# Source paper archive - Source title: NcSnRK1and NcRbohC1 regulate pollen germination on waterlily stigma after pollination - Author(s): Jing Cao, Xin-Rui He, Fan-Rui Min, Chun-Qing Sun - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.28.762668v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.10.28.762668v1.full.pdf - PDF SHA-256: f5c2cb76047344f3fef28c1a1c1acc8601a72516675b6a6d8ff6984c5c5a6c5c - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1 NcSnRK1 and NcRbohC1 regulate pollen germination on waterlily stigma after 2 pollination 3 # 4 Jing Cao [1], Xin-Rui He [2], Fan-Rui Min [2], Chun-Qing Sun [1*] 5 6 7 _1_ _Zhenjiang Institute of Agricultural Science in Jiangsu Hilly Areas, Jurong, 212400, China._ 8 _2_ _College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China_ 9 10 11 **Corresponding author: Chun-Qing Sun** 12 Tel: +86 051180978053 13 E-mail: scqsunchunqing@126.com 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 32 **NcSnRK1and** **NcRbohC1** **regulate** **pollen** **germination** **on** **waterlily** **stigma** **after** 33 **pollination** 34 **Abstract** : Pre-fertilization barrier is the primary cause of intersubgeneric 35 hybridization barrier in waterlily. The research results indicate that reactive oxygen 36 species (ROS) are involved in regulating the germination process of pollen on the 37 stigma of waterlily after pollination, and candidate genes _NcSnRK1_ and _NcRbohC1_ 38 have been identified; Transient transgenic experiments were used to validate the 39 functions of NcSnRK1 and NcRbohC1 on the waterlily stigma. Through yeast 40 two-hybrid assays, luciferase complementation, and Pull-down experiments, the 41 interaction between NcSnRK1 and NcRbohC1 was confirmed. The above results 42 indicate that NcSnRK1 may phosphorylate certain N-terminal sites of NcRbohC1 43 after pollination in waterlily, thereby activating ROS burst in the stigmatic papilla 44 cells, disrupting ROS homeostasis on stigma of waterlily, and consequently inhibiting 45 pollen germination on the stigma. The research results can provide theoretical basis 46 for taking effective measures to overcome pre-fertilization barrier and germplasm 47 innovation in waterlily. 48 **Keywords** : _Nymphaea_ ; Sexual hybridization; Pollen-stigma recognition; NADPH 49 oxidase; _NcSnRK1_ ; _NcRbohC1_ 50 **Introduction** 51 Waterlily is a perennial herbaceous flowering plant belonging to the genus 52 _Nymphaea_ in the family Nymphaeaceae (Wang et al., 2025). According to the 53 presence or absence of their carpel appendages, waterlily can be divided into 54 subgenus _Nymphaea_, subgenus _Brachyceras_, subgenus _Anecphya_, subgenus _Lotos_, 55 and subgenus _Hydrocallis_ . Among them, the subgenus _Nymphaea_ is hardy waterlily, 56 while the other four subgenus are tropical waterlily (Sun et al., 2021). At present, 57 cross breeding is still the most common and effective method used in waterlily 58 breeding (Sun et al., 2018; Zhou et al., 2024). However, in the process of cross 59 breeding of waterlily, many breeders have found that intersubgeneric hybridization 60 (hardy waterlilies and tropical waterlilies) has poor seed setting rates, and some 61 hybrid combinations even cannot obtain seeds (Wang et al., 2025; Sun et al., 2019; 62 Sun et al., 2023). This problem greatly affects the efficiency of waterlily breeding and 63 restricts the development and utilization of some excellent color and flower type [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 64 genes in tropical waterlily. For example, breeders hope to transfer the blue flower 65 gene of tropical waterlily to hardy waterlily in order to obtain blue flower waterlily 66 with cold resistant traits, but most of them have not been successful (Sun et al., 2018). 67 Previous studies have shown that pre-fertilization barrier is the main cause of 68 intersubgeneric hybridization barrier in waterlily (Sun et al., 2019; Hao et al., 2022; 69 Sun et al., 2023). However, the specific reasons for the intersubgeneric hybridization 70 barrier of waterlily have not been reported. 71 In the early stage of this study, transcriptome and proteomic analysis were 72 conducted on the stigma of waterlily after intersubgeneric hybridization and 73 self-pollination. It was found that ROS was involved in regulating the pollen 74 germination on the stigma of waterlily after pollination, and candidate genes 75 _NcSnRK1_ and _NcRbohC1_ were identified. This study will focus on the candidate 76 genes _NcSnRK1_ and _NcRbohC1_, and conduct in-depth research on the mechanism of 77 intersubgeneric hybridization barrier in waterlily from functional analysis and 78 mechanism of action. It can provide theoretical and technical support for breaking 79 down intersubgeneric hybridization barrier in waterlily and has important guiding 80 significance for cultivating new waterlily varieties. 81 **Materials and Methods** 82 **Plant materials** 83 Artificial hybridization was carried out using the hardy waterlily 84 ( _N_ .