The AP2/ERF transcription factorHcERF5confers drought tolerance via ABA-mediated signaling in kenaf

31 The APETALA2/ethylene response factor (AP2/ERFs) are pivotal in regulating 32 abiotic stress responses in plants. However, the specific role of ERFs in kenaf’s 33 response to drought stress remains unclear. In this study, a transcription factors 34 HcERF5 was isolated from kenaf and its role in drought stress tolerance was analyzed. 35 HcERF5 was found to localize in both nucleus and cytoplasm and could be 36 significantly induced by polyethylene glycol -6000 (PEG -6000) and abscisic acid 37 (ABA) in kenaf seedlings. In transgenic Arabidopsis expressing the HcERF5 38 promoter-driven β-glucuronidase (GUS), strong GUS activity was observed in roots, 39 stems, and leaves. Overexpression of HcERF5 in Arabidopsis enhanced seed 40 germination rates under drought or ABA stress and improved drought tolerance in 41 seedlings by increasing antioxidant enzyme activities , whereas aterf5 knockout lines 42 exhibited the opposite trend. Additionally, HcERF5 overexpressing Arabidopsis 43 showed significantly increased drought tolerance and reduced sensitivity to ABA. 44 Furthermore, virus -induced gene silencing (VIGS) of HcERF5 in kenaf reduced 45 drought tolerance, as evidenced by decreased antioxidant enzyme activity, increased 46 stomatal apert ure, and elevated levels of malondialdehyde (MDA), reactive oxygen 47 species (ROS), and proline under drought stress. RNA-seq analysis further revealed 48 that HcERF5 directly regulated ABA signaling pathway. Yeast-two-hybrid (Y2H) 49 assays revealed 29 proteins t hat interact with HcERF5. Among them, the expression 50 of downstream drought stress -related genes HcPRK, HcRD22, HcMAP2, HcCAB, 51 HcCS, and HcCCoAOMT3 were significantly reduced in HcERF5-silenced plants. 52 Overall, this study highlights the significant potential of HcERF5 in enhancing 53 drought tolerance in kenaf. 54 55 56

Kenaf; HcERF5; Drought stress; ABA sensitivity; ROS scavenging 57 system 58 59 60 61 Abbreviations: GUS, β -glucuronidase; ABA, abscisic acid; PEG, polyethylene 62 glycol; MS, Murashige and Skoog ; OE, overexpressing ; GFP, green fluorescent 63 protein; ROS, reactive oxygen species; VIGS, Virus‑induced gene silencing; WT, wild 64 type; CAT, Catalase; MDA, malondia ldehyde; POD, peroxidase; SOD, s uperoxide 65 dismutase; qRT-PCR, quantitative real -time PCR; DAB, 3, 3′ -diaminobenzidine; 66 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 3 NBT, nitroblue tetrazolium; H2O2, hydrogen peroxide; O2 -, superoxide anion radical. 67 68 69 Highlights： 70 1. HcERF5 overexpression enhanced drought tolerance in kenaf, however silencing 71 increased drought sensitivity. 72 2. HcERF5 regulates ABA synthesis and increases kenaf stomatal conductance and 73 density under drought stress. 74 3. HcERF5 regulates plant hormone signal transduction, MAPK signalling, and 75 phenylpropanoid biosynthesis in kenaf under drought stress. 76 4. Protein interaction revealed HcERF5 interacts with six stress-response genes. 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 4 106 107 108 109

110 Drought stress is one of the significant abiotic factor s that negatively impacts 111 plant growth and productivity, posing a threat to sustainable crop production globally 112 (Farooq et al., 2017; Wang et al., 2023) . Its detrimental effects on crop yield and 113 development are exacerbated by factors such as population increase, global water 114 scarcity, and climate change. Understanding the mechanisms behind drought 115 resistance in crops is crucial for addressing and mitigating the challenges posed by 116 drought stress (Yang et al., 2017) . Plants have developed intricate and sophisticated 117 signalling networks to withstand prolonged drought stress (Meena et al., 2017; Lu et 118 al., 2023) . These networks encompass a variety of biological processes, including 119 physiological, biochemi cal, and molecular adaptations. Key responses include 120 enhanced antioxidant production, stomatal closure, osmotic adjustment, as well as 121 hormonal and transcriptional regulation. These responses collectively involve the 122 alteration of thousands of gene expres sion patterns to protect the normal cellular 123 functions (Udawat et al., 2016; Haider et al., 2017; Udawat et al., 2017; Gong et al., 124 2020; Challabathula et al., 2022). 125 Transcription factors families are crucial for signal transduction and the 126 regulation of gene expression, with the ethylene response factor (ERF) genes being a 127 notable subfamily. The ERF family has a unique AP2 domain, making AP2/ERF an 128 important class of transcription factors found in all plants. Based on their structural 129 characteristics, AP2/ERF is categorized into four subgroups: ERF, DREB, AP2, and 130 RA V . These subgroups play vital roles in plant growth and development, 131 morphogenesis, responses to external damage, pathogen resistance, stress responses, 132 metabolic biosynthesis regulation, and plant hormone-mediated regulation (Tang et al., 133 2005; Wu et al., 2007; Xu et al., 2007; Zhang et al., 2008; Liu et al., 2014; Do et al., 134 2020; Feng et al., 2020). Plant hormones are important for plant adaptation to adverse 135 biotic and abiotic stress conditions (Khan et al., 2023) . Among different plant 136 hormones, abscisic acid (ABA) is a key phytohormone produced in response to 137 osmotic stress, regulates plant growth and development, and is vital for adaptation to 138 various environmental stresses (Raghavendra et al., 2010; Vishwakarma et al., 2017; 139 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 5 Yoshida et al., 2019). The regulation of ABA signal ing expression can improve plant 140 tolerance (Lim et al., 2015) and ABA-mediated stomatal movement is essential for 141 transpiration regulation under water stress (Kollist et al., 2014; Qiu et al., 2020) . 