Differential roles of duplicate genes OsATG9a and OsATG9b in development and drought stress response in rice

Differential roles of duplicate genes OsATG9a and OsATG9b in development and drought stress response in rice | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 February 2025 V1 Latest version Share on Differential roles of duplicate genes OsATG9a and OsATG9b in development and drought stress response in rice Authors : Yiming Li , Yuantai Liu , Mengzhao Shi , Xiaoyun Luo , Yanshu Huang , Hao Zeng , Yunfeng Liu , … Show All … , Yifeng Huang , Peng Xu , Yangwen Qian , Xixian Li , Jieying Wang , Qingjun Xie 0000-0002-6372-3260 , and Qianying Yang [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.173856974.49208859/v1 384 views 231 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Gene duplication events frequently occur during eukaryotic genome evolution, often leading to functional redundancy for organism survival in complex environments. However, whether duplicate genes evolve diverse functions remains unclear. In this study, we explored the roles of autophagy-related gene 9 OsATG9a and OsATG9b in rice development and drought stress responses. Autophagy, an evolutionarily conserved degradation pathway, plays a critical role in multiple biological processes by recycling cellular components. We found both OsATG9a and OsATG9b involved in autophagy, with functional redundancy affecting traits like grain size, plant height, tiller number, primary branch number, and panicle length. Notably, OsATG9b exhibited a distinct response to drought stress. The osatg9a mutant displayed a lower survival rate than wild type (WT) after drought stress, similar to other osatg mutants, while the osatg9b mutant showed the opposite. Moreover, autophagy flux decreased in osatg9a mutant but increased in osatg9b , surpassing WT response. Overexpression of OsATG9b resulted in lower survival rates and reduced autophagy induction under drought stress. Moreover, the response of ABA related genes in osatg9a and in osatg9b were opposite compared with WT. These suggest that OsATG9a promotes autophagy during drought stress, while OsATG9b negatively impacts it, representing a newly evolved function in rice by differently regulating ABA pathway. Our findings provided insights into the functional divergence of duplicate genes during evolution. Differential roles of duplicate genes OsATG9a and OsATG9b in development and drought stress response in rice Yiming Li 1,† , Yuantai Liu 1,† , Mengzhao Shi 1,† , Xiaoyun Luo 1 , Yanshu Huang 1 , Hao Zeng 1 , Yunfeng Liu 2 , Yifeng Huang 3 , Peng Xu 4 , Yangwen Qian 5 , Xixian Li 1 , Jieying Wang 1 , Qingjun Xie 1,* , Qianying Yang 1,* 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Key Laboratory for Enhancing Resource Use Efficiency of Crops in South China, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University, Guangzhou 510642, China. 2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences and Technology, Guangxi University, Nanning 530004, China. 3 Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Science, Hangzhou, 310001, China. 4 CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, The Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, China. 5 WIMI Biotechnology Co. Ltd., Changzhou, 213000, China. * Corresponding author: Dr. Qianying Yang ( [email protected] ) and Dr. Qingjun Xie ( [email protected] ) † These authors contributed equally to this work. Short informative title: Function differences between OsATG9a and OsATG9b Abstract Gene duplication events frequently occur during eukaryotic genome evolution, often leading to functional redundancy for organism survival in complex environments. However, whether duplicate genes evolve diverse functions remains unclear. In this study, we explored the roles of autophagy-related gene 9 OsATG9a and OsATG9b in rice development and drought stress responses. Autophagy, an evolutionarily conserved degradation pathway, plays a critical role in multiple biological processes by recycling cellular components. We found both OsATG9a and OsATG9b involved in autophagy, with functional redundancy affecting traits like grain size, plant height, tiller number, primary branch number, and panicle length. Notably, OsATG9b exhibited a distinct response to drought stress. The osatg9a mutant displayed a lower survival rate than wild type (WT) after drought stress, similar to other osatg mutants, while the osatg9b mutant showed the opposite. Moreover, autophagy flux decreased in osatg9a mutant but increased in osatg9b , surpassing WT response. Overexpression of OsATG9b resulted in lower survival rates and reduced autophagy induction under drought stress. Moreover, the response of ABA related genes in osatg9a and in osatg9b were opposite compared with WT. These suggest that OsATG9a promotes autophagy during drought stress, while OsATG9b negatively impacts it, representing a newly evolved function in rice by differently regulating ABA pathway. Our findings provided insights into the functional divergence of duplicate genes during evolution. gene duplication; autophagy; grain development; OsATG; rice; stress response 1 | INTRODUCTION Gene duplication plays a pivotal role in the evolution of genome and species. A significant portion of protein-coding genes as well as numerous non-coding regulatory elements have originated through duplication events (Magadum et al., 2013; Ohta, 2000). Following duplication, a subset of duplicated genes retains the ancestral function alongside the original gene or acquiring a novel function (Maskalenka et al., 2023). Consequently, the emergence of duplicated genes or regulatory sequences equips organisms with the capacity to exploit new competitive advantages and to adapt to changing environments (Fenech et al., 2020). Autophagy is an evolutionarily conserved catabolic process essential for the degradation and recycling of cellular components. In plants, autophagy plays a critical role in development and is typically induced by stress (Yagyu et al., 2023). Drought is a common environmental stress that plants encounter. Numerous studies have shown that autophagy is triggered in response to drought stress in various plants, including Arabidopsis (Hachez et al., 2014; Liu et al., 2009), Medicago (Yang et al., 2021), tomato (Wang et al., 2015), apple (Jia et al., 2021b; Jia et al., 2021a; Sun et al., 2018; Xiang et al., 2024), banana (Li et al., 2019a), barley (Zeng et al., 2017), emmer wheat (Kuzuoglu-Ozturk et al., 2012), tomato (Zhu et al., 2018), and peach (Wang et al., 2019). In Arabidopsis, mutants of ATG genes (AuTophaGy related genes), such as atg5 , atg7 , and RNAi-ATG18a, which cannot activate autophagy under drought conditions, exhibit hypersensitivity to drought stress (Liu et al., 2019; Zhou et al., 2013). Arabidopsis PIP2;7 (Plasma Membrane Intrinsic Protein 2;7) interacts with TSPO (Tryptophan-rich Sensory Protein) and is selectively degraded by macroautophagy. This selective autophagy helps to prevent water loss from the cell by degrading PIP2;7s, thereby enhancing drought tolerance (Hachez et al., 2014). However, the regulatory mechanisms of autophagy in rice under drought stress are still not well understood. The formation of autophagy is regulated by numerous ATGs (Liu et al., 2021). These ATGs, along with other components, orchestrate the canonical autophagic pathway. Initially, ATG13 undergoes rapid dephosphorylation, promoting interactions between ATG1 and ATG13, as well as between ATG13 and ATG17, thereby forming the ATG1 kinase complex. Once activated, this complex recruit downstream ATG proteins to initiate autophagosome formation (Li et al., 2014; Yao et al., 2023). ATG9 is essential for ER-derived autophagosome formation in plants, with its deficiency causing accumulation of autophagosome-related tubules (Zhuang et al., 2017). Subsequently, the autophagosome is adorned with ATG8 conjugated to phosphatidylethanolamine (PE) to form the ATG8-PE adduct (Marshall and Vierstra, 2018). Concurrently, ATG8 interacts with cargo receptors or proteins containing either the Atg8-interacting motif (AIM) or the newly defined ubiquitin-interacting motif (UIM), facilitating their delivery into the autophagosome for subsequent degradation within the vacuole (Gassmann et al., 2013; Marshall et al., 2022; Xie et al., 2016). ATG genes have been found to have novel functions beyond their canonical role in autophagy; in Arabidopsis, ATG8 interacts with various proteins such as ABS3 to induce senescence and protein degradation independently of canonical autophagy (Jia et al., 2019), while also participating in non-autophagic processes like Golgi recovery under heat stress through interactions with Clathrin light chain 2 (CLC2) (Gouveia et al., 2023; Zheng et al., 2023; Zhou et al., 2023), and ATG5 interacts with stress-response factors, the ubiquitin-proteasome system, and endomembrane trafficking proteins, indicating regulatory roles beyond autophagy (Elander et al., 2023). Except in plant, the autophagy proteins and other components of the autophagic machinery additionally participate in cellular repro­gramming, such as Atg5 not only promotes autophagy but also sensitizes cells to apoptotic stimuli, with calpain-mediated cleavage of Atg5 triggering mitochondrial translocation and apoptosis (Yousefi et al., 2006). The functions of ATGs beyond autophagy may stem from evolutionary duplication. While yeast