‘Arc-EN-Ciel’) as the female parent and the tropical waterlily ( _N_ . ‘Nine Grade 85 Blue’) as the male parent. The parent plants were bagged and isolated one day before 86 flowering. During self-pollination and cross-pollination, the pistils of the female 87 parent plants were emasculated on the day of flowering, and fresh pollen from the 88 male parent plants was injected into the stigma liquid of the female parent plants. 89 After pollination, the flowers were bagged and isolated again. Waterlily plants are 90 preserved in the aquatic flower resource garden of Zhenjiang Academy of Agricultural 91 Sciences in hilly areas of Jiangsu Province. 92 **Pollen germination on stigma** 93 The germination and growth of pollen on the stigma were observed using the 94 aniline blue staining method. After 6 h pollination, the entire stigma tissue was 95 quickly cut off with a knife and placed in the FAA (70% ethanol : acetic acid: 96 formaldehyde solution = 90 : 5 : 5) fixative solution for more than 12 h of treatment. 97 The fixed stigma tissue was longitudinally cut and soaked in 70% ethanol, 50% [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 98 ethanol, 30% ethanol, and water in sequence for 10 minutes each, then the stigma 99 tissue was placed in 8 mol/L NaOH solution for softening for 12 h. After softening, it 100 was rinsed with distilled water 3 times and stained with 0.1% aniline blue solution in 101 the dark for 4 h. The pollen tube growth was observed under a upright fluorescence 102 microscope (Ex375-328/DM415/BA351p), and photos were taken using a DSRi2 103 digital camera (Sun et al., 2015). Each experiment was conducted with three 104 biological replicates. 105 **Determination of ROS content in stigma** 106 After 0.5 h pollination, the stigma was quickly cut off and immersed in the 107 MES-KCl buffer solution (10 mM MES, 5 mM KCl, 50 mM CaCl 2, pH 6.15) for 30 108 minutes. The stigma was then longitudinally cut with a knife, and stained in the dark 109 with 50 mM H 2 DCFDA fluorescent dye for 1.5 h. The sample was then rinsed three 110 times with the MES-KCl buffer solution (Zhang et al., 2021; Wang et al., 2025). After 111 cleaning, the sample was placed on a glass slide and observed and photographed using 112 an upright fluorescence microscope (with the same exposure value of 200) for the 113 analysis of the experimental results. 114 **Vector construction and transient transgene** 115 The transient gene interference or overexpression of waterlily was achieved by 116 using the improved double virus vector system (IL-60-BS/IR) of tomato yellow leaf 117 curl virus (Ofer et al., 2014; Yuvalet al., 2007). The full-length _NcRbohC1_ ( _NcSnRK1_ ) 118 gene was inserted into the PIR-X empty vector to form the PIR- _NcRbohC1_ 119 (PIR- _NcSnRK1_ ) overexpression vector; the conserved region of approximately 300 bp 120 in the _NcRbohC1_ ( _NcSnRK1_ ) gene was inserted into the IR-X-RI empty vector to 121 form the IR- _NcRbohC1_ (IR- _NcRbohC1_ ) interference vector. The overexpression 122 vector PIR- _NcRbohC1_ (PIR- _NcSnRK1_ ) or the interference vector IR- _NcRbohC1_ 123 (IR- _NcRbohC1_ ) was mixed with the IL-60-1 plasmid at a ratio of 1:1 and injected into 124 the young leaves of waterlily at a concentration of 800 ng/100 μL, with 200 μL 125 injected each time and the infection performed once a week (Li et al., 2024). 126 Cross-pollination and self-pollination were conducted 14 days after infection. The 127 primers used for gene cloning and vector construction are shown in Table S1, and the 128 gel imaging results of the bacterial liquid detection are shown in Figure S1. 129 **RNA extraction and real-time quantitative PCR** 130 RNA extraction was performed using FastPure Universal Plant Total RNA 131 Isolation Kit (Vazyme Biotech Co., Ltd; Nanjing); Reverse to cDNA using HiScript [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 132 III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd; Nanjing); The 133 ChamQ SYBR qPCR Master Mix was selected as the fluorescent quantitative reagent 134 and measured using the QuantStudioTM real-time fluorescent quantitative PCR 135 system. The relative expression levels of genes were calculated using the ∆CT method, 136 and the Actin11 gene of waterlily was used as an internal reference gene (Luo et al., 137 2010). The relevant primer designs are shown in Table S1. 138 **Subcellular localization** 139 For the subcellular localization of proteins in _Nicotiana_ _benthamiana_, the 140 Agrobacterium GV3101 strain carrying pCambia1300-35S:: _NcRbohC1-_ GFP 141 (pCambia1300-35S:: _NcSnRK1_ -GFP) was cultured at 28°C and 200 rpm/min until OD 142 600=0.8, and then resuspended until OD 600=1.0. The resuspension was prepared by 143 adding 1 mL 1mol/L MES, 1 ml 1mol/L MgCl 2 and 100 μL 2 mol/L acetosyringone 144 (AS) to 100 mL distilled water. The resuspension was still in the dark for 2-3 h, and 145 then was immersed into the leaves of _Nicotiana_ _benthamiana_ with a sterile syringes. 146 The leaves were cultured overnight in the dark and then cultured under normal light 147 for 48-72 h. The GFP fluorescence signal position was observed using a confocal laser 148 scanning microscope (Leica TCS SP8) to determine the subcellular localization of 149 _NcRbohC1_ ( _NcSnRK1_ ). 