome development share hormonal and transcription factor regulation. J Exp Bot 67(5):1209\\u0026ndash;1219\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMen X, Shi J, Liang W, Zhang Q, Lian G, Quan S, Zhu L, Luo Z, Chen M, Zhang D (2017) Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3) is required for anther development and male fertility in rice. J Exp Bot 68(3):513\\u0026ndash;526\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNafisi M, Stranne M, Fimognari L, Atwell S, Martens HJ, Pedas PR, Hansen SF, Nawrath C, Scheller HV, Kliebenstein DJ et al (2015) Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses. Front Plant Sci 6:550\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M (2013) MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. Plant Cell 25(5):1609\\u0026ndash;1624\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOhtani M, Demura T (2019) The quest for transcriptional hubs of lignin biosynthesis: beyond the NAC-MYB-gene regulatory network model. Curr Opin Biotechnol 56:82\\u0026ndash;87\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePetit J, Bres C, Mauxion J-P, Wong Jun Tai F, Martin LBB, Fich EA, Joub\\u0026egrave;s J, Rose JKC, Domergue F, Rothan C (2016) The glycerol-3-phosphate acyltransferase GPAT6 from tomato plays a central role in fruit cutin biosynthesis. Plant Physiol 171(2):894\\u0026ndash;913\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePhilippe G, Geneix N, Petit J, Guillon F, Sandt C, Rothan C, Lahaye M, Marion D, Bakan B (2020) Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides. New Phytol 226(3):809\\u0026ndash;822\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePollard M, Beisson F, Li Y, Ohlrogge JB (2008) Building lipid barriers: biosynthesis of cutin and suberin. Trends Plant Sci 13(5):236\\u0026ndash;246\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSuo B, Seifert S, Kirik V (2013) Arabidopsis GLASSY HAIR genes promote trichome papillae development. J Exp Bot 64(16):4981\\u0026ndash;4991\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTraw MB, Bergelson J (2003) Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Plant Physiol 133(3):1367\\u0026ndash;1375\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang Q, Lei S, Yan J, Song Y, Qian J, Zheng M, Hsu YF (2023) UBC6, a ubiquitin-conjugating enzyme, participates in secondary cell wall thickening in the inflorescence stem of Arabidopsis. Plant Physiol Biochem 205:108152\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang H, Zhao Q, Chen F, Wang M, Dixon RA (2011) NAC domain function and transcriptional control of a secondary cell wall master switch. Plant J 68(6):1104\\u0026ndash;1114\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang Q, Liu N, Yang R, Zhang X, Wang Y, Li Y, Prusky D, Bi Y, Han Y (2024a) Essential role of ABA signaling and related transcription factors in phenolic acid and lignin synthesis during muskmelon wound healing. Front Plant Sci 15:1404477\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang Y, Li P, Sun W, Zhang T (2024b) Plant cell walls: Emerging targets of stomata engineering to improve photosynthesis and water use efficiency. New Crops 1:100021\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWaszczak C, Carmody M, Kangasj\\u0026auml;rvi J (2018) Reactive oxygen species in plant signaling. Annu Rev Plant Biol 69:209\\u0026ndash;236\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWellesen K, Durst F, Pinot F, Benveniste I, Nettesheim K, Wisman E, Steiner-Lange S, Saedler H, Yephremov A (2001) Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development. Proc Natl Acad Sci U S A 98(17):9694\\u0026ndash;9699\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eXu H, Dong C, Wu Y, Fu S, Tauqeer A, Gu X, Li Q, Niu X, Liu P, Zhang X et al (2024) The JA-to-ABA signaling relay promotes lignin deposition for wound healing in Arabidopsis. Mol Plant 17(10):1594\\u0026ndash;1605\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang J, Song J, Feng Y, Cao Y, Fu B, Zhang Z, Ma N, Li Q, Hu T, Wang Y et al (2023) Osmotic stress-induced lignin synthesis is regulated at multiple levels in alfalfa (Medicago sativa L). Int J Biol Macromol 246:125501\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang W, Simpson JP, Li-Beisson Y, Beisson F, Pollard M, Ohlrogge JB (2012) A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution. Plant Physiol 160(2):638\\u0026ndash;652\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhan J, Di T, Chen X, Zheng T, Sun W, Yang M, Zhou M, Shen Z, Chen H, Su N (2024) CbMYB108 integrates the regulation of diterpene biosynthesis and trichome development in Conyza blinii against UV-B. Plant Cell Environ 47(4):1300\\u0026ndash;1318\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang X, Gao H, Liu Y, Zhao H, L\\u0026uuml; S (2024) Function identification of Arabidopsis GPAT4 and GPAT8 in the biosynthesis of suberin and cuticular wax. Plant Sci 339:111933\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhong R, Ye ZH (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav 4(11):1028\\u0026ndash;1034\\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\":\"info@researchsquare.com\",\"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\":\"Mt-GPAT, cutin, trichome, secondary cell wall, water balance\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6445935/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6445935/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eTrichomes and cuticles are critical epidermal adaptations that serve protective roles in plants. The cuticle functions as a barrier, allowing for controlled interactions between the plant and its environment. Cutin synthesis is crucial for plants to withstand various external stresses. In this study, we report on the Arabidopsis mutant \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, which exhibits a highly permeable cuticle and defects in trichome development. Mutation of \\u003cem\\u003eGPAT1\\u003c/em\\u003eand \\u003cem\\u003eGPAT2\\u003c/em\\u003e resulted in a reduction of cutin monomer. In \\u003cem\\u003egpat1 gpat2\\u003c/em\\u003e, the structure of the cuticular layer of the cell wall is notably altered. Additionally, \\u003cem\\u003eGPAT1 \\u003c/em\\u003eand \\u003cem\\u003eGPAT2\\u003c/em\\u003e are found to negatively regulate the synthesis of lignin and cellulose, which are related to secondary cell wall (SCW) formation. The dysfunction of \\u003cem\\u003eGPAT1\\u003c/em\\u003e and \\u003cem\\u003eGPAT2\\u003c/em\\u003e disrupted the water balance of the plant. Our findings reveal a network where mitochondrial GPAT1 and GPAT2 play roles in maintaining water balance by participating in both Arabidopsis cutin synthesis and SCW formation.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Mitochondrial GPATs protect plants from water stress by impacting cuticle and secondary cell wall development\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-07 18:52:06\",\"doi\":\"10.21203/rs.3.rs-6445935/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"268f2e69-93ed-49ef-9eb2-91dad2c16f59\",\"owner\":[],\"postedDate\":\"May 7th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-05-26T16:13:59+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-05-07 18:52:06\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6445935\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6445935\",\"identity\":\"rs-6445935\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}