142 Numerous studies have explored the regulation of ABA content by ERF transcription 143 factors during drought stress. For instance, AtERF4 as a negative regulator of ethylene 144 and ABA signaling pathways, while AtERF1 enhances drought stress tolerance by 145 regulating stress -specific gene s and integrating ethylene, jasmonic acid, and ABA 146 signalling in Arabidopsis (Yang et al., 2011; Cheng et al., 2013) . In another study, it 147 was found that guard cells overexpressing AtERF7 exhibited reduced sensitivity to 148 ABA and increased water loss, whereas AtERF7 RNA interference lines showed high 149 sensitivity to ABA (Song et al., 2005) . AhDREB1, a member of the ERF5 family, 150 enhances drought tolerance in Arabidopsis through the ABA -dependent signalling 151 pathway. Overexpression of AhDREB1 in Arabidopsis increases ABA sensitivity, 152 modifies ABA signaling pathways, and elevates the expression of downstream 153 drought stress -related genes such as RD29A, P5CS1, P5CS2, and NCED1. The 154 transcript levels of ABA signaling pathway -associated genes AtPYL2, AtPP2C5, 155 AtSnRK2.2, AtSnRK2.4, AtAREB3, and AtABF4 significantly increased under normal 156 growth conditions and after exogenous ABA application (Zhang et al., 2018) . 157 Moreover, OsERF71 regulates genes associated with ABA response and proline 158 biosynthesis in rice. This regulation results in increased sensitivity to exogenous ABA 159 treatment and proline accumulation, thereby improving drought tolerance of plants (Li 160 et al., 2018) . In soybea n plants, the expression of GmERF4 is upregulated by cold, 161 salt, and drought but inhibited by ABA (Zhang et al., 2010) . In cotton, GhERF2, 162 GhERF3, and GhERF6 respond to ABA stress in upland cotton (Jin et al., 2010) . 163 Reactive oxygen species (ROS) are highly reactive molecules that function as 164 signalling molecules and induce various stress responses, such as stomatal closure 165 (Medeiros et al., 2020) . ROS generated under drought stress interact with ABA to 166 regulate the plant response and enhance its drought tolerance (Geng et al., 2023). 167 Kenaf ( Hibiscus cannabinus L.), a member of the Malvaceae family, is a n 168 important bast fiber crop globally. It is mainly cultivated in temperate, subtropical and 169 tropical areas of Asia and Africa (Dubois et al., 2013) . Kenaf is currently used 170 commercially in over 20 countries, especially in China, Bangladesh, India, and 171 Thailand (FAO, 2021) accounting for 90% of the global kenaf cultivation area and 172 over 95% of global kenaf production (Ding et al., 2016). It is characterized by drought 173 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 6 tolerance, salt tolerance, ease of cultivation, marginal land use, high fiber yields, and a 174 variety of industrial applications (Chen et al., 2018) . However, the regulatory 175 mechanisms controlling the drought resistance of kenaf are not yet clear and the role 176 of kenaf ERF genes is also poorly understood. 177 Our earlier transcriptome and qRT -PCR analysis revealed that HcERF5 was 178 significantly up -regulated in leaves of kenaf seedling s after PEG-induced drought 179 stress, indicating a possible regulatory role of HcERF5 in drought stress of kenaf (Luo 180 et al., 2023) . In this study, to elucidate the role of HcERF5 in ABA signaling and 181 drought resistance, HcERF5 was identified and systematically investigated its role in 182 kenaf drought tolerance, via subcellular localization, protein interactions, 183 heterologous overexpression and VIGS analysis. Our evidence suggests that 184 overexpression of HcERF5 confers ABA insensitivity and enhances drought 185 resistance in plants by enhancing the antioxidant enzyme system. In addition, 186 HcERF5 affects ABA synthesis and stomatal conductance in HcERF5 silenced plants 187 under drought stress. The present study provides important and new insights into the 188 drought resistance mechanism of HcERF5 in kenaf. 189

190 Identification and characterization of HcERF5 in kenaf 191 The coding sequence (CDS) of HcERF5 is 981 bp in length, encoding a protein of 192 327 amino acids with an isoelectric point of 5.08 and a molecular weight of 80.8 kDa. 193 Multiple sequence alignment indicated that HcERF5 shares high similarity with 194 HsERF5, GhERF5, and GrERF5, and notably, it has a conserved AP2/ERF domain 195 composed of 58 amino acids (Fig. 1A) . Phyre2 predictions suggest ed that HcERF5 196 has a single transmembrane α-helix (Fig. 1B) . Phylogenetic analysis revealed that 197 HcERF5 is most closely related to Hibiscus syriacus based on amino acid sequences 198 (Fig. 1C) . Subcellular localization experiments dem onstrated that HcERF5 -GFP 199 fluorescence was detected in both the cytoplasm and nucleus, while GFP alone or the 200 nuclear localization marker control showed fluorescence throughout the cell and 201 nucleus (Fig. 1D) . These findings indicate that HcERF5 is localize d in both the 202 cytoplasm and nucleus. 