possesses a single ATG8, the Arabidopsis genome boasts nine AtATG8s, each displaying distinct expression patterns, hinting at their diverse functions (Yoshimoto et al., 2004). Mammals possesses two homologs of Atg9, ATG9A and ATG9B (Yamada et al., 2005). ATG9A and ATG9B are homologs with similar sizes and functions in autophagy, but they show distinct tissue expression patterns (Wang et al., 2017; Chiduza et al. 2023). ATG9A is ubiquitously expressed, particularly in the brain and spinal cord (Tamura, Shibata, Koike, Sasaki, & Uchiyama, 2010; Yamada et al., 2005), while ATG9B is mainly found in the placenta and pituitary gland, with low expression in the testis and uterus (Yamada et al., 2005). ATG9B can rescue ATG9A-deficient cells in some cases, although this effect is limited by its restricted tissue distribution (Chiduza et al. 2023). Despite their functional overlap, recent studies suggest that ATG9B may have evolved novel roles in placental development, while ATG9A retains more conserved functions (Chiduza et al. 2023). However, not all species have two ATG9 genes; for example, yeast and Arabidopsis only have one ATG9 (Lai et al., 2020; Matoba et al., 2020). In rice, OsATG9b emerged via segmental duplication events from OsATG9a, with a Ka/Ks ratio > 1 implying positive selection on this duplicated pair (Xia et al., 2011). In our previous study, we observed that OsATG9b plays a role in determining grain size and quality in rice (Liu et al., 2023). Mutations of OsATG9b result in smaller grains and increased chalkiness, whereas its overexpression enhances grain size and quality. Considering OsATG9b is the duplication gene from OsATG9a and they experienced positive selection, we hypothesized potential divergent functions of these genes during rice development and in response to environmental stressors. Our investigation revealed that both OsATG9a and OsATG9b participated in autophagy processes and exhibited functional redundancy in development. However, under drought stress conditions, OsATG9b demonstrated a distinct negative impact on autophagy, suggesting a specialized role in the rice drought response. In summary, we revealed that duplication and neo-functionalization of OsATG9 play important roles in balancing development and drought stress response. 2 | MATERIALS AND METHODS 2.1 | Plant materials and growth conditions Oryza sativa subsp . japonica cv Zhonghua 11 (ZH11, WT) was used in this study. The knockout mutants osatg9a and osatg9a osatg9b were generated by CRISPR/Cas9-mediated gene editing (Figure S1, Table S1). The detailed editing sequences were shown in Table S2. Other materials used in this study, including osatg9b , osatg5 , osatg7 , OsATG8b-OE , and ATG9b-OE , were previously published by our lab (Liu et al., 2023). The functionality of the osatg9b line used in this study was confirmed in our earlier work by observing the agronomic traits of the complementary line osatg9b-C (Liu et al., 2023). In transgenic plants overexpressing OsATG5 , the transgenes were driven by the maize Ubiquitin promoter. All transgenes and corresponding binary plasmids were sequenced and verified, then transformed into Agrobacterium tumefaciens strain EHA105 before being introduced into ZH11. Twelve independent positive calli (T0) were obtained, and line no. 1 of OE-OsATG5 T2 homozygous was selected as the representative for this study due to its highest expression level of OsATG5 (Fig. S2). Plants were grown and routinely managed at the paddy fields in the South China Agricultural University Wushan Campus Teaching & Research Base (Guangzhou, China, 113°21’E, 23°9’N), Zengcheng Campus Teaching & Research Base (Guangzhou, China, 113°49’E, 23°18’N), and Lingshui (Hainan, China, 18°22’E, 109°45’N). Rice seeds were surface-sterilized using 2.5% (v/v) NaClO and washed several times, germinated, and cultivated in soil mix or Yoshida’s nutrition solution in a growth room with a cycle of 16 h light at 30℃ and 8 h dark at 28℃. Rice etiolated seedlings used for protoplasts isolation were grown on ½-strength Murashige and Skoog (½MS) medium with 3% (w/v) sucrose and 0.8% (w/v) agar in the dark at 28°C for 10 days. 2.2 | Plasmid construction To generate the overexpression lines of the Ubipro:OsATG5-OE , the full-length coding sequence (CDS) of OsATG5 ( Os02g02570 ) was amplified from ZH11 seedlings and cloned into the binary vector pRHVnGFP (He et al., 2018). For subcellular localization of OsATG9a and OsATG9b, the CDSs of OsATG9a ( Os03g14380 ) and OsATG9b ( Os10g0163100 ) were cloned and inserted separately into the pCAMBIA2300-35S-EGFP-N vector. Additionally, the OsATG9b CDS was inserted into the pRGV-RFP vector (He et al., 2018). The 2000 bp promoter sequences upstream of the coding regions of OsATG9a and OsATG9b were cloned to create plant expression vectors, pCAM-OsATG9apro and pCAM-OsATG9bpro, which contained the fusion genes OsATG9apro::GUS and OsATG9bpro::GUS , respectively. These constructs were introduced into rice plants via Agrobacterium-mediated genetic transformation. The primers used for vector construction were shown in Table S3. 2.3 | Subcellular localization Protoplasts were isolated from 10-day-old rice seedlings. GFP-OsATG9s were cotransfected with mCherry-AtRER1B (Golgi marker) and Ubipro::RFP-OsATG8b, respectively (Liu et al., 2023). To detect whether OsATG9a and OsATG9b can colocalize, the GFP-OsATG9a and RFP-OsATG9b were cotransfected into rice protoplasts. Fluorescence expression was subsequently observed using a laser scanning confocal microscope (Leica STELLARIS 5; Leica, Wetzlar, Germany). 2.4 | Transient investigation of autophagy activity In terms of transient investigation of autophagy activity, the autophagy marker RFP-OsATG8b was introduced into protoplasts extracted from WT, osatg9a , osatg9b , and osatg9a osatg9b mutants. Rice protoplasts were cultured in W5 solution containing 1 μM concanamycin A (ConA; BVT-0237-M001; Adipogen, San Diego, CA, USA) for 12 h in the dark according to previous report (Liu et al., 2023). 2.5 | Agronomic traits analysis Grain size was measured using Microtek Scan-Wizard EZ scanner V-2.140 and Wan Shen grain analyzer. Grain size and the thousand-grain weight were measured in > 20 replicates, with each replicate consisting of > 100 grains. not-yet-known not-yet-known not-yet-known unknown 2.6 | Plant treatments For dehydration treatment, uniformly germinated seeds of the WT, OsATG-OE lines, and osatg mutans were transplanted into 96-well plant hydroponic culture boxes and hydroponically grown using 800×Yoshida rice nutrient salts solution (NS1040-1L; Coolaber, Beijing, China). Two-week-old plants were treated with 20% (w/v) PEG6000 solution for about 7 d and recovered with water for 5 d. The survival ratio (the number of surviving plants divided by the total number of treated plants) of each line was calculated. For soil drought tolerance assays, uniformly germinated seeds of the WT and transgenic lines were transplanted into soil and grown for three weeks under normal watering conditions. Drought stress treatment was then applied by stopping irrigation for about 10 days. When all leaves had completely rolled, watering was resumed for 7 d. The survival ratio (the number of surviving plants divided by the total number of treated plants in the pot) of each line was calculated. To examine the transcriptional levels of target genes under drought conditions, WT and osatg mutants were hydroponically grown in a light incubator for 2 weeks and then treated with 20% PEG. Leaf samples were collected at 0 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 1 d, 3 d, 5 d, and 7 d after treatment, frozen in liquid nitrogen, and stored at -80 ℃ until further analysis. Each stress test was repeated three times. 2.7 | Physiological and biochemical analysis After 7 days of PEG treatment, leaves were collected from two-week-old seedlings to measure proline content, soluble sugar content, superoxide dismutase (SOD) activity, catalase (CAT) activity, malondialdehyde (MDA) content, and hydrogen peroxide (H 2 O 2 ) content. Proline concentration was measured using the proline determination kit (BC0295; Solarbio, Beijing, China). Approximately 0.1 g of the sample was weighed, 1 mL of extract solution was added, and the mixture was homogenized in an ice bath. It was then boiled for 10 minutes, followed by centrifugation at 10,000 g at room temperature for 10 minutes. The supernatant was collected, cooled, and tested by measuring absorbance at 520 nm using a spectrophotometer, with L-Pro as the standard for calculation. Soluble sugars were extracted using a plant soluble sugar detection kit (A145-1-1; Nanjing Jiancheng, Nanjing, China). The sample was ground with liquid nitrogen, 0.1 g was weighed, and 1 mL of distilled water was added. The mixture was boiled for 10 minutes, cooled, and centrifuged at 4000 rpm for 10 minutes. The supernatant was diluted 10 times with distilled water, mixed well, and tested by adding substrate solution and concentrated sulfuric acid. The mixture was incubated in a boiling water bath (95–100°C) for 10 minutes, cooled, and absorbance was measured at 620 nm using a 1 cm optical path cuvette. For SOD activity, 0.1 g of the ground sample was mixed with 0.9 mL of phosphate-buffered saline (0.1 mol/L, pH 7.4), vortexed for 3 minutes, and centrifuged at 3500 rpm for 10 minutes. The supernatant was used to measure SOD activity by incubating at 37°C for 20 minutes and reading absorbance at 450 nm using a microplate reader following the SOD detection kit (A001-3; Nanjing Jiancheng, Nanjing, China). For CAT activity, 0.1 g of the sample was weighed, substrate solution was added, and the reaction was carried out for 1 minute at 37°C. The absorbance was measured at 405 nm following the CAT detection kit (A007-1-1; Nanjing Jiancheng, Nanjing, China). For MDA content, 0.1 g of plant tissue was weighed, 0.9 mL of reagent extraction solution was added, and the mixture was vortexed, centrifuged at 3500–4000 rpm for 10 minutes, and the supernatant was collected. The working solution was added, the mixture was incubated in a boiling water bath for 20 minutes, cooled, and absorbance was measured at 530 nm following the MDA detection kit (A003-3-1; Nanjing Jiancheng, Nanjing, China). For H 2 O 2 content, 0.1 g of tissue was weighed, 0.9 mL of PBS buffer (pH 7.0-7.4, 0.1 mol/L) was added, and the mixture was vortexed, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. Absorbance was measured at 405 nm following the H 2 O 2 detection kit (A064-1-1; Nanjing Jiancheng, Nanjing, China). 