150 **Yeast two hybridization** 151 The yeast two-hybrid bait vector pGBKT7- _NcRbohC1_ -N (BD- _NcRbohC1_ -N) 152 was constructed. Combined with BD-empty and pcl1 plasmids, self-activation 153 verification was conducted respectively in single-missing plate SD/-Trp or SD/-Leu. 154 Using pGADT7 (AD) prey in yeast library and BD- _NcRbohC1_ -N, x-α-gal dye was 155 used to screen out the genes that might interact with BD- _NcRbohC1_ -N in 156 SD/-Trp-Leu and SD/-Trp-Leu-His-Ade. The gene vector AD- _NcSnRK1_, which may 157 interact with BD- _NcRbohC1_ -N, was constructed and verified by yeast one-to-one 158 interaction using x-α-gal dye in SD/-Trp-Leu and SD/-Trp-Leu-His-Ade, respectively. 159 **Luciferase Complementation Assay** 160 The vectors of pCAMBIA1300-nluc- _NcRbohC1_ -N (nluc- _NcRbohC1_ -N) and 161 pCAMBIA1300-cluc- _NcSnRK1_ (cluc- _NcSnRK1_ ) were constructed, and the plasmids 162 were transferred into Agrobacterium GV3101 strain with OD 600=1.0. The 163 supernatant was removed by centrifugation, and resuspended to OD 600=0.4-0.6 with 164 the resuspension. After standing at 28℃in the dark for 3 h, the supernatant was 165 inserted into the leaves of _Nicotiana benthamiana_ . After dark culture for 1 d and light [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 166 culture for 2 d, the luciferase (D-luciferase potassium salt) was used to measure 167 luciferase activity using NightOWL II LB983 in vivo imager (Berthold, Stuttgart, 168 Germany). The suspension solution was prepared according to 2.5. 169 **Protein induction and pull-down experiments** 170 The _NcRbohC1_ -N gene was inserted into the pGEX-4T-1 vector with GST tag, 171 and the _NcSnRK1_ gene was inserted into the pCold vector with His tag. The 172 constructed recombinant plasmid was transferred into Escherichia coli BL21 cells to 173 form the _NcRbohC1_ -N-GST and _NcSnRK1_ -His fusion proteins. 174 The bacterial liquid was propagated at 37℃and 200rpm until OD 600=0.6-0.8. 175 500 μL of bacterial solution was absorbed, centrifuged at 5000 g for 15 min at 4℃, 176 and stored at -80℃for later use. IPTG was added to the remaining bacterial solution 177 (the final concentration was 0.1 mM) for protein induction. SDS loading buffer was 178 added to 200 uL bacterial solution and then boiled in boiling water at 100℃for 10 179 min. SDS-PAGE electrophoresis and protein staining analysis were performed. After 180 successful induction, the protein was centrifuged to obtain the supernatant. The 181 bacterial pellet was resuspended in 1×PBS and separated using an ultrasonic 182 disintegrator (with ice bath throughout, approximately 25 min). After the treatment, 183 the sample was centrifuged at 10000 rpm for 10 minutes at 4℃. 184 The two proteins that had been ultrasonically broken were mixed and incubated 185 at 4℃for 2 h in an incubator. 50-100 μL of the product was aspirated and stored at 186 -80℃for future use. The washed Anti-GST magnetic beads were added to the 187 remaining product and incubated for more than 8 h. The protein magnetic beads that 188 have been incubated were washed with 1×PBS (this process was repeated 3 times). 189 After washing, they were placed in a multi-functional rotating shaker at room 190 temperature for a brief incubation. Then, SDS loading buffer was added, and the 191 mixture was boiled in boiling water for 10 min to denature. Subsequently, 192 polyacrylamide gel electrophoresis and Western blotting were performed. 193 **Western blot assay** 194 The treated protein samples were separated by 12% polyacrylamide gel 195 electrophoresis for 80 min. The protein glue was imprinted on the PVDF membrane 196 (the PVDF membrane needed to be activated in advance). After transferring the 197 membrane, the membrane was placed into a Petri dish with blocking solution, placed 198 in a shaker at 75 rpm, and blocked at 4℃for more than 1 h. The membrane was 199 cleaned with 1×PBST for 3 times. The washed membranes were placed in the primary [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 200 antibody buffer solution, incubated at 75 rpm and 4℃for 12 h, and then washed 5-6 201 times. The washed membrane was placed in the secondary antibody buffer, incubated 202 at 75 rpm at 4℃for 4 h. After washing the membrane 5-6 times again, it was 203 observed using a universal molecular imager. 204 **Data analysis** 205 All data are presented in the form of mean ± standard deviation. Data analysis 206 was conducted using Microsoft Office Excel. For the analysis of significant 207 differences, one-way analysis of variance was selected, and Tukey's multiple 208 comparison test or independent sample T-test was used. GraphPad Prism 8 was used 209 for plotting. 210 **Results** 211 **Pre-fertilization barrier between subgenera** 212 After 6 h of self-pollination of _N_ .‘Arc-EN-Ciel’, a large number of pollen tubes 213 germinated and penetrated the stigma disk to reach the ovule for fertilization (Fig. 1A). 214 In the _N_ .‘Arc-EN-Ciel’× _N_ . ‘Nine Grade Blue’, the pollen grains only adhered to the 215 stigma disk but did not germinate (Fig. 1B).The results indicate that there is a 216 pre-fertilization barrier in intersubgeneric hybridization of waterlily. 