203 HcERF5 can be induced by osmotic stress and ABA treatment 204 To investigate the role of HcERF5 in drought and ABA phytohormone signaling, 205 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 7 the expression levels of HcERF5 in kenaf leaves treated with 20% PEG 6000 or 100 206 μM ABA were evaluated using qRT -PCR. During drought conditions, HcERF5 207 expression increased from 2 hours and peaked at 12 hours, reaching 3.1 times the 208 level observed at 0 hours. Although the expression declined after 12 hours, it 209 remained significantly highe r than the baseline level before treatment (Fig. 2A). The 210 expression level of HcERF5 did not show a significant change after 2 hours in ABA 211 treated plants compared to the control group. However, after 4 hours, the expression 212 levels increased significantly, peaking at 12 hours with a 23.2 -fold increase from the 213 initial level. With ABA treatment, the expression level of HcERF5 did not show a 214 significant change after 2 hours compared to the control group. However, after 4 215 hours, the expression started to incre ase significantly, peaking at 12 hours with a 216 23.2-fold increase from the initial level. Following this increase, the expression levels 217 began to decline, but remained 18.2 and 13.4 times higher at 24 and 48 hours 218 respectively, compared to the levels at 0 hours (Fig. 2B). These findings suggest that 219 HcERF5 is induced and expressed under both drought stress and ABA treatment, 220 indicating its important role in the stress response. 221 The expression patterns of HcERF5 genes were investigated in the different organ 222 tissues of kenaf. HcERF5 is expressed in leaves, petioles, stems, and roots, with the 223 highest expression in kenaf leaves, followed by petioles and roots, and the lowest 224 expression in stems (Fig. S2), suggesting that the HcERF5 have distinct and typical 225 tissue-specific expression patterns. To further investigate the spatial expression of 226 HcERF5, the positively transformed Pro HcERF5:GUS Arabidopsis plants were 227 identified (Fig. S3), and then the GUS activity of 25 -day-old plants e xposed to 400 228 mM mannitol or 100 μM ABA for 0, 6, and 12 h was determined . Histochemical 229 staining showed that there is no GUS activity in WT, but in Pro HcERF5:GUS 230 Arabidopsis plants, GUS activity increases with increasing mannitol treatment time 231 and spreads throughout the plant (Fig. 2C). Besides, GUS activity showed the same 232 change trend under ABA treatment (Fig. 2C). These results indicate that the HcERF5 233 gene promoter is highly induced by ABA and mannitol treatments. Collectively, this 234 strongly suggests that HcERF5 plays a role in ABA signalling and drought stress 235 responses. 236 Overexpression of HcERF5 increased Arabidopsis seed germination performance 237 to drought and ABA stress 238 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 8 To elucidate the function of AtERF5 in Arabidopsis, a mutant line named 239 SALK_208574 (referred to as aterf5) was identified. In this mutant line, T-DNA was 240 inserted into the fourth exon (Fig. S4A) and genomic DNA PCR was performed to 241 confirm the homozygous lines (Fig. S4B). 242 HcERF5 was constructed using the expression vector PBI121 (Fig. S5A) and 243 introduced into wild-type Arabidopsis (WT) as background material. The positive 244 seedlings of the T1 generation were screened on the Kan resistance plates (Fig. S5B), 245 and the homozygous T3 generation of the transgenic materia l was obtained for 246 phenotype analysis. The expression levels of HcERF5 in the transgenic Arabidopsis 247 of the T3 generation and in the aterf5 mutant were analyzed by semi quantitative PCR. 248 The results showed that HcERF5 was not expressed in WT and in the aterf5 mutant of 249 Arabidopsis, but only in the overexpressed HcERF5 lines OE1 and OE2; in contrast, 250 ATERF5 gene was expressed only in WT (Fig. S5C). Therefore, HcERF5 was 251 successfully expressed heterologous in Arabidopsis and AtERF5 was silenced in 252 aterf5 lines. 253 To investigate the role of HcERF5 in seed germination under drought and ABA 254 stress, the germination of WT, aterf5 mutants, and HcERF5-OE lines was evaluated 255 over a 7 d ays period under these stresses (Fig. 3A) . The results showed that under 256 normal growth conditions, the germination rate of WT, aterf5 mutant and 257 HcERF5-OE seeds on 1/2 MS media had no obvious differences (Fig. 3B). However, 258 under drought stress, the germination rate of both WT and aterf5 mutant seeds on 1/2 259 MS medium supplemented with 200 mM mannitol was slightly delayed (Fig. 3C) . 260 When the concentration of mannitol was increased to 400 mM, the germination rate of 261 WT was higher than that of the aterf5 mutants, but lower than that of the two 262 overexpressing strains, and the germination rate of the aterf5 mutants remained low 263 even seven day after germination (Fig. 3D). Seed germination of the aterf5 mutants 264 was significantly delayed on 1/2MS medium supplemented with 2 μM ABA, and only 265 38.3% and 70.3% of the seeds of aterf5 mutants germinated on day 3 and 7 266 respectively, while about 50.1% and 88.6% o f the seeds of HcERF5-OE germinated 267 on day 3 and 7 and approximatel y 40.5% and 78.4 % o f the seeds of WT germinated 268 on day 3 and 7 respectively (Fig. 3E). Their germination rates showed a similar trend 269 at concentrations of 4 μM ABA (Fig. 3F) . It can be concluded that aterf5 in 270 Arabidopsis can increase the sensitivity of Arabidopsis seeds to drought and ABA 271 stress compared with WT, while overexpression of HcERF5 can decrease the 272 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 9 sensitivity of Arabidopsis seeds to drought and ABA stress. These results indicate that 273 HcERF5 has a positive effect on seed germination under drought and ABA stress. 