2.8 | Protein isolation and immunoblot analysis Total protein extraction was conducted using 2×SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200 mmol -1 β-ME (β-Mercaptoethanol). The anti-NBR1 antibody, previously used in another published study (Guo et al., 2023), was purchased from Agrisera (AS142805; Agrisera, Sweden) and applied in this study. To detect OsATG8a-PE (the lipidated form), protein samples were separated by 15% SDS-PAGE containing 6 mol/L urea, as described by Qi et al. (2023). Anti-OsATG8a (Cat. No. ab77003, 1:1,000; Abcam, UK) was used for the detection. The anti-TGW6 antibody in previous published paper (Liu et al., 2023) was used in this study. Quantification of the protein band intensity from immunoblots was performed with the software Image J. Each experiment was performed at least three times, and one representative result is shown. 2.9 | RT-qPCR analysis Total RNA was extracted with Trizol reagent (P118-05; GenStar, Beijing, China) and first-strand cDNAs were synthesized using an Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728; Accurate Biology, ChangSha, Hunan, China). qPCR was performed using 2×SYBR ® Green Pro Taq HS Premix (AG11701; Accurate Biology, ChangSha, Hunan, China) with three biological replicates. Data are presented as means ± standard deviation (SD). The relative expression level of the tested genes was normalized to that of OsUBIQUITIN1 ( OsUBI1 , LOC_Os06g46770) and calculated by the 2 -∆∆Ct method. The primers used for qPCR are listed in Table S4. 2.10 | SEM observation of stomata and glumes in rice For the observation of stomatal conductance, 14-day-old rice seedlings were treated with 20% PEG6000 for 3 days. The leaves were detached and immediately fixed in FAA solution (containing 2% formaldehyde, 5% acetic acid, 63% alcohol, and 5% glycerol) at 4℃ for 24 h. After dehydration in an ethanol series (ranging from 60% to 100%, with 5% as a gradient interval), the leaves were submerged in a graded series of tert-butyl alcohol (TBA) solutions (ethanol: TBA [v/v], ratios of 3:1, 2:2, 1:3) for 15 minutes each, and then twice in 100% TBA. Subsequently, the materials were transferred into 100% t-butanol. After incubation in t-butanol at 4℃ for 30 minutes, they were freeze-dried using a VFD-21S t-BuOH freeze-dryer (SHINKKU VD brand). The materials were then stuck onto the sample stage and sprayed with gold using the MSP-1S magnetron sputter (also SHINKKU VD brand). Stomata were imaged using a scanning electron microscope according to the manufacturer’s instructions. One hundred stomata from each line were observed, and the completely open, partially open, and completely closed stomata were analyzed as described previously (Copenhaver et al., 2015; Zhang et al., 2011). For the observation of rice epidermal cells, mature dry rice seeds were stuck onto the sample stage and sprayed with gold as previously described. Epidermal cells were then imaged using a scanning electron microscope. 2.11 | GUS staining Tissue-specific expression patterns of OsATG9a and OsATG9b were then analyzed in the OsATG9apro::GUS and OsATG9bpro::GUS transgenic lines by vacuum-infiltrating intact tissues from GUS buffer (SL7160-100 mL; Coolaber, Beijing, China). After incubating the tissues in the dark at 37°C for 24 hours and removing chlorophyll through ethanol treatment, ATG9pro::GUS activity was observed. not-yet-known not-yet-known not-yet-known unknown 2.11 | Statistical analysis All the data were analyzed by the statistical package SPSS version 22.0 (IBM Corporation, Armonk, NY, USA). Data obtained were statistically analyzed according to the Student’s t -test and the probability value of *P < 0.05 or **P < 0.01 was considered as significant difference. One-way analysis of variance (ANOVA) was completed, and the treatment means were compared using the LSD (least significant difference) test (at P < 0.05). not-yet-known not-yet-known not-yet-known unknown 3 | RESULTS 3.1 | Evolution analysis of OsATG9s To explore the evolutionary relationship between OsATG9 and ATG9 proteins in other organisms, we constructed a phylogenetic tree using the amino acid sequences of OsATG9a and OsATG9b, along with ATG9 proteins from 20 other species (Figure S3). The results show that OsATG9a and OsATG9b belong to different clusters, with OsATG9a being most close homology to OgATG9a ( Oryza glaberrima ATG9a), while OsATG9b to OgATG9b. The phylogenetic tree reveals that ten organisms have only one ATG9, while eleven organisms possess multiple ATG9 copies (Figure S3). The analysis also indicates sequence divergence among certain members of the ATG9 family between monocotyledonous, dicotyledonous species and Saccharomyces cerevisiae (Figure S3). Focusing on monocotyledon, we found that the open reading frames (ORFs) of OsATG9a and OsATG9b are 2709 bp and 2784 bp long, encoding proteins of 902 and 927 amino acids, respectively (Figure S4). These two protein sequences share 64.72% identity (Figure S4). Additionally, the superfamily domain of ATG9 is highly conserved across different species (Figure S4). 3.2 | Both OsATG9a and OsATG9b positively regulate autophagy Our previous study has already demonstrated that OsATG9b positively regulates autophagy (Liu et al., 2023). Considering the high identity of amino acid sequences between OsATG9a and OsATG9b, we speculated that OsATG9a also plays a positive role in autophagy. Firstly, we examined the subcellular localization of OsATG9a to determine if it aligns with that of OsATG9b. Both GFP-OsATG9a and GFP-OsATG9b showed colocalization with the Golgi marker, mCherry-AtRER1B (Sato et al., 1999; Takeuchi et al., 2000) (Figure 1a), consistent with previous findings (Liu et al. , 2023; Zhuang et al. , 2017). Furthermore, OsATG9a was colocalized with OsATG9b (Figure 1b). In our previous study, we demonstrated that OsATG9b colocalizes with the autophagosome marker OsATG8b and contributes to the initiation and formation of autophagosomes in the cytoplasm (Liu et al., 2023). In this study, we further confirmed that both GFP-OsATG9b and GFP-OsATG9a colocalize with RFP-OsATG8b (Figure S5). We therefore propose that OsATG9a is likely involved in the cytoplasmic biogenesis of autophagosomes in rice, serving a role similar to that of OsATG9b. To prove the above hypothesis, we transiently transfected protoplasts with the autophagic body marker GFP-OsATG8b in the osatg9a or osatg9b or osatg9a osatg9b . We detected fewer GFP positive puncta in all of osatg9a , osatg9b and osatg9a osatg9b than that of WT following ConA treatment which stabilize autophagic bodies in the vacuole and thus aids in their detection (Izumi et al., 2015) (Figure 2a, b). NBR1 is a selective autophagy substrate and it also act as cargo receptors for degradation of other substrates (Gassmann et al. , 2013; Svenning et al., 2011), thus NBR1 can be used as the autophagy markers (Bassham, 2015). Here, we detected the NBR1 protein levels in WT, osatg9a , osatg9b and osatg9a osatg9b . The protein levels of NBR1 in osatg9 , osatg9b and osatg9a osatg9b mutants were significantly higher than that in WT, indicating that the autophagic flux were lower in the osatg9 , osatg9b and osatg9a osatg9b mutants (Figure 2c, d). All the results indicated that both OsATG9a and OsATG9b participate in the autophagy process in agreement with previous studies (Chung et al., 2010; Zhuang et al., 2017). not-yet-known not-yet-known not-yet-known unknown 3.3 | Contribution of OsATG9s to rice development In a previous study, OsATG9b was identified as a key player in the development of grain size and quality in rice (Liu et al., 2023). Here, we hypothesize that OsATG9a may have a similar role in rice grain development, given its participation in the autophagy process. To explore this, we firstly examined the spatial and temporal expression patterns of OsATG9a and OsATG9b in rice. Both genes exhibited comparable expression patterns, being constitutively expressed from seedling to maturity, with peak expression in the leaves during the second tillering, jointing and mature stages (Figure S6a, b). Additionally, GUS staining revealed high expression levels of both genes in panicles and leaves (Figure S6c-n), suggesting their potential roles in regulating grain development. To validate our hypothesis, we assessed the grain size in several