217 **ROS is the key regulatory factor** 218 In the preliminary stage of this study, transcriptome and proteomics analyses 219 were conducted on the stigma of waterlily after cross-pollination and self-pollination. 220 It was found that ROS was involved in regulating pollen germination on stigma after 221 pollination in waterlily. The ROS content at 0.5 h in the _N_ .‘Arc-EN-Ciel’ × _N_ . ‘Nine 222 Grade Blue’ (Fig. 1D) was significantly higher than that in the self-pollination of 223 _N_ .‘Arc-EN-Ciel’ (Fig. 1E). This indicates that ROS may play an important role in the 224 regulation of pollen germination on stigma after pollination in waterlily. Half an hour 225 before pollination of _N_ .‘Arc-EN-Ciel’ × _N_ . ‘Nine Grade Blue’, an antioxidant DMTU 226 (N, N’-Dimethylthiourea) was added to the stigma liquid. The ROS level in the 227 stigma significantly decreased, and a large number of pollen grains germinated on the 228 stigma after cross-pollination (Fig. 2A and D); while half an hour before 229 self-pollination of _N_ . ‘Arc-EN-Ciel’, xanthine/xanthine oxidase (X/XO) was added to 230 the stigma liquid. The ROS level in the stigma significantly increased, and it 231 effectively inhibited the germination of pollen on the stigma (Fig. 2B and E). The 232 above experiment demonstrated that ROS is involved in regulating the germination of 233 pollen on the stigma after pollination in waterlily. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 234 **NcRbohC1 associated with ROS generation** 235 Half an hour before pollination, DPI (NADPH oxidase inhibitor) was added to 236 the stigma fluid of _N_ .‘Arc-EN-Ciel’ × _N_ . ‘Nine Grade Blue’. The ROS level in the 237 stigma decreased significantly, and a large number of pollen germinated on the stigma 238 after cross-pollination (Fig. 2C and F). This indicates that NADPH oxidase is the 239 main source of ROS in the stigma of waterlily. Based on the previous transcriptome 240 data and the verification of stigma gene expression levels after cross-pollination and 241 self-pollination, we found that the expression level of NcRbohC1 showed significant 242 differences (Fig. 1C), suggesting that NcRbohC1 may play an important role in the 243 ROS metabolism process of the stigma of waterlily. 244 **Functional verification of NcRbohC1** 245 Subcellular localization indicated that NcRbohC1 was located on the cell 246 membrane (Fig. 3). The waterlily plants were treated with interference (IR- _NcRbohC1_ ) 247 or overexpression (PIR- _NcRbohC1_ ). After the expression level of NcRbohC1 showed 248 significant changes, cross-pollination and self-pollination were performed (Fig. S2). 249 The qRT-PCR experiment proved that after cross-pollination, the expression level of 250 NcRbohC1 in the stigma of the interference treatment group decreased significantly, 251 while that in the overexpression treatment group increased significantly (Fig. 4A); 252 after self-pollination, the expression levels of the interference and overexpression 253 groups showed opposite trends compared to those after cross-pollination (Fig. 4B). 254 After the transient transgenic treatment for cross-pollination, the pollen of stigma in 255 the interference group germinated and the ROS content decreased significantly 256 (Fig.5A and E), while the over-expressed pollen did not germinate, and the ROS 257 content was high (Fig.5B and F). After transient transgene treatment for 258 self-pollination, the pollen germination quantity in the overexpression group was 259 significantly lower than that in the interference group (Fig. 5C and D), and the ROS 260 content also increased (Fig. 5G-H). Combined with the results of gene function 261 verification (Fig. 4A), it indicates that NcRbohC1 plays an important role in 262 regulating the ROS content in the stigma of waterlily, and NcRbohC1 participates in 263 regulating the germination process of pollen on the stigma after pollination by 264 regulating the ROS content. 265 **Screening of regulatory proteins of NcRbohC1** 266 To further explore the regulatory mechanism of NcRbohC1 during pollen 267 germination, the pGBKT7- _NcRbohC1_ -N (BD- _NcRbohC1_ -N) vector was constructed [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 268 (the N-terminal region of the _NcRbohC1_ gene is the functional site), and 269 self-activation verification was performed (Fig. 6A). The existing yeast library was 270 screened. A target gene _NcSnRK1_, which might interact with it and be related to 271 pollen germination, was identified (Fig. S3). The pGADT7- _NcSnRK1_ (AD- _NcSnRK1_ ) 272 vector was constructed, and one-to-one yeast two-hybrid interaction verification 273 experiment was conducted with pGBKT7- _NcRbohC1_ -N. The results showed that 274 BD- _NcRbohC1_ -N interacted with AD- _NcSnRK1_ (Fig. 6B). The pull-down (Fig. 6C) 275 and luciferase complementation (Fig. 6D) experiments also demonstrated that there is 276 an interaction between NcRbohC1-N and NcSnRK1. 277 **Functional verification of** _**NcSnRK1**_ 278 After self-pollination, the expression level of the _NcSnRK1_ gene significantly 279 decreased in _N_ .