274 HcERF5 enhances drought tolerance in Arabidopsis 275 To investigate the role of HcERF5 in root growth under drought stress and ABA 276 treatment, one-week-old seedlings of WT, aterf5 mutants, and HcERF5-OE 277 Arabidopsis seedlings were subjected to normal growth conditions , drought stress 278 (200 or 400 mM mannitol in the 1/2 MS medium) and ABA treatment (2 or 4 μM 279 ABA in the 1/2 MS medium) . After one week of growth, no significant differences 280 were observed between WT, aterf5 mutants, and HcERF5-OE under normal 281 conditions (Fig. 4A) . Under drought conditions , the primary root length of aterf5 282 mutants was significantly reduced compared to WT, whereas the HcERF5-OE lines 283 exhibited significantly longer root lengths than WT (Fig. 4B). Similarly, the primary 284 root length of the aterf5 mutants was significantly shorter than WT, while 285 HcERF5-OE lines displayed longer roots compared to both WT and aterf5 mutants 286 (Fig. 4C) . These findings suggest that drought stress and ABA treatment markedly 287 influence root length, and that HcERF5 is a key regulator in the response to these 288 stresses. Additionally, three-week-old Arabidopsis seedlings were subjected to natural 289 drought stress in soil for one week. There was no notable difference between WT and 290 HcERF5-OE plants under normal conditions. However, WT and aterf5 mutant leaves 291 turned yellow and showed severe wilting symptoms under drought stress. Most leaves 292 of HcERF5-OE plants remained green and had a higher survival rate after rewatering 293 (Fig. 4D). 294 The chlorophyll content in HcERF5 -OE under drought conditions was 295 approximately 0.72 mg/g, which was significantly higher than in WT (0.52 mg/g) and 296 aterf5 mutant (0.36 mg/g) (Fig. 4E). The mean fresh weight of HcERF5-OE lines was 297 17.2 mg, which was significantly greater than that of the WT (14.3 mg) and aterf5 298 (10.4 mg) (Fig. 4F). Similarly, HcERF5-OE had a considerably higher RWC (51.36%) 299 than the WT (40.4%) or the aterf5 mutant (30.3%) (Fig. 4G) . The survival rate of 300 HcERF5-OE lines was 83.7%, surpassing that of WT (63.2%) and aterf5 mutants 301 (43.5%) (Fig. 4H). The results suggests that HcERF5-OE Arabidopsis plants displays 302 significantly enhanced drought resistance compared to WT and aterf5 mutants. 303 To investigate whether the drought resistance phenotype of the transgenic 304 Arabidopsis lines is caused by changes in ROS homeostasis, antioxidant enzyme 305 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 10 activity and ROS a ccumulation were compared in WT, aterf5 mutant, and 306 HcERF5-OE lines under normal and drought stress conditions. The activity of SOD 307 was significantly increased by 96.5%, 67.2%, 124.3%, and 119.1% in WT, aterf5 308 mutant, OE1, and OE2, respectively compared to the control (Fig. 5A). Similarly, the 309 POD activity was significantly increased by 559.1%, 454.0%, 637.1%, and 608.6% in 310 WT, aterf5 mutant, OE1, and OE2, respectively compared to the control (Fig. 5 B). 311 Furthermore, significant increase in CAT activity was 82.8%, 32.2%, 104.5%, and 312 138.3% in WT, aterf5 mutant, OE1, and OE2, respectively compared to the control 313 (Fig. 5C) . Moreover, MDA contents were significantly increased by 70.9%, 95.2%, 314 26.9%, and 26.3% in WT, aterf5 mutant, OE1, and OE2, respectively compared to the 315 control (Fig. 5D). Quantitative measurements of H 2O2 and O2 - showed no significant 316 differences in the accumulation of H 2O2 and O 2 - content in leaf tissues of WT and 317 aterf5 mutants, and HcERF5-OE Arabidopsis under normal growth conditions. 318 However, after drought treatment, the content of H 2O2 and O2 - increased significantly 319 in WT and aterf5 mutants, and HcERF5-OE Arabidopsis. However, the extent of 320 accumulation in WT and aterf5 mutants was significantly higher than in HCERF5-OE 321 (Fig. 5E and F) . Therefore, HcERF5-OE Arabidopsis plants exhibit lower H 2O2 and 322 O2 - accumulation than WT and aterf5 mutant Arabidopsis plants. 323 Virus‑induced gene silencing of HcERF5 decreased kenaf drought stress capacity 324 To further confirm the involvement of HcERF5 in response to drought stress, 325 HcERF5 was knock ed-down by the VIGS technology. After the successful 326 construction of the recombinant vector pRTV2-HcERF5 (Fig. S7), the kenaf chloroplast 327 thioredoxin (HcTrx) served as positive control, which was used as a reporter gene to 328 detect the status of gene silencing. After 10 days of growth, the HcTrx silenced kenaf 329 plants,exhibited a variegated leaf phenotype from the second or th ird true leaf (Fig. 330 6A), indicating the reliability of VIGS technology and the down-regulation of 331 HcERF5 was verified by qRT -PCR in HcERF5 silenced leaves (Fig. S8 ). After 332 drought stress, the pTRV2 -HcERF5 silenced seedlings showed severe wilting effect 333 compared with the pTRV2 seedlings (Fig. 6B). 