mutants: osatg9a-1, osatg9a-2, osatg9b and osatg9a osatg9b-1, osatg9a osatg9b-2 double knockout mutants (Figure 3a). Phenotypic analysis revealed a significant reduction in grain length across all mutants compared to the WT (Figure 3a, b), suggesting that both OsATG9a and OsATG9b contribute to grain length determination. However, only the osatg9a-1 and osatg9a-2 mutants exhibited a decrease in grain width, while osatg9b did not affect grain width (Figure 3a, b), suggesting a specific role for OsATG9a in grain width determination. Additionally, the thousand-grain weight of all the mutants was lower compared to WT (Figure 3b). Given the importance of spikelet hulls in determining final grain size (Li et al., 2019b), we conducted cytological analyses of mutant spikelet hulls. The results demonstrated significantly shorter epidermal cell lengths in all mutans compared to WT (Figure 3c, d). However, there was no significant difference in the number of longitudinal cells between all mutants and the WT (Figure 3c, d), leading to shorter grain length in all mutants. Interestingly, only osatg9b mutant displayed a significant reduction in cell width, but the total cell number in the cross-section increased (Figure 3c, d), resulting in unchanged grain width in osatg9b . In contrast, the cell width of osatg9a-1 and osatg9a-2 mutants was unaltered, but their total cell number in the cross-section decreased significantly (Figure 3c, d), leading to reduced grain width (Figure 3c, d). These findings suggest slight differences in the roles of OsATG9a and OsATG9b in grain development. To further understand how OsATG9a regulates grain development, we monitored grain growth in osatg9a, osatg9b and osatg9a osatg9b mutants across nine developmental stages of spikelet, ranging from 3-5 to 23-25 cm in length. We observed that grain length in all mutants was significantly shorter than WT starting from the 10-13 cm stage (Figure 3e). Grain width in osatg9a was reduced from the 10-13 cm stage compared with WT (Figure 3e), ultimately resulting in shorter grain width in osatg9a mutants. Grain width in osatg9b and osatg9a osatg9b mutants did not significantly differ from WT (Figure 3e). Further analysis demonstrated that the shape of the endosperm in osatg9a, osatg9b and osatg9a osatg9b mutants was normal, suggesting normal endosperm development (Figure S7a). Previous studies demonstrated that autophagy regulates grain development by controlling the degradation of THOUSAND-GRAIN WEIGHT 6 (TGW6) (Liu et al., 2023). We therefore examined TGW6 protein levels in osatg9a, osatg9b and osatg9a osatg9b grains at 3 d after fertilization (DAF). TGW6 proteins were more abundant in all mutants compared with WT (Figure S7b), indicating that OsATG9a also regulates grain development through autophagic degradation of TGW6. Additionally, osatg9a-1, osatg9a-2, osatg9b and osatg9a osatg9b-1, osatg9a osatg9b-2 double knockout mutants exhibited significantly reduced plant height, panicle length and tiller number compared to WT (Figure S8). Moreover, the primary branch number was significantly decreased in osatg9a-1, osatg9b and osatg9a osatg9b-1 mutants compared to WT (Figure S8b, e), indicating similar roles for OsATG9a and OsATG9b in rice development. 3.4 | OsATG9a and OsATG9b play opposite roles in response to drought stress Given that OsATG9b originated from duplication events of OsATG9a with a Ka/Ks ratio > 1 (Xia et al. , 2011), it is plausible that OsATG9b has evolved distinct functions compared to OsATG9a. As depicted in Figure 3, Figure S7, and Figure S8, both OsATG9a and OsATG9b contributed partially similarly to rice development, hence we speculated that OsATG9b might play a different role in stress response. To explore this hypothesis, we subjected WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 to 20% PEG treatment to simulate drought stress. Surprisingly, osatg9a-1 and osatg9a-2 exhibited significantly lower survival rates than WT, whereas osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 mutants displayed higher survival rates than WT (Figure 4a-f). These results suggested that OsATG9a positively regulates drought stress response, while OsATG9b negatively regulated it, with OsATG9b potentially exerting an epistatic effect over OsATG9a. To further validate these findings, we conducted soil experiments, which yielded consistent results: osatg9a-1 and osatg9a-2 mutants had lower survival rates, while osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 mutants had higher survival rates compared to WT (Figure 4g, h). Drought stress impacts several physiological parameters in plants, including stomatal behavior, which is closely related to a plant’s water retention capacity (Jia et al., 2021b). Under normal conditions, osatg9a-1 and osatg9a-2 mutants exhibited a higher percentage of completely open stomata and a lower percentage of partially and fully closed stomata compared to WT (Figure 5a). In contrast, osatg9b mutants showed a lower percentage of partially open stomata and a higher percentage of fully closed stomata compared to WT (Figure 5a). No significant differences in stomatal characteristics were observed between osatg9a osatg9b-1 , osatg9a osatg9b-2 and WT (Figure 5a). Upon PEG treatment, WT plants showed a decrease in the percentage of fully and partially open stomata compared to normal conditions (Figure 5a). The stomatal characteristics of the other genotypes remained largely consistent with WT under both PEG and normal conditions, except for a lower percentage of fully and partially open stomata in osatg9b , osatg9a osatg9b-1 , and osatg9a osatg9b-2 mutants (Figure 5a). This suggests that stomatal behavior contributes to the differing drought resistance capacities of osatg9a and osatg9b . Consistent with the stomatal opening rates, osatg9a-1 and osatg9a-2 mutants showed higher water loss rates than WT, whereas osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 mutants showed lower water loss rate than WT (Figure 5b). Proline and soluble sugars are key metabolites that help plants resist drought stress (Altaf et al., 2022). Under drought conditions, both proline and soluble sugar contents were lower in osatg9a mutants but higher in osatg9b and osatg9a osatg9b mutants compared to WT (Figure 5c). To analyze the oxidative status in osatg9 mutants under drought stress, we measured the activities of major antioxidant enzymes. Superoxide dismutase (SOD) and catalase (CAT) activities were lower in osatg9a mutants but higher in osatg9b and osatg9a osatg9b mutants compared to WT (Figure 5c). Drought damage was further evaluated by measuring malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) concentrations in the leaves (Sun et al., 2018). Both MDA and H 2 O 2 contents were higher in osatg9a mutants but lower in osatg9b and osatg9a osatg9b mutants compared to WT under drought stress (Figure 5c). The relative expression levels of drought-responsive genes, OsERD1 ( Early-Responsive to Dehydration stress ) and OsP5CS1 ( Δ1-pyrroline-5-carboxylate synthetase ) (Yuan et al., 2019) were also assessed. They were downregulated in osatg9a mutants but upregulated in osatg9b and osatg9a osatg9b mutants compared to WT under drought stress (Figure 5d). These results indicated that osatg9a mutants were more susceptible to drought stress, whereas osatg9b and osatg9a osatg9b mutants exhibited greater resistance compared to WT. To elucidate whether the functional difference between OsATG9a and OsATG9b in drought resistance are related to the autophagy process, we first confirmed the role of autophagy in drought resistance. The expression levels of OsERD1 were induced at 6 h after 20% PEG treatment and gradually decreased by day 7 (Figure S9a, b), suggesting a short-term induction of drought response that diminished over time. Similarly, the relative expression levels of OsATG9a were induced in the short term but gradually decreased over the long time (Figure 6a, b). In contrast, OsATG9b was induced in the short term but showed no response to drought stress after 24 h (Figure 6a, b). We also monitored NBR1 protein levels in WT plants under short- and long-term PEG treatment. NBR1 levels decreased continuously for the first 24 hours (Figure 6c, d) but significantly increased after three days, surpassing baseline levels. (Figure 6e, f). Furthermore, we examined the expression of various OsATG genes at 6 h post-PEG treatment and found that all OsATG genes were upregulated (Figure S10), indicating that autophagy was induced in the short term but suppressed after one day. Given that OsATG5 , OsATG7 and OsATG8 were reported to be involved in autophagy (Falk et al., 2022; Fan et al., 2020; Guo et al., 2017; Hanamata et al., 2020; Wada et al., 2015; Yu et al., 2019; Zhang et al., 2023), we used osatg5 , OsATG5-OE , osatg7 and OsATG8b-OE to explore the role of autophagy in response to drought stress. The survival rates of osatg5 and osatg7 were lower than WT after PEG treatment, while overexpressing OsATG5 or OsATG8b significantly increased survival rates (Figure S11a-d). In addition, proline content, soluble sugar content, SOD activity, and CAT activity were lower in osatg5 and osatg7 mutants but higher in OsATG5-OE and OsATG8b-OE lines compared to WT under drought stress (Figure S12). Conversely, MDA and H 2 O 2 contents showed the opposite trend (Figure S12). Consistent with these drought response phenotypes, drought-resistant genes were