‘Arc-EN-Ciel’, but it did not increase significantly after pollination in 280 the _N_ .‘Arc-EN-Ciel’ × _N_ . ‘Nine Grade Blue’ (Fig. 1F). Subcellular localization 281 indicated that NcSnRK1 was located in the nucleus (Fig. 3). Using transient 282 transgenic technology to treat waterlily plants, the cross-pollination and 283 self-pollination were performed after the expression level of _NcSnRK1_ changed (Fig. 284 S4). The results showed that the expression level of NcSnRK1 in the interference 285 group decreased significantly (Fig. 4C), while it increased significantly in the 286 overexpression group (Fig. 4D). Compared with the hybridization after 287 overexpression of _NcSnRK1_, the pollen germination was significantly increased after 288 interference (Fig. 7A and B), and the ROS content was significantly decreased (Fig. 289 7E and F); while the number of self-pollen germination after overexpression of 290 _NcSnRK1_ was significantly lower than that after interference (Fig. 7C and D), and the 291 ROS showed the opposite trend (Fig. 7G and H). This indicates that NcSnRK1 and 292 NcRbohC1 have a synergistic effect on the pollen germination of the stigma. 293 **The regulatory relationship between NcRbohC1 and NcSnRK1** 294 Cross-poliination after plant interference/overexpression of _NcSnRK1_ had a 295 relatively small effect on the expression level of _NcRbohC1_ (Fig.8A), and no 296 significant difference was observed compared with the control group (Cp). Although 297 self-pollination showed a significant difference compared with the control group (Sp) 298 (Fig.8 B), it might be because self-pollination itself can cause changes in ROS content. 299 After overexpression of _NcRbohC1_, both cross-pollination and self-pollination 300 experiments showed a significant increase in _NcSnRK1_ expression (Fig. 8 C-D). 301 Conversely, after interfering with _NcRbohC1_, the expression of _NcSnRK1_ decreased [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 302 significantly (Fig. 8 C-D). This indicates that NcSnRK1 may be upstream of 303 NcRbohC1, and may indirectly regulate ROS expression through NcRbohC1. 304 **Discussion** 305 Most of the existing molecular mechanisms of prefertilization barrier in plant 306 distant hybridization are based on the self-incompatibility (SI) system, which is a 307 typical interspecific hybridization barrier in which the stigma maintains outcrossing 308 by rejecting its own pollen (Huang et al., 2023; Qi et al., 2023). For instance, the SI 309 receptor (S-locus receptor kinase, SRK) on the stigma of Chinese cabbage can 310 recognize self-pollen and activate the ROS pathway on the stigma, increasing the 311 ROS level on the stigma and thereby preventing self-pollen from entering the ovary 312 (Huang et al., 2023; Cao et al., 2025). Similarly, the S-RNase gene expressed in the 313 pistil of pear can recognize the F-box expressed in self-pollen and prevent the pollen 314 tube from extending into the ovary (Qi et al., 2023). Not all plants have the SI system, 315 and the SI systems of plants from different families and genera also vary significantly. 316 Therefore, the molecular mechanism of incompatibility in distant hybridization based 317 on the SI system can only explain some of the hybridization barriers between plant 318 species, and does not have broad applicability. 319 After the recognition barrier of pollen and stigma, the downstream pathways of 320 stigmatic papillar cells can be further activated, among which the ROS metabolic 321 pathway is more common (Zhang et al., 2021; Huang et al., 2023; Liu et al., 2021.). 322 ROS plays an important regulatory role in self-incompatibility systems of the 323 Papaveraceae (Wilkins et al., 2011; Eaves et al., 2014), _Brassica_ (Zhang et al., 2021; 324 Huang et al., 2023), and the genus _Pyrus_ (Jiang et al., 2014). Similarly, this study also 325 demonstrates that ROS is involved in regulating the germination of pollen on the 326 stigma after pollination in _Nymphaea_ . The NADPH oxidase RBOHs is also known as 327 the respiratory burst oxidase homolog. It is the main source of ROS production in 328 plants. RBOHs have been extensively studied in the process of pollen and stigma 329 recognition (Zhang et al., 2021). This study also demonstrated that NcRbohC1 is a 330 key protein regulating the ROS content in the stigma of waterlily. 