334 DAB and NBT staining revealed that ROS accumulation in pRTV2 -HcERF5 335 plants was greater than in pRTV2 plants, indicating increased ROS production under 336 drought stress in pRTV2-HcERF5 plants (Fig. 6C). Furthermore, the rate of water loss 337 in detached leaves and the relative water content of kenaf seedling leaves were 338 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 11 examined to determine whether drought -sensitive phenotypes are linked to reduced 339 water retention. The results showed that leaves from pRTV2-HcERF5 plants exhibited 340 a significantly higher water loss rate compared to control seedlings (Fig. 6F). Since 341 there is a direct correlation between stomatal regulation and leaf water loss , the size 342 and density of stomata on the abaxial epidermis of leaves from both pRTV2 and 343 pRTV2-HcERF5 plants were analyzed using microscopy under drought conditions 344 (Fig. 6D and E). Measurement of stomatal aperture showed that there was no 345 remarkable difference between pRTV2 and pRTV2 -HcERF5 plants in terms of 346 average stomatal size and stomatal density. However, both the average stomatal size 347 and stomatal density were significantly lower in pRTV2 plants compared with 348 pRTV2-HcERF5 plants (Fig. 6G and H), indicating that stomatal size and stoma tal 349 density are linked to HcERF5-mediated leaf water loss. These findings suggested that 350 inhibition of HcERF5 expression could increase stomatal density and prevent stomatal 351 closure, which promote water loss and reduce tolerance to drought stress. 352 Silencing of HcERF5 increased the accumulation of ROS under drought stress 353 The plant height and fresh weight of pRTV2 -HcERF5 plants were significantly 354 reduced by 17.2% and 17.9%, respectively, compared to pTRV2 plants after 7 days of 355 drought treatment, although the stem diameter remained unchanged (Fig. 7A and B) . 356 The results showed that the activities of SOD, POD, CAT, and GR significantly 357 decreased by 28.8%, 13.1%, 25.7%, and 32.9% respectively, in pRTV2 -HcERF5 358 plants, compared to pTRV2 control (Fig. 7E-H). However, further quantitative results 359 showed that there was a significant increase of 29.3%, 346.1%, 185.8%, and 47.2% in 360 the content of MDA, H 2O2, O2 -, and proline respectively, in pRTV2 -HcERF5 plants, 361 compared to pTRV2 control (Fig. 7I-L). Overall, silencing of HcERF5 weakened the 362 antioxidant capacity, and led to excessive ROS accumulation in kenaf, which 363 decreased its tolerance to drought stress, suggesting that HcERF5 plays a necessary 364 role in kenaf drought-tolerance. 365 ABA content and sig nal transduction are crucial factors in regulating stomatal 366 opening (Jurca et al., 2022) . ABA-dependent signal transduction pathways are 367 activated by drought stress (Medeiros et al., 2020) . Analysis of ABA content in the 368 leaves revealed a significant decrease in ABA synthesis in pRTV2-HcERF5 plants 369 under drought stress (Fig. 7D), suggesting that this reduction in ABA content might 370 contribute to the reduced stress resistance observed in HcERF5-silenced plants. 371 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 12 Transcriptome profiling reveals differentially expressed genes regulated by 372 HcERF5 373 To explore the regulatory network of HcERF5 in response to drought stress, a 374 comparative transcriptome analysis of HcERF5-silenced and pTRV2 control plants 375 was performed by RNA sequencing (RNA-seq). The clean reads were submitted with 376 accession number PRJNA1061751 to the Sequence Read Archive (SRA) database. 377 There were 2,489 differentially expressed genes (DEGs) between HcERF5-silenced 378 and pTRV2 control plants (|log2FC| ≥ 1 and p < 0.05). In the HcERF5-silenced line, 379 722 genes were upregulated and 1767 genes were downregulated in comparison to 380 pTRV2 (Fig. 8A, Table S3) . GO enrichment analysis with a p-value < 0.05 was 381 conducted on these DEGs to investigate their putative functions . As a result, these 382 DEGs were classified into 45 functional groups, including ‘biological process’ (BP, 19 383 subcategories), ‘cellular component’ (CC, 12 subcategories) and ‘molecular function’ 384 (MF, 13 subcategories) (Fig. 8B, Table S4). Specifically, 188, 44, 6 and 3 DEGs were 385 annotated with the terms ‘response to stimulus’ (GO: 0050896), ‘antioxidant activity’ 386 (GO: 0016209), ‘detoxification’ (GO: 0098754) and ‘signaling’ (GO: 0023052), 387 respectively. All of which are known to play crucial roles in plant stress tolerance. To 388 gain insight into the biological functions of DEGs in drought tolerance, a KEGG 389 pathway analysis was conducted. This analysis identified 233 DEGs enriched in 14 390 significant KEGG pathways ( p value < 0.05 ) (Fig. 8C, Table S5) . These findings 391 suggest that these pathways are crucial for the molecular mechanisms by which 392 HcERF5 regulates other drought defens e genes in kenaf. Notably, 53 genes were 393 enriched in plant hormone signal transduction, 38 genes in the MAPK signaling 394 pathway, and 28 genes in phenylpropanoid biosynthesis. The expression heatmaps of 395 DEGs participating in these three metabolic pathways revealed that these genes were 396 highly differentially expressed (Fig. S9). In addition, the expression levels of genes 397 related to ABA signalling were analysed in HcERF5-silenced and pTRV2 plants . 398 Compared to pTRV2, silencing of HcERF5 altered the expression of several ABA 399 signalling pathway genes, including PYR/PYLs, PP2Cs and SnRK2s (Fig. 8D) . In 400 addition, silencing of HcERF5 also decreased the expression of some genes related to 401 antioxidant enzyme activity, such as POD (Hc.06G002730, Hc.06G003530, 402 Hc.07G015280 and Hc.10G023380), GR (Hc.18G003420), and increased ROS 403 expression (Hc.02G012210 and Hc.03G037930) (Fig. S10). 