expressed at lower levels in osatg5a and osatg7 mutants but at higher levels in OsATG5-OE and OsATG8b-OE lines compared to WT under drought stress (Figure S13). To confirm that these differences were due to variations in autophagy levels, we measured NBR1 abundance before and after PEG treatment. The value of PEG:CK ratio of WT was lower than 1, suggesting autophagy was induced after drought stress. The PEG:CK ratios in osatg5 and osatg7 were higher than in WT, while those in OsATG5-OE and OsATG8b-OE were lower than WT (Figure S11e-h), indicating that autophagy plays a positive role in response to drought stress. To further investigate whether the negative role of OsATG9b in drought stress was due to autophagy, we examined the expression patterns of autophagy-related genes in osatg9a and osatg9b mutants. The heatmap showed that OsATG s genes were either upregulated or downregulated in osatg9a mutants under PEG treatment, suggesting that autophagy flux may be decreased in osatg9a due to the disordered OsATG genes expression (Figure S14). In contrast, all OsATG genes were upregulated in osatg9b mutants, with particularly stronger induction of genes like OsATG1a , OsATG1c , OsATG2 , OsATG5 , OsATG6b , OsATG7 , OsATG8a , OsATG8b , OsATG8d , OsATG9a , OsATG12 and OsATG13b than WT (Figure S14), suggesting that autophagy flux was increased in osatg9b . We further assessed NBR1 protein levels in osatg9a-1 , osatg9a-2 and osatg9b and osatg9a osatg9b-1 , osatg9a osatg9b-2 mutants. The PEG:CK ratios of osatg9a-1 and osatg9a-2 were higher than in WT (Figure 6g-l), consistent with the observations in osatg5 and osatg7 (Figure S11e-h), indicating a reduction in autophagy activity in osatg9a in response to drought stress. However, the PEG:CK ratios in osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 were significantly lower than in WT (Figure 6g-l), suggesting a much higher induction of autophagy flux in osatg9b and osatg9a osatg9b compared to WT. Detection of ATG8-PE (phosphatidylethanolamine) is another stringent criterion for assessing autophagic flux (Marshall & Vierstra, 2018). Using the ATG8 antibody, our immunoblotting analyses revealed that the OsATG8-PE: OsATG8 ratio increased after PEG treatment in all lines (Figure 6m-n). Under normal conditions, the OsATG8-PE:OsATG8 ratio was lower in all mutants compared to WT, indicating reduced autophagic flux in the osatg9 mutants under normal condition. However, after PEG treatment, the ratio was lower in osatg9a mutants but higher in osatg9b mutant compared to WT (Figure 6m-n), indicating a much stronger induction of autophagy flux in osatg9b . To further confirm the negative role of OsATG9b in drought response, we overexpressed OsATG9b in rice to examined its phenotype. The survival rate of OsATG9b-OE was significantly lower than WT under PEG treatment, while the survival rate of osatg9b was higher (Figure 7a-d). Consistent results were obtained from soil experiments, where OsATG9b-OE exhibited lower survival rates (Figure 7e, f). Under both normal and PEG conditions, the percentages of completely open stomata and partially open stomata in OsATG9b-OE were higher than in WT, opposite to the trend observed in osatg9b (Figure 7g), which led to higher water loss in OsATG9b-OE compared to WT (Figure 7h). The ratio of NBR1 protein abundance under PEG treatment versus control conditions showed that overexpression of OsATG9b downregulated autophagy flux under PEG treatment, opposite to the effect of OsATG9a (Figure 7i, j). The OsATG8-PE: OsATG8 ratio revealed that under normal conditions, the ratio was higher in OsATG9b-OE line compared to WT (Figure 7k-l), indicating increased autophagic flux in the OsATG9b-OE line under normal condition. However, after PEG treatment, the ratio in the OsATG9b-OE line did not significantly differ from that in WT, but was lower than under normal condition (Figure 7k-l), suggesting a suppression of autophagic flux in the OsATG9b-OE line following drought stress. Collectively, these results demonstrate that OsATG9a plays a positive role in response to drought stress, similar with OsATG5, OsATG7 and OsATG8b, while OsATG9b negatively regulates drought stress response by downregulating autophagy flux. Abscisic acid (ABA) is a key phytohormone that regulates plant drought resistance and response (Shinozaki & Yamaguchi-Shinozaki, 2007). To investigate whether the different roles of OsATG9a and OsATG9b in regulating drought stress are linked to the ABA pathway, we examined the expression levels of ABA-related genes in osatg9a and osatg9b mutants. OsDREB2A ( AP2/EREBP transcription factor ) and Os LEA3 ( late embryogenesis abundant 3 ) are ABA regulatory and signaling genes responsible for stomatal closure and regulation of drought tolerance (Yang, Dai, & Zhang, 2012). OsbZIP23 ( bZIP transcription factor ) and MYB2 ( MYB Domain Protein 2 ), an R2R3-type MYB transcription factors, mediate ABA-dependent pathways by upregulating ABA signaling genes, including LEA3 ( Late Embryogenesis Abundant 3 ) and OsPYL7 ( Pyrabactin Resistance-like Abscisic Acid Receptor ) (He et al., 2014; Lim et al., 2022). Our results showed that these ABA-related drought-resistant genes were upregulated in WT, osatg9a and osatg9b after PEG treatment (Figure 8). However, the foldchanges of these genes in osatg9a were lower than in WT, while in osatg9b , they were higher than in WT (Figure 8), suggesting that OsATG9a and OsATG9b play distinct roles in regulating ABA pathway to modulate drought stress tolerance. not-yet-known not-yet-known not-yet-known unknown 4. | DISCUSSION Whole-genome duplications are widespread in plants and lead to gene duplications. Many duplicated genes maintain functional redundancy, allowing plants to survive in complex environments (Qiao et al., 2022). In this study, we discovered that OsATG9a and OsATG9b share functional redundancy in rice development, both being involved in autophagy. However, OsATG9a and OsATG9b have evolved opposite functions in response to drought stress: OsATG9a induces autophagy to combat drought stress, while OsATG9b suppresses autophagy, playing a negative role in drought response. This suggests that OsATG9b represents a novel function that emerged during rice evolution. Our findings provide valuable insights into the process of neofunctionalization following gene duplication, contributing to both evolutionary biology and the understanding of drought stress responses. Evolutionary processes offer three potential outcomes for duplicated homologues: non-functionalization, neofunctionalization, and subfunctionalization. Neofunctionalization and subfunctionalization can lead to the retention of duplicated genes, fostering biological novelty and diversity (Conant et al., 2014; Lynch and Conery, 2000; Van de Peer et al., 2017). Analysis of intragenomic and intergenomic synteny in the rice (Oryza sativa ) genome has identified two ancient whole-genome duplication (WGD) events, referred to as σ and ρ. The σ duplications are estimated to have taken place approximately 130 million years ago (Mya) (Tang et al., 2009), while the ρ duplications are estimated to have occurred around 70 Mya (Paterson et al., 2004; Wang et al., 2005). The divergence between OsATG9a and OsATG9b can be traced back to 18.82 Mya (Xia et al., 2011). Moreover, the pineapple (Ananas comosus (L.) Merr.), which diverged from rice after the σ duplication but before the ρ duplication, has only one ATG9 (Ming et al., 2015), indicating that the diversification between ATG9a and ATG9b occurred after the ρ duplication. However, the evolutionary fate of ATG9 homologues remains largely unexplored. The ρ event preceded the diversification of major grass lineages, suggesting that the gene arrangements in rice and sorghum are representative of most grass genomes, although they have been further modified by additional duplications and gene losses. The ρ duplication affected all modern chromosomes of rice and sorghum, covering much of the euchromatin. Even duplications previously thought to be recent are results of the ρ event followed by concerted evolution (Ming et al., 2015; Tang et al., 2009). In this study, we determined that the sequence identity between OsATG9b and SbATG9b was 73.55%, even higher than that between OsATG9b and OsATG9a (Figure S3 and Figure S4). Unfortunately, the function of SbATG9b has not yet been reported. Whether SbATG9b plays a negative role in drought stress response warrants further investigation. Similar duplication outcomes have been observed in various gene families. For example, in the Leguminosae, type II chalcone isomerase (CHI ) genes are divided into CHI1A and CHI1B clades, which exhibit functional divergence in root nodule symbiosis. Knocking down CHI1B significantly reduces nodulation in soybean and Medicago truncatula, whereas CHI1A knockdown has no effect (Liu et al., 2024). Similarly, the E1 gene family, a legume-specific transcription factor and core regulator of flowering in soybeans, has undergone subfunctionalization. This allows for self-suppression and mutual inhibition, balancing the total activity of E1 homologues to optimize flowering and adaptation (Fang et al., 2024). NAC transcription factors, including vascular-related NAC domain proteins (VNDs) and xylem NAC domain proteins (XNDs), also show functional divergence. In Arabidopsis thaliana, VND6 and VND7 are key regulators of xylem vessel differentiation, activating secondary