331 ROS production by RBOHs is mainly caused by Calcium-dependent protein 332 kinase (CDPK/CPK) (Kaur and Pati, 2018; Yu et al., 2018), ROP (Rho-related [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 333 GTPases from plants) protein (Huang et al., 2013), RAC (Ras-related C3 botulinum 334 toxin substrate) protein (Potocký et al., 2012;), etc. Recent studies have found that 335 RBOHs on the stigma are mainly regulated by ROPs to produce ROS. For example, in 336 Chinese cabbage, the FER-RAC/ROPs-RBOHs signaling module can regulate the 337 increase in ROS content on the self-pollination stigma, leading to self-incompatibility 338 (Zhang et al., 2021; Huang et al., 2023). Similarly, in Arabidopsis, the 339 FER/ANJ/LLG1-ROP2-RBOHD signaling module regulates the changes in ROS 340 content (Liu et al., 2021). However, in waterlily, using yeast two-hybrid technology to 341 further screen the interacting proteins of NcRbohC1, it was found that NcRbohC1 342 does not interact with ROPs, indicating that the regulation mechanism of ROS in the 343 stigma of waterlily is different from that of Chinese cabbage and Arabidopsis. The 344 regulatory mechanism still needs further study. The yeast two-hybrid technique was 345 used to screen the interacting proteins of NcRbohC1. It was found that NcSnRK1 346 interacted with NcRbohC1. Pull-down and LCA experiments also proved that there 347 was an interaction between NcRbohC1 and NcSnRK1. Moreover, our previous 348 research revealed that NcSnRK1 could regulate the ROS content on the stigma after 349 pollination in waterlily (Zhou et al., 2025). This indicates that NcSnRK1 may regulate 350 the ROS content on the stigma after pollination in waterlily by regulating 351 NcRBOHC1. 352 The SnRK1 of plants is a homolog of the yeast SNF1 complex and the 353 mammalian AMPK complex (Baena-González and Lunn, 2020). The SnRK1 protein 354 kinase is a heterotrimeric complex composed of the α catalytic subunit and the 355 regulatory subunits β and γ (Baena-González and Lunn, 2020). The function of 356 SnRK1 is mainly determined by the α catalytic subunit, and the function of the α 357 subunit is crucial for understanding the function of the SnRK1 protein kinase 358 (Martinez-Barajas and Coello, 2020). The α subunit contains a kinase domain, and the 359 N-terminal kinase domain and the C-terminal regulatory domain play a key role in the 360 function of SnRK1 (Broeckx et al., 2016; Margalha et al., 2016). SnRK1 has been 361 reported to be involved in regulating the sexual reproduction process of plants. 362 Previous studies have shown that SnRK1 had an impact on the directional growth of 363 pollen, thereby affecting the fertilization process. For instance, in _Arabidopsis_ [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 364 _thaliana_, the overexpression of SnRK1 inhibits the growth of pollen tubes, thereby 365 affecting fertilization (Baena-Gonzalez et al., 2007). The SnRK1 mutant kinβγ plants 366 of Arabidopsis exhibited pollen abortion, the number of mitochondria and 367 peroxisomes in the pollen was significantly less than that of the wild type, and the 368 level of ROS in the pollen was significantly reduced, which led to the failure of pollen 369 to absorb water and germinate on the stigma (Gao et al., 2016). The SnRK1 mutant 370 kinβγ plants of Arabidopsis exhibited pollen abortion, with significantly fewer 371 mitochondria and peroxisomes in pollen than the wild type, and significantly lower 372 levels of ROS in pollen, resulting inthe failure of pollen to absorb water and 373 germinate on the stigma (Gao et al., 2016). Furthermore, SnRK1 can participate in the 374 regulation of ROS homeostasis. For example, SnRK1α in tomato can phosphorylate 375 serine residues Ser188, Ser189 and Ser308 on RBOH1 protein, and activate RBOH1 376 to produce apoplasmic ROS (Zheng et al., 2025). 377 Based on the above analysis, we infer that after cross-pollination among the 378 subgenera of _Nymphaea_, the pollen and stigma encounter recognition barrier, 379 resulting in the extensive expression of NcSnRK1 and NcRbohC1. NcSnRK1 380 phosphorylates several sites at the N-terminal of NcRBOHC1, thereby activating the 381 ROS burst in the stigmatic papilla cells of the waterlily, leading to the disruption of 382 the ROS homeostasis in the waterlily stigma, and subsequently hindering the 383 germination of pollen on the stigma. 384 385 **Author contributions** 386 **Jing Cao:** Investigation, Methodology, Writing – review & editing. **Xin-Rui He:** 387 Data curation, Validation. **Fan-Rui Min:** Data curation, Formal analysis. **Chun-Qing** 388 **Sun:** Methodology, Supervision, Writing – review & editing. 389 **Declaration of Competing Interest** 390 The authors declare no conflict of interest. 391 **Acknowledgments** 392 This study was supported by the National Natural Science Foundation of China [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 393 (31701948) and the Fund of Independent Innovation of Agricultural Sciences in Jiang 394 Su Province of China [CX(19)3039]. 395 **Abbreviations** 396 ROS, reactive oxygen species; Rboh, Respiratory burst oxidase homolog protein ; 397 qRT-PCR, Quantitative Real-time PCR; NADPH, Nicotinamide Adenine Dinucleotide 398 Phosphate Oxidase; DPI, Diphenyliodonium Chloride; DMTU: Dimethylthiourea; 399 X/XO, xanthine/xanthine oxidase; CDPK, Calcium-dependent protein kinase; RAC, 400 Ras-related C3 botulinum toxin substrate; ROP, Rho-related GTPases from plants; SI, 401 Self-incompatibility. 402 403 **References** 404 Baena-González E, Lunn JE. SnRK1 and trehalose 6-phosphate–two ancient 405 pathways converge to regulate plant metabolism and growth. Current Opinion in 406 Plant Biology, 2020, 55: 52-59. 407 Baena-González E, Rolland F, Thevelein JM, et al. A central integrator of 408 transcription networks in plant stress and energy signalling. Nature, 2007, 448: 409 938-942. 