404 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 13 To investigate the different gene expression profiles are regulated by HcERF5 405 under drought stress , K-means clustering analysis was performed . The result showed 406 that 2,451 DEGs were distributed among the top 3 K subclusters, accounting for 98.5% 407 of the total DEGs (Fig. S11). 408 Screening of HcERF5 interaction proteins 409 To further investigate the function of the HcERF5 gene, screening of the HcERF5 410 gene from a kenaf yeast library was performed. Dot plate analysis revealed that yeast 411 Y2HGold strain containing recombinant plasmids pGBKT7 -HcERF5, empty 412 pGBKT7 plasmid, and pGBKT7 -53+pGADT7-T was grown normally on SD/ -Trp 413 medium, indicating that the expression of recombinant plasmids pGBKT7-HcERF5 in 414 yeast cells is no n toxic. At the same time, only the positive control 415 PGBKT7-53+PGADT7-T was detected on SD/-TDO+X-α-gal. The growth on the gal 416 plate and the blue colony indicates that the recombinant plasmid pGBKT7-HcERF5 417 has no transcriptional activation activity (Fig. 7A). These results indicate that yeast 418 hybridization technology can be used to screen proteins that interact with the HcERF5 419 protein. 420 After the mixture of the bait vector pGBKT7 -HcERF5 and the library was first 421 screened on SD/-TDO medium, the colonies were further identified on SD/ -DDO and 422 SD/-QDO+X-α-Gal. The results showed that the positive control 423 pGBKT7-53+pGADT7-T grow n normally on SD/ -DDO and SD/ -QDO+X-α-Gal 424 medium and turned blue on SD/ -QDO+X-α-Gal medium . The negative control 425 pGADT7+pGBKT7-HcERF5 grew normally on SD/ -DDO medium, but failed to 426 grow normally on SD/-QDO+X-α-Gal medium without showing blue spots (Fig. 7B). 427 PCR was used to screen the positive strains, and the amplification results showed that 428 most of the bands were about 1000 bp in length (Fig. S12 ), indicating the 429 effectiveness of screening with kenaf library. These positive clones were sequenced 430 and compared, and unknown proteins were removed. In addition to duplicate clones, 431 29 proteins that significantly interacted with HcERF5 and had functional annotations 432 were examined (Table S2). According to these annotations, HcERF5 interacted with a 433 number of protein s involved in growth metabolism, stress tolerance, and 434 photosynthesis. These putative proteins have roles in signal transduction or immune 435 processes, indicating that HcERF5 plays an important role in plant stress signal 436 transduction, downstream gene transcriptional regulation, and translation. 437 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 14 To further investigate the interaction between HcCAB and HcERF5. 438 Point-to-point analysis showed that HcCAB interacts with HcERF5 (Fig. 9A) . To 439 further verify the interaction between HcERF5 and HcCAB protein, a BIFC method 440 was used based on transient expression in tobacco leaves. The tobacco leaves 441 co-transformed with cYFP-HcERF5 and nYFP -HcCAB showed a clear interaction 442 fluoresence in the plant live imaging system (Fig. S13 ), and furthermore, a strong 443 yellow fluorescence was detected at the impregnated site, while none was found in the 444 control. This is further demonstrated that HcERF5 and HcCAB can interact in plants 445 (Fig. 9B). 446 Expression of stress-response genes in HcERF5-silenced plants 447 The antioxidant system of the HcERF5-silenced plants was severely damaged, 448 leading to a reduction in plant tolerance to drought stress. To clarify the mechanism, 449 the transcription level of phosphoribulokinase (HcPRK), BURP domain protein 450 RD22-like (HcRD22), mitogen-activated protein kinase homolog MMK2 -like 451 (HcMAPK2), chlorophyll a-b binding protein of LHCII type 1 -like (HcCAB), cysteine 452 synthase-like (HcCS) and caffeoyl-CoA O-methyltransferase 3 (HcCCoAOMT3) were 453 monitored, based on the interaction genes of the yeast two hybrid . The expression 454 levels of these six genes were significantly lower in the leaves of HcERF5-silenced 455 kenaf plants when exposed to salt and drought stress conditions (Fig. 11A-F). These 456 findings imply that HcERF5 controls the transcriptional activity of these genes to 457 respond drought stress. 458

459 HcERF5 confer drought tolerance in Arabidopsis and kenaf 460 In recent years, drought stress has significantly impacted crop productivity, 461 making it a serious concern f or the sustainability of global agriculture (Morgil et al., 462 2019). ERFs are key regulators that play a positive role in controlling physiological 463 functions, growth, and stress responses in plants under stress, as part of the ethylene 464 signaling pathway. Various studies have shown that the overexpression of ERF5 465 transcription factors can enhance resistance in a variety of plant species (Fujimoto et 466 al., 2000; Fischer and Dröge-Laser, 2004; Jin et al., 2009; Chuang et al., 2010; Moffat 467 et al., 2012; Pan et al., 2012; Son et al., 2012; Dubois et al., 2013; Severo et al., 2015; 468 Li et al., 2023) . However, the role of the ERF5 gene from kenaf in drought tolerance 469 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 15 has not been well explored. In this study, HcERF5 gene was cloned, which has a 470 typical conserved AP2/ERF domain and subcellular localization showing its 471 expression in the cytoplasm and nucleus (Fig. 1). Therefore, we hypothesized that it 472 has a similar function to other ERF5 genes. The changes of ERF5 expression in 473 tamarix hispida, tomato and