wall deposition in xylem vessels (Hirai et al., 2019; Ohashi-Ito et al., 2010; Yamaguchi et al., 2008; Yamaguchi et al., 2011). In contrast, XND1 negatively regulates this process by interacting with secondary wall NACs’ DNA-binding domains and sequestering VND6 in the cytoplasm, thereby preventing gene activation and secondary wall deposition (Zhong et al., 2021). Among SG2-type R2R3-MYBs transcription factors in Arabidopsis, MYB15 and MYB30 control basal immunity through lignification or by functioning in very long chain fatty acid transport, respectively. MYB13 and MYB14, however, are not involved in defense-induced lignification. Despite these differences, all of these MYB transcription factors play roles in regulating plant growth. These diversities likely help balance plant growth and adaptation. Plants regulate their responses to environmental stress through a complex mechanism that balances growth and drought tolerance. In our study, we observed that autophagy levels increased within the first 24 hours of PEG treatment but dropped significantly after 1 day (Figure 6c-f), suggesting that autophagy cannot be sustained at high levels over time. Notably, OsATG9b was found to suppress autophagy under drought conditions (Figures 6, 7), suggesting its role in balancing plant growth and drought tolerance in rice. ABA, a key phytohormone, regulates stomatal closure and enhances drought resistance (Lim et al., 2022). In our findings, ABA related genes were similarly expressed between WT and osatg9 mutants under normal conditions (Figure 8). However, they were more highly induced in osatg9b mutants than in wild-type plants under drought condition (Figure 8), which likely explains the higher percentage of fully closed stomata and reduced water loss observed in osatg9b (Figures 5a, b). In Arabidopsis, the protein COST1 (constitutively stressed 1) is crucial for balancing drought tolerance and plant growth. Under normal conditions, COST1 interacts with ATG8 to suppress drought stress responses, promoting plant growth. Under stress, COST1 is degraded by both the 26S proteasome and autophagy, releasing ATG8 and enabling autophagy, which enhances drought tolerance (Bao and Bassham, 2020; Bao et al., 2020). Referring to the case in Arabidopsis, we hypothesize that OsATG9b may influence the ABA pathway in different conditions, and that a key factor in this pathway may, in turn, regulate autophagic activity. Protein structure modification can influence function. ATG9, the only integral membrane protein in the autophagy core machinery, is crucial for autophagosome formation by providing an essential membrane source (Lai et al., 2020). The homotrimeric architecture of ATG9 is evolutionarily conserved across species, including human ATG9A/ATG9B (Chiduza et al., 2024; Guardia et al., 2020a; Guardia et al., 2020b; Maeda et al., 2020), fission yeast ATG9 (Matoba et al., 2020) and Arabidopsis ATG9 (Lai et al., 2020). These proteins share two transmembrane helices with neighboring protomers within the trimer and possess two distinct pores that mediate the scramblase activity essential for autophagosome formation (Maeda et al., 2020; Matoba et al., 2020). It remains unknown whether the distinct pores of OsATG9b can be modified under drought stress, potentially leading to its negative effect on autophagy. While autophagy is generally conserved in eukaryotes, the underlying systems are subject to modifications and refinements in different clades (Zhang et al., 2022). Various Arabidopsis ATG8 isoforms have been shown to be differentially cleaved by ATG4b (Woo et al., 2013), suggesting that OsATG9b may also undergo different modifications under drought conditions. Further study on the structure and modification of OsATG9b is worthy. In summary, our study reveals that the OsATG9 gene family in rice has undergone neofunctionalization. While both OsATG9a and OsATG9b regulate rice development in a similar manner, OsATG9a positively influences drought stress response by inducing autophagy, whereas OsATG9b negatively regulates drought response by suppressing autophagy. These findings offer new insights into the fate of duplicated genes and the balance between plant growth and stress response. 5. | ACKNOWLEDGEMENTS This work was supported by the STI 2030 - Major Projects (2023ZD040710104), National Natural Science Foundation of China (32301829), Natural Science Foundation of Guangdong Province (2024A1515010610, 2024A1515012928), the Science and Technology Planning Project of Guangzhou (2024A04J4039), the open competition program of top 10 critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG05), Guangdong province rural revitalization strategy special fund seed industry revitalization project (2022-NJS-15-001), the Special Plan for Key Laboratory of “Western Light Western cross team” (xbzg-zdsys-202111), the ”Top Young Scientist of the Pearl River Talent Plan” (No. 20170104) and the Double first-class discipline promotion project (2021B10564001). 6. | REFERENCES Altaf, M.A., Shahid, R., Ren, M.X., Naz, S., Altaf, M.M., Khan, L.U. et al. 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YF.Liu assisted on the microscope analyses. Y.H. and P.X. conducted the materials cultivation and paddy field management. Y.Q. produced most of the transgenic plants. Q.X., Q.Y., and Y.L. analyzed the data, wrote and revised the manuscript. All authors read and commented on the paper. DECLARATION OF INTERESTS The authors declare no competing interests. FIGURE LEGENDS FIGURE 1. Subcellular localization analysis of OsATG9a and OsATG9b in rice protoplasts through transient transformation. (a) Co-localization of mCherry-AtRER1B with OsATG9a or OsATG9b in rice protoplasts. mCherry-AtRER1B was used as a Golgi marker. (b) Co-localization analyses of OsATG9a and OsATG9b. PC: Pearson’s Correlation. OC: Overlap Coefficient. Scale bars, 10 μm. Experiments were repeated three times with consistent results. not-yet-known not-yet-known not-yet-known unknown FIGURE 2. OsATG9a and OsATG9b positively regulate autophagy in rice. (a) GFP-OsATG8b fusion protein was transient expressed in rice protoplasts derived from WT, osatg9a, osatg9b and osatg9a osatg9b mutants. After treatment with ConA or DMSO for 12 hours in the dark, the protoplasts were observed under confocal microscopy. White arrows indicate GFP-OsATG8b-labeled autophagic bodies, visible as green puncta within the vacuole. Scale bars, 10 μm. (b) Quantification of autophagic puncta in the protoplasts of (a). A minimum of 10 protoplasts per transgenic line and condition were analyzed. (c) Assessment of autophagy levels by Anti-NBR1 immunoblotting. Protein extracts from WT and osatg mutants (osatg9a, osatg9b and osatg9a osatg9b ) seedlings were analyzed via anti-NBR1 immunoblotting. Two-week-old seedlings grown under normal conditions were used. Anti-Actin immunoblots were used as loading controls. (d) Quantification of the protein bands shown in (c). Each column represents the mean ± SD (n = 3). Different letters indicate statistically significant differences at the P < 0.05 level, determined by one-way ANOVA followed by post hoc multiple comparisons. not-yet-known not-yet-known not-yet-known unknown FIGURE 3. Characterization of grain traits in osatg9a, osatg9b and osatg9a osatg9b mutants. (a) Representative images of grain length and grain width in WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 . Scale bars, 1 cm. (b) Quantification of grain length, grain width, and thousand-grain weight of the grains shown in (a). Data represent measurements from over 20 plants, with at least 100 grains per plant. (c) Scanning electron micrographs of epidermal cells from the spikelet hulls of WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 at the mature stage. Scale bars, 20 μm. (d) Quantification of cell length, cell width, and cell number in both the longitudinal and transverse directions in the grain shown in (c). Error bars represent SD (n ≥ 3 grains). (e) Grain size analysis at different developmental stages of osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 mutants. Quantification of grain length (left) and grain width (right) in WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 at various lengths of florescence. Each column represents the mean ± SD (n = 3), with different letters indicating statistically significant differences (P < 0.05) as determined by one-way ANOVA followed by post hoc multiple comparisons. Statistical significance of differences between WT and mutants at the same time point was determined using Student’s t -test. * P < 0.05, ** P < 0.01 and *** P < 0.001. FIGURE 4. The phenotypes of osatg9a , osatg9b and osatg9a osatg9b mutants in response to drought stress. (a) Representative images of WT, osatg9a-1 and osatg9a-2 plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (b) Quantification of the survival rates of WT, osatg9a-1 and osatg9a-2 plants after recovery shown in (a). (c) Representative images of WT and osatg9b plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (d) Quantification of the survival rates of WT and osatg9b plants after recovery shown in (c). (e) Representative images of WT, osatg9a osatg9b-1 and osatg9a osatg9b-2 plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (f) Quantification of the survival rates of WT, osatg9a osatg9b-1 and osatg9a osatg9b-2 plants after recovery shown in (e). (g) Representative images of WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 