410 Broeckx T, Hulsmans S, Rolland F. The plant energy sensor: evolutionary 411 conservation and divergence of SnRK1 structure, regulation, and function. 412 Journal of Experimental Botany, 2016, 67: 6215-6252. 413 Cao YY, Cui XS, Yang YQ, Pan LH, Li SY, et al. Pan-family pollen signals control an 414 interspecific stigma barrier across Brassicaceae species. Science, 2025, 391. 415 Eaves DJ, Flores-Ortiz C, Haque T, Lin Z, Teng N, Franklin-Tong VE. 416 Self-incompatibility in _Papaver_ : advances in integrating the signalling network. 417 Biochemical Society Transactions, 2014, 42: 370-376. 418 Gao XQ, Liu CZ, Li DD, et al. The Arabidopsis KINβγ subunit of the SnRK1 419 complex regulates pollen hydration on the stigma by mediating the level of 420 reactive oxygen species in pollen. PLoS Genetics, 2016, 12: e1006228. 421 Hao Q, Xu LH, Wang H, Liu QH, Wang KL. Evaluation of pollen viability, stigma 422 receptivity, and the cross barrier between tropical and hardy water lily cultvars. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 423 Flora, 2022, 290: 152046. 424 Huang GQ, Li E, Ge FR, Li S, Wang Q, Zhang CQ, Zhang Y. Arabidopsis RopGEF4 425 and RopGEF10 are important for FERONIA-mediated developmental but not 426 environmental regulation of root hair growth, New Phytologist, 2013, 200(4): 427 1089-1101. 428 Huang JB, Yang L, Yang L, Wu XY, Cui XS, et al. Stigma receptors control 429 intraspecies and interspecies barriers in Brassicaceae. Natue, 2023, 614: 430 303-335. 431 Jiang X, Gao Y, Zhou H, Chen J, Wu J, Zhang S. Apoplastic calmodulin promotes 432 self-incompatibility pollen tube growth by enhancing calcium influx and reactive 433 oxygen species concentration in _Pyrus_ _pyrifolia_ . Plant Cell Reports, 2014, 33: 434 255-263. 435 Li XHS, Xu YC, Wei ZY, Kuang JY, She MZ, Wang YJ, Jin QJ. 436 NnSnRK1-NnATG1-mediated autophagic cell death governs flower bud 437 abortion in shaded lotus, The Plant Journal, 2024, 117(4): 979-998. 438 Liu C, Shen L, Xiao Y, Vyshedsky D, Peng C, et al. Pollen PCP-B peptides unlock a 439 stigma peptide-receptor kinase gating mechanism for pollination. Science, 2021, 440 372: 171-175 441 Luo H, Chen SM, Wan HJ, Chen FD, Gu CS, Liu ZL. Candidate reference genes for 442 gene expression studies in water lily. Analytical Biochemistry, 2010, 404(1): 443 100-102. 444 Kaur G, and Pati PK. In silico insights on diverse interacting partners and 445 phosphorylation sites of respiratory burst oxidase homolog (Rbohs) gene 446 families from Arabidopsis and rice. BMC Plant Biology, 2018, 18(1): 161. 447 Margalha L, Valerio C, Baena-González E. Plant SnRK1 kinases: structure, regulation, 448 and function. AMP-activated protein kinase, 2016: 403-438. 449 Martínez-Barajas E, Coello P. How do SnRK1 protein kinases truly work?. Plant 450 Science, 2020, 291: 110330. 451 Ofer G,Yuval P,Rita MK, Eyal M, Haim DR, Ilan S. Only minimal regions of tomato 452 yellow leaf curl virus (TYLCV) are required for replication, expression and 453 movement. Archives of virology, 2014, 159(9): 2263-2274. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 454 Potocký M, Pejchar P, Gutkowska M, et al. NADPH Oxidase Activity in Pollen Tubes 455 Is Affected by Calcium Ions, Signaling Phospholipids and Rac/Rop GTPases. 456 Journal of Plant Physiology, 2012, 169, 16: 1654-1663. 457 Qi YJ, Gao ZH, Ma N, Lu LQ, Ke FJ, Zhang SL, Xu YL. Specific functions of single 458 pistil S-RNases in S-gene homozygous Pyrus germplasm. BMC Plant Biology, 459 2023, 23: 578. 460 Sun CQ, Ma ZH, Sun GS, Dai ZL, Teng NJ, Pan YP. Cellular mechanism of 461 reproductive barrier in some crosses of water lily ( _Nymphaea_ spp.) cultivars. 462 2015, HortScience, 50(1): 30-35. 463 Sun CQ, Ma ZH, Zhang ZC, Sun GS, Dai ZL. Factors influencing cross barriers in 464 interspecific hybridizations of water lily. Journal of the American Society for 465 Horticultural Science, 2018, 143, 130-135. 466 Sun CQ, Cao J, Wang JH, Zhou P, Xu YC, Chen FD. Pectin methylesterase regulates 467 pollen germination on stigma after pollination in water lily. Scientia 468 Horticulturae, 2023, 320. 469 Sun CQ, Chen FD, Teng NJ, Xu YC, Dai ZL. Comparative analysis of the complete 470 chloroplast genome of seven _Nymphaea_ species. Aquatic Botany, 2021, 170. 471 Sun CQ, Chen FD, Teng NJ, Yao YM, Shan X, Dai ZL. Transcriptomic and 472 proteomic analysis reveals mechanisms of low pollen-pistil compatibility during 473 water lily cross. BMC Plant Biology, 2019, 19: 542. 474 Wang JH, CaoJ, Yang ZJ, Jin QJ, Wang YJ, Su Q, Wang HY, Sun CQ, Xu YC. 475 NcFER1 and NcRbohC1 Regulate Pollen Germination on Waterlily Stigma After 476 Pollination. Physiologia Plantarum, 2025, 177: e70318. 477 Wilkins KA, Bancroft J, Bosch M, Ings J, Smirnoff N, Franklin-Tong VE. Reactive 478 Oxygen Species and Nitric Oxide Mediate Actin Reorganization and 479 Programmed Cell Death in the Self-Incompatibility Response of Papaver. Plant 480 Physiology, 2011, 156: 404-416. 481 Yu HX, Xiao AF, Dong R, Fan YQ, Zhang XP, Liu C, Wang C, Zhu H, Duanmu DQ, 482 Cao YR, Zhang ZM. Suppression of innate immunity mediated by the 483 CDPK-Rboh complex is required for rhizobial colonization in Medicago 484 truncatula nodules, New Phytologist, 2018, 220(2): 425-434. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 485 Yuval P, Rita MK, Fuad A, Edna T, Henryk C, Ilan S. A universal 486 expression/silencing vector in plants. Plant physiology, 2007, 145(4): 1251-63. 487 Zhang L, Huang J, Su S, Wei X, Yang L, Zhao H, Yu J, Wang J, Hui J, Hao S, Zhang 488 X, Cheung A Y, Duan Q. FERONIA receptor kinase-regulated reactive oxygen 489 species mediate self-incompatibility in _Brassica_ rapa. Current Biology, 2021, 490 14(31) 3004-3016.e4. 491 Zheng XL, Yang HF, Zou JP, Jin WD, Qi ZY, Yang P, Yu JQ, Zhou J. 492 SnRK1α1-mediated RBOH1 phosphorylation regulates reactive oxygen species 493 to enhance tolerance to low nitrogen in tomato. The Plant Cell, 2025, 37: 494 koae321. 495 Zhou P, Li J, Jiang HY, Yang ZJ, Sun CQ, Wang HY, Su Q, Jin QJ, Wang YJ, Xu 496 YC. Hormone and transcriptomic analysis revealed that ABA and BR are key 497 factors in the formation of inter-subgeneric hybridization barrier in water lily. 498 Physiol Plantarum, 2024, 176: e14177. 499 Zhou P, Li JW, Jiang HY, Yang ZJ, Sun CQ, Wang HY, Su Q, Jin QJ, Wang YJ, Xu 500 YC. NpCIPK6-NpSnRK1 module facilitates intersubgeneric hybridization 501 barriers in water lily（ _Nymphaea_ ）by reducing abscisic acid content. Horticulture 502 Research, 2025, 12: uhae289. 503 504 # 505 Figure legends 506 Fig.1 Pollen germination, ROS content and the relative gene expression level of the stigma after 507 cross-pollination and self-pollination of waterlilies. 508 Note: Sp: self-pollination, Cp: cross-pollination, Mock: control. 509 510 511 Fig.2 The effects of different active substances on pollen germination and the ROS content in the 512 stigma 513 Note: DMTU: N, N’-Dimethylthiourea; X+XO: Xanthine /Xanthine Oxidase; 514 DPI:Diphenyleneiodonium chloride 515 516 517 Fig.3 Subcellular localization of NcRbohC1 and NcSnRK1 518 Note: The subcellular localization of NcRbohC1 used cell membrane marker and NcSnRK1 used 519 cell nucleus marker [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.28.762668; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.28.762668) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 520 521 Fig.4 The gene expression level of _NcRbohC1_ and _NcSnRK1_ after different treatments 522 523 524 Fig.5 The effects of different treatments on pollen germination and the ROS content in the stigma 525 526 527 Fig.6 Yeast two-hybrid, pull-down and luciferase complementation assay 528 A: BD- _NcRbohC1_ -N yeast self-activation and toxicity detection; B: One-to-one interaction 529 verification; C: pull-down assay; D: Luciferase complementation assay. 530 Note: Positive is the positive control; Negative is the negative control 531 532 533 Fig.7 The effects of different treatments on pollen germination and the ROS content in the stigma 534 535 536 Fig.8 The relative expression level of gene NcRbohC1 and gene NcSnRK1 with different 537 treatments 538 Note: PIR-S:over-expression NcSnRK1;PIR-R:over-expression NcRbohC1;IR-S:interfering 539 NcSnRK1；IR-R:interfering NcRbohC1；IR-R+IR-S:interfering NcRbohC1+interfering NcSnRK1； 540 PIR-R+PIR-S：over-expression NcRbohC1+over-expression NcSnRK1 541 542 543 Fig.S1 The gene cloning of _NcRbohC1, NcRbohC1_ -N and _NcSnRK1_ 544 545 Fig.S2 The gene expression level of _NcRbohC1_ after transient transfection 546 547 Fig.S3 Yeast two-hybrid screening followed by gel electrophoresis detection 548 549 Fig.S4 The gene expression level of _NcSnRK1_ after transient transfection 550 551 Table S1 The primers used in the experiment 552 553 Fig.1 Pollen germination, ROS content and the relative gene expression level of the stigma after cross-pollination and self-pollination of waterlilies. Note: Sp: self-pollination, Cp: cross-pollination, Mock: control. Fig.2 The effects of different active substances on pollen germination and the ROS content in the stigma Note: DMTU: N, N’-Dimethylthiourea; X+XO: Xanthine /Xanthine Oxidase; DPI:Diphenyleneiodonium chloride Fig.3 Subcellular localization of NcRbohC1 and NcSnRK1 Note: The subcellular localization of NcRbohC1 used cell membrane marker and NcSnRK1 used cell nucleus marker Fig.4 The gene expression level of _NcRbohC1_ and _NcSnRK1_ after different treatments Fig.5 The effects of different treatments on pollen germination and the ROS content in the stigma Fig.6 Yeast two-hybrid, pull-down and luciferase complementation assay A: BD- _NcRbohC1_ -N yeast self-activation and toxicity detection; B: One-to-one interaction verification; C: pull-down assay; D: Luciferase complementation assay. Note: Positive is the positive control; Negative is the negative control Fig.7 The effects of different treatments on pollen germination and the ROS content in the stigma Fig.8 The relative expression level of gene _NcRbohC1_ and gene _NcSnRK1_ with different treatments Note: PIR-S:over-expression _NcSnRK1_ ;PIR-R:over-expression _NcRbohC1_ ;IR-S:interfering _NcSnRK1_ ； IR-R:interfering _NcRbohC1_ ； IR-R+IR-S:interfering _NcRbohC1_ +interfering _NcSnRK1_ ； PIR-R+PIR-S ： over-expression _NcRbohC1_ +over-expression _NcSnRK1_