cotton were significantly induced by drought or ABA (Jin 474 et al., 2009; Pan et al., 2012; Liu et al., 2014) . In this study, HcERF5 expression in 475 kenaf was significantly upregulated by drought and ABA, and high expression levels 476 in the leaves, petioles, stems, and roots of transgenic Arabidopsis, indicating a 477 potential role in the development of these organs (Fig. 2) . The heterologous 478 expression of HcERF5 in Arabidopsis was found to enhance seed germination rates 479 under drought stress and decrease seed sensitivity to ABA. Conversely, aterf5 480 Arabidopsis mutants exhibited reduced seed germination rates under drought stress 481 and increased seed sensitivity to ABA (Fig. 3). Heterologous expression of HcERF5 482 in Arabidopsis can significantly enhance the drought resistance of seedlings, while the 483 aterf5 mutant of Arabidopsis significantly reduced drought tolerance (Fig. 4) . It is 484 well established that increased lipid peroxidation exacerbates cellular oxidative 485 damage in plants under drought stress (Tang et al., 2013) . This phenomenon results 486 from the excessive accumulation of ROS such as H 2O2 and O2 - radicals. Antioxidants 487 are essential for mitigating oxidative damage induced by drought (Wu et al., 2015) . 488 Under drought conditions, the levels of H2O2 and O 2 - in the leaves of the aterf5 489 Arabidopsis mutant were significantly higher than those in WT, wh ereas WT plants 490 exhibited higher levels of H2O2 and O 2 - than transgenic plants (Fig. 5E and F). This 491 suggests that the transgenic lines experienced slightly less cellular oxidative damage 492 compared to WT plants. The higher MDA content in plants indicates higher degree of 493 lipid peroxidation and consequently, more extensive cell membrane damage (Sun et 494 al., 2014) . Under drought stress, the aterf5 Arabidopsis mutant showed the highest 495 MDA levels in its leaves, followed by the WT, w ith the transgenic plants having the 496 lowest levels (Fig. 5D), indicates that the aterf5 Arabidopsis mutant experienced the 497 most severe damage, followed by the WT, with the transgenic plants showing the 498 least damage. Additionally, the activities of SOD, POD, and CAT, which are crucial 499 for ROS degradation, were measured. Arabidopsis plants under drought conditions 500 exhibited a significant increase in SOD, POD, and CAT levels. However, the enzyme 501 activity was significantly higher in transgenic plants compare d to WT plants, and WT 502 plants had higher enzyme activity than the aterf5 Arabidopsis mutant (Fig. 5A -C). 503 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 16 This demonstrates that overexpression of HcERF5 can mitigate cellular oxidative 504 damage under stress by enhancing ROS scavenging capacity. Mutation of AtERF5 in 505 Arabidopsis led to reduced antioxidant enzyme activity, resulting in decreased 506 drought resistance. These findings suggest that HcERF5 enhances drought tolerance 507 by regulating ROS scavenging. 508 The growth and development of HcERF5-silenced plants were significantly 509 inhibited under drought stress, including plant height and fresh weight (Fig. 7A and 510 B). At the same time, there was a significant decrease in antioxidant enzyme activities 511 (Fig. 7E -H), and increase in MDA, H 2O2, and O 2 - contents (Fig. 7 I-K). Consist ent 512 with our observation, increased ROS accumulation was recently reported in 513 HcERF4-silenced kenaf plants under drought stress (Yue et al., 2022) . In addition, 514 accumulation of proline under abiotic stress protects cells from damage by osmotic 515 regulation and free radical scavenging (Kollist et al., 2014) . Proline can serve as an 516 indicator of the degree of stress -induced damage (Auriga and Wrobel, 2018) , and 517 silencing of HcERF5 resulted in a significant increase in proline content (Fig. 7H), 518 indicating that silencing of HcERF5 led to severe drought damage in kenaf plants. 519 Notably GO enrichment analysis of transcriptome DEGs revealed that there is a wide 520 array of FB processes, including immune system process es, detoxification, signaling, 521 and response to stimul i etc. (Fig. 8B, Table S4) . The expression of some antioxidant 522 enzymes or genes also changed significantly (Fig. S10). Taken together, this means 523 that HcERF5 can regulate the expression of related genes, and then improve the 524 physiological indexes such as antioxidant enzyme activity of plants under drought 525 stress, and is a factor that responds positively to drought tolerance. 526 HcERF5 is involved in regulating th e ABA signaling pathway affects ABA 527 content and regulates stomatal conductance 528 ABA plays a pivotal role in plant responses to abiotic stresses, including 529 drought (Hirayama and Shinozaki, 2007; Liu et al., 2023) . Under challenging 530 conditions such as drought and high temperatures, there is a notable increase in 531 ABA levels within plants. This elevation triggers the rapid activation of the 532 ABA signalling pathway, which in turn enhances the plant's ability to withstand 533 stress (Cutler et al., 2010; Wang et al., 2023) . A substantial number of 534 transcription factors have been documented to respond to osmotic stress 535 through ABA -dependent manner (Jardak-Jamoussi et al., 2016; Chen et al., 2022) . 536 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 17 The ABA signal transduction process is the most widely researched and earliest 537 known signaling pathway involved in PP2Cs. In higher plants, the ABA signaling 538 module mainly consist of three parts, namely ABA receptor PYR/PYL/RCARs, PP2C 539 members of A subfami ly, and SnRK2s (Fujii et al., 2009) . Protein phosphatase 2C 540 (PP2Cs) serve as a key regulators in plant responses to abiotic stresses. In Arabidopsis 541 thaliana, PP2CA acts as a central regulator of ABA signaling , negatively influencing 542 plant growth, development, and responses to various stre sses (Baek et al., 2019) . Our 543 study identified a total of 13 PP2C transcription factors that were significantly 544 up-regulated in pTRV2 -HcERF5 plants (Fig. 8D) , highlighting their crucial role in 545 kenaf's response to drought stress. 546 Stomata play a n important role in plants by regulating water loss and gas 547 exchange. During drought periods, there is a positive correlation between stomatal 548 density and the transpiration rate. ABA influences stomatal conductance by inducing 549 stomatal closure, thereby reducing transpiration water loss and triggering the 550 expression of drought-responsive genes. This mechanism ultimately enhances drought 551 resistance in plants (Lim et al., 2015; Saradadevi et al., 2017) . Under drought stress, 552 overexpression of AP2/ERF gene, IbRAP2-12 Arabidopsis thaliana lines can 553 upregulate ABA signaling genes, significantly increase ABA content and decrease 554 water loss rate , thereby enhancing the plant tolerance (Li et al., 2019) . Our findings 555 are similar to those of previous studies, particularly in observing stomatal closure in 556 both pTRV2 and pTRV2-HcERF5 plants when subjected to normal and drought stress 557 conditions. Under normal conditions, stomata in both pTRV2 and pTRV2 -HcERF5 558 plants remained open, with no significant difference observed in the stomatal 559 width-to-length ratio between the two plant types . However, stomatal closure was 560 more pronounced in pTRV2 plants under drought than in pTRV2-HcERF5 plants (Fig. 561 6D and G). The changes in stomatal density showed the same trend (Fig. 6E and H) . 562 These results demonstrate that HcERF5 promotes stomatal closure and stomatal 563 density in response to drought, thus reducing the rate of water loss (Fig. 6F). ABA 564 content under drought stress was also measured and found that the accumulation of 565 ABA significantly increased in both pTRV2 and pTRV2-HcERF5 plants after drought 566 treatment, and the ABA content in pTRV2 plants w as higher than that in 567 pTRV2-HcERF5 plants (Fig. 6G). These findings showed that ABA synthesis under 568 drought is inhibited by silencing of HcERF5. Therefore, it is speculated that HcERF5 569 enhances drought resistance through ABA signaling. 570 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 18 HcERF5 interact with stress-responsive related genes in drought tolerance 571 Plants have evolved sophisticated protective systems to recognize and respond 572 to adverse environmental conditions, such as the expression of stress -related genes 573 (Nevo et al., 2010) . For instance, the expression of PRK in rice is regulated by ABA, 574 gibberellin (GA) and methyl jasmonate (MeJA) (Chen et al., 2005) . The 575 overexpression of myo -inositol-1-phosphate synthase ( IbMIPS1) in sweet potato has 576 been shown to significantly upregulate the photosphoribulokinase ( PRK) gene, 577 thereby enhancing the plant's salt tolerance (WANG et al., 2016) . In soybeans, the 578 expression of the stress -induced RD22-like protein ( GmRD22) can mitigate salt and 579 osmotic stress (Wang et al., 2012) . Similarly, the grapev ine RD22 gene has been 580 found to confer drought resistance when expressed in tobacco (Jardak-Jamoussi et al., 581 2016). Mitogen-activated protein kinase (MAPK) cascades play crucial roles in 582 responding to both biotic and abiotic stresses, including drought. Notably, SlMAPK1 583 is activated by various abiotic stresses and hormone treatments, and its overexpression 584 in transgenic tomato plants has been li nked to improved drought tolerance (Wang et 585 al., 2018). VvCCoAOMT is a multifunctional O-methyltransferase, potentially playing 586 an important role in the methylation of anthocyanins in grape berries, particularly 587 under conditions of drought stress (Giordano et al., 2016) . Cysteine synthase (CS) is 588 an enzyme that catalyzes the biosynthesis of cysteine in plants. Cysteine serves as a 589 precursor for the synthesis of various sulfur -containing metabolites, the most 590 important of which is glutathione, which is used as a universal antioxidant and 591 detoxifying agent to cope with various stresses (Yang et al., 2007). Overexpression of 592 CS has been shown to significantly enhance the tolerance of tobacco plants to heavy 593 metals and sulfur containing pollutants (Noji et al., 2001; Kawashima et al., 2004). In 594 our study, we observed that drought stress induces the expression of HcERF5 (Fig. 2), 595 and that HcERF5 interact with various proteins, including HcPRK, HcRD22, 596 HcMAP2, HcCS, HcCCoAOMT3, among others, as demonstrated by the yeast 597 two-hybrid system (Fig. 8, Table S2) . The expression of these proteins in 598 HcERF5-silenced kenaf plants had significantly changed after silencing under drought 599 stress treatment (Fig. 9) , suggesting that HcERF5 was involved in stress signal 600 transduction and transcriptional regulation of downstream genes. In addition, the 601 chlorophyll a-b binding protein is an important product of chlorophyll synthesis and 602 photosynthesis. Overexpression of apple MdLhcb