after rehydration in soil pot experiments. (h) Quantification of the survival rates of WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 after rehydration in soil pot experiments shown in (g). Each column represents the mean ± SD ( n = 3) and different letters indicating statistically significant differences ( P < 0.05) as determined by one-way ANOVA followed by post hoc multiple comparisons. Statistical significance of differences between WT and mutants was determined using Student’s t -test. ** P < 0.01. FIGURE 5. Physiological parameters and relative expression levels of drought related genes of osatg9a , osatg9b and osatg9a osatg9b mutants in response to drought stress. (a) Scanning electron micrographs of completely open, partially open, and completely closed stomata. Scale bars, 10 μm. Percentages of completely open, partially open, and completely closed stomata in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Results are represented as means ± SD ( n = 50). Statistical significance between WT and mutant lines was determined using Student’s t -test. ** P < 0.01. (b) Water loss rates in detached leaves from 70-day-old WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants. Results are presented as means ± SD ( n = 3). Statistical significance between WT and mutant lines at the same time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (c) Measurement of proline content, soluble sugar, SOD activity, CAT activity, MDA content and H 2 O 2 content in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (d) Relative expression levels of drought related genes in autophagy related plants. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000 for 6 hours. The relative expression levels of drought related genes were measured in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 6. The role of autophagy in drought stress response in rice. (a, b) Relative expression levels of OsATG9a and OsATG9b after PEG6000 treatment. Leaves were harvested at 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 5 d or 7 d post-treatment for RT-qPCR analysis. The expression levels at the “0 h” time point were normalized to 1, and fold changes relative to the “0 h” time point were calculated. Results are presented as means ± SD ( n = 3). Statistical significance between “0 h” time point and other time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (c, e) Anti-OsNBR1 immunoblot analysis of protein extracts from WT seedlings. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000. Leaves were harvested at 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 5 d or 7 d time points for immunoblot analysis. Protein abundances at the “0 h” time point were set to 1, and fold changes in protein abundance relative to “0 h” time point were calculated as the fold change. Anti-Actin immunoblots were used as loading controls. (d, f) Quantification of the protein bands shown in (c, e). Each column represents the mean ± SD ( n = 3). Statistical significance between “0 h” time point and other time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (g, i, k) Relative OsNBR1 levels in leaves of WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 after 1 day of 20% PEG6000 treatment. OsNBR1 protein abundance was determined by immunoblotting, with Actin serving as a loading control. (h, j, l) Quantification of OsNBR1 levels based on immunoblot density analysis using Image J. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (m) The ratio of OsATG8a:PE/OsATG8a in rice leaves after 1 day of 20% PEG6000 treatment. The proteins were detected with anti‐OsATG8a antibody. Actin was used as the loading control. (n) Statistical analysis of the ratio of OsATG8a-PE/OsATG8a shown in (m). Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 7. The role of OsATG9b in drought stress response in rice. (a, c) Representative images of WT, osatg9b and OsATG9b-OE plants from hydroponic experiments before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (b, d) Quantification of the survival rates for WT, osatg9b and OsATG9b-OE plants after recovery shown in (a, c). Each column represents the mean ± SD ( n = 3). Statistical significance between WT and osatg9b or OsATG9b-OE were determined using Student’s t -test. ** P < 0.01. (e) Representative images of WT and OsATG9b-OE after rehydration in soil pot experiments. (f) Quantification of the survival rates of WT and OsATG9b-OE after rehydration in soil pot experiments shown in (e). Each column represents the mean ± SD ( n = 3). Statistical significance between WT and OsATG9b-OE were determined using Student’s t -test. ** P < 0.01. (g) Percentages of completely open, partially open, and completely closed stomata in WT, osatg9b and OsATG9b-OE plants under normal and PEG6000 treatment conditions. Results are represented as means ± SD ( n = 50). Statistical significance between WT and mutant lines was determined using Student’s t -test. ** P < 0.01. (h) Water loss rates in detached leaves from 70-day-old WT, osatg9b and OsATG9b-OE plants. Results are presented as means ± SD ( n = 3). Statistical significance between WT and mutant lines at the same time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (i) Relative OsNBR1 levels in leaves of WT, osatg9b and OsATG9b-OE after 1 day of 20% PEG6000 treatment. OsNBR1 protein abundance was determined by immunoblotting, with Actin serving as a loading control. (j) Quantification of OsNBR1 levels based on immunoblot density analysis using Image J. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (k) The ratio of OsATG8a:PE/OsATG8a in rice leaves under drought stress for 1 d. The proteins were detected with anti‐OsATG8a antibody. Actin was used as the loading control. (l) Statistical analysis of the ratio of OsATG8a-PE/OsATG8a shown in (k). Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 8. Relative expression levels of ABA related genes in autophagy related plants. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000 for 6 hours. Measurement of relative expression levels of ABA related genes in WT, osatg9a-1 , and osatg9b plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 1. Subcellular localization analysis of OsATG9a and OsATG9b in rice protoplasts through transient transformation. (a) Co-localization of mCherry-AtRER1B with OsATG9a or OsATG9b in rice protoplasts. mCherry-AtRER1B was used as a Golgi marker. (b) Co-localization analyses of OsATG9a and OsATG9b. PC: Pearson’s Correlation. OC: Overlap Coefficient. Scale bars, 10 μm. Experiments were repeated three times with consistent results. FIGURE 2. OsATG9a and OsATG9b positively regulate autophagy in rice. (a) GFP-OsATG8b fusion protein was transient expressed in rice protoplasts derived from WT, osatg9a , osatg9b and osatg9a osatg9b mutants. After treatment with ConA or DMSO for 12 hours in the dark, the protoplasts were observed under confocal microscopy. White arrows indicate GFP-OsATG8b-labeled autophagic bodies, visible as green puncta within the vacuole. Scale bars, 10 μm. (b) Quantification of autophagic puncta in the protoplasts of (a). A minimum of 10 protoplasts per transgenic line and condition were analyzed. (c) Assessment of autophagy levels by Anti-NBR1 immunoblotting. Protein extracts from WT and osatg mutants ( osatg9a , osatg9b and osatg9a osatg9b ) seedlings were analyzed via anti-NBR1 immunoblotting. Two-week-old seedlings grown under normal conditions were used. Anti-Actin immunoblots were used as loading controls. (d) Quantification of the protein bands shown in (c). Each column represents the mean ± SD ( n = 3). Different letters indicate statistically significant differences at the P < 0.05 level, determined by one-way ANOVA followed by post hoc multiple comparisons. not-yet-known not-yet-known not-yet-known unknown FIGURE 3. Characterization of grain traits in osatg9a, osatg9b and osatg9a osatg9b mutants. (a) Representative images of grain length and grain width in WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 . Scale bars, 1 cm. (b) Quantification of grain length, grain width, and thousand-grain weight of the grains shown in (a). Data represent measurements from over 20 plants, with at least 100 grains per plant. (c) Scanning electron micrographs of epidermal cells from the spikelet hulls of WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 at the mature stage. Scale bars, 20 μm. (d) Quantification of cell length, cell width, and cell number in both the longitudinal and transverse directions in the grain shown in (c). Error bars represent SD (n ≥ 3 grains). (e) Grain size analysis at different developmental stages of osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 mutants. Quantification of grain length (left) and grain width (right) in WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 at various lengths of florescence. Each column represents the mean ± SD (n = 3), with different letters indicating statistically significant differences (P < 0.05) as determined by one-way ANOVA followed by post hoc multiple comparisons. Statistical significance of differences between WT and mutants at the same time point was determined using Student’s t -test. * P < 0.05, ** P < 0.01 and *** P < 0.001. FIGURE 4. The phenotypes of osatg9a , osatg9b and osatg9a osatg9b mutants in response to drought stress. (a) Representative images of WT, osatg9a-1 and osatg9a-2 plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (b) Quantification of the survival rates of WT, osatg9a-1 and osatg9a-2 plants after recovery shown in (a). (c) Representative images of WT and osatg9b plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (d) Quantification of the survival rates of WT and osatg9b plants after recovery shown in (c). (e) Representative images of WT, osatg9a osatg9b-1 and osatg9a osatg9b-2 plants from hydroponic experiments are shown before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (f) Quantification of the survival rates of WT, osatg9a osatg9b-1 and osatg9a osatg9b-2 plants after recovery shown in (e). (g) Representative images of WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 after rehydration in soil pot experiments. (h) Quantification of the survival rates of WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 after rehydration in soil pot experiments shown in (g). Each column represents the mean ± SD ( n = 3) and different letters indicating statistically significant differences ( P < 0.05) as determined by one-way ANOVA followed by post hoc multiple comparisons. Statistical significance of differences between WT and mutants was determined using Student’s t -test. ** P < 0.01. FIGURE 5. Physiological parameters and relative expression levels of drought related genes of osatg9a , osatg9b and osatg9a osatg9b mutants in response to drought stress. (a) Scanning electron micrographs of completely open, partially open, and completely closed stomata. Scale bars, 10 μm. Percentages of completely open, partially open, and completely closed stomata in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Results are represented as means ± SD ( n = 50). Statistical significance between WT and mutant lines was determined using Student’s t -test. ** P < 0.01. (b) Water loss rates in detached leaves from 70-day-old WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants. Results are presented as means ± SD ( n = 3). Statistical significance between WT and mutant lines at the same time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (c) Measurement of proline content, soluble sugar, SOD activity, CAT activity, MDA content and H 2 O 2 content in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (d) Relative expression levels of drought related genes in autophagy related plants. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000 for 6 hours. The relative expression levels of drought related genes were measured in WT, osatg9a-1 , osatg9a-2 , osatg9b , osatg9a osatg9b-1 and osatg9a osatg9b-2 plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. not-yet-known not-yet-known not-yet-known unknown FIGURE 6. The role of autophagy in drought stress response in rice. (a, b) Relative expression levels of OsATG9a and OsATG9b after PEG6000 treatment. Leaves were harvested at 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 5 d or 7 d post-treatment for RT-qPCR analysis. The expression levels at the “0 h” time point were normalized to 1, and fold changes relative to the “0 h” time point were calculated. Results are presented as means ± SD (n = 3). Statistical significance between “0 h” time point and other time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (c, e) Anti-OsNBR1 immunoblot analysis of protein extracts from WT seedlings. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000. Leaves were harvested at 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 5 d or 7 d time points for immunoblot analysis. Protein abundances at the “0 h” time point were set to 1, and fold changes in protein abundance relative to “0 h” time point were calculated as the fold change. Anti-Actin immunoblots were used as loading controls. (d, f) Quantification of the protein bands shown in (c, e). Each column represents the mean ± SD (n = 3). Statistical significance between “0 h” time point and other time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (g, i, k) Relative OsNBR1 levels in leaves of WT, osatg9a-1, osatg9a-2, osatg9b, osatg9a osatg9b-1 and osatg9a osatg9b-2 after 1 day of 20% PEG6000 treatment. OsNBR1 protein abundance was determined by immunoblotting, with Actin serving as a loading control. (h, j, l) Quantification of OsNBR1 levels based on immunoblot density analysis using Image J. Each column represents the mean ± SD (n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (m) The ratio of OsATG8a:PE/OsATG8a in rice leaves after 1 day of 20% PEG6000 treatment. The proteins were detected with anti‐OsATG8a antibody. Actin was used as the loading control. (n) Statistical analysis of the ratio of OsATG8a-PE/OsATG8a shown in (m). Each column represents the mean ± SD (n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 7. The role of OsATG9b in drought stress response in rice. (a, c) Representative images of WT, osatg9b and OsATG9b-OE plants from hydroponic experiments before treatment, after 7 days of exposure to 20% (w/v) PEG6000, and after 5 days of recovery in water. (b, d) Quantification of the survival rates for WT, osatg9b and OsATG9b-OE plants after recovery shown in (a, c). Each column represents the mean ± SD ( n = 3). Statistical significance between WT and osatg9b or OsATG9b-OE were determined using Student’s t -test. ** P < 0.01. (e) Representative images of WT and OsATG9b-OE after rehydration in soil pot experiments. (f) Quantification of the survival rates of WT and OsATG9b-OE after rehydration in soil pot experiments shown in (e). Each column represents the mean ± SD ( n = 3). Statistical significance between WT and OsATG9b-OE were determined using Student’s t -test. ** P < 0.01. (g) Percentages of completely open, partially open, and completely closed stomata in WT, osatg9b and OsATG9b-OE plants under normal and PEG6000 treatment conditions. Results are represented as means ± SD ( n = 50). Statistical significance between WT and mutant lines was determined using Student’s t -test. ** P < 0.01. (h) Water loss rates in detached leaves from 70-day-old WT, osatg9b and OsATG9b-OE plants. Results are presented as means ± SD ( n = 3). Statistical significance between WT and mutant lines at the same time point was determined using Student’s t -test. * P < 0.05 and ** P < 0.01. (i) Relative OsNBR1 levels in leaves of WT, osatg9b and OsATG9b-OE after 1 day of 20% PEG6000 treatment. OsNBR1 protein abundance was determined by immunoblotting, with Actin serving as a loading control. (j) Quantification of OsNBR1 levels based on immunoblot density analysis using Image J. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. (k) The ratio of OsATG8a:PE/OsATG8a in rice leaves under drought stress for 1 d. The proteins were detected with anti‐OsATG8a antibody. Actin was used as the loading control. (l) Statistical analysis of the ratio of OsATG8a-PE/OsATG8a shown in (k). Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. FIGURE 8. Relative expression levels of ABA related genes in autophagy related plants. Fourteen-day-old seedlings grown in hydroponic were treated with 20% PEG6000 for 6 hours. Measurement of relative expression levels of ABA related genes in WT, osatg9a-1 , and osatg9b plants under normal and PEG6000 treatment conditions. Each column represents the mean ± SD ( n = 3), with different letters indicating statistically significant differences at the P < 0.05 level, as determined by one-way ANOVA followed by post hoc multiple comparisons. Information & Authors Information Version history V1 Version 1 03 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords gene duplication genetic variation grain development osatg stress response Authors Affiliations Yiming Li South China Agricultural University College of Agriculture View all articles by this author Yuantai Liu South China Agricultural University College of Agriculture View all articles by this author Mengzhao Shi South China Agricultural University College of Agriculture View all articles by this author Xiaoyun Luo South China Agricultural University College of Agriculture View all articles by this author Yanshu Huang South China Agricultural University College of Agriculture View all articles by this author Hao Zeng South China Agricultural University College of Agriculture View all articles by this author Yunfeng Liu Guangxi University College of Life Science and Technology View all articles by this author Yifeng Huang Zhejiang Academy of Agricultural Sciences View all articles by this author Peng Xu Xishuangbanna Tropical Botanical Garden View all articles by this author Yangwen Qian WIMI Biotechnology Co Ltd View all articles by this author Xixian Li South China Agricultural University College of Agriculture View all articles by this author Jieying Wang South China Agricultural University College of Agriculture View all articles by this author Qingjun Xie 0000-0002-6372-3260 South China Agricultural University College of Agriculture View all articles by this author Qianying Yang [email protected] South China Agricultural University College of Agriculture View all articles by this author Metrics & Citations Metrics Article Usage 384 views 231 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yiming Li, Yuantai Liu, Mengzhao Shi, et al. Differential roles of duplicate genes OsATG9a and OsATG9b in development and drought stress response in rice. Authorea . 03 February 2025. DOI: https://doi.org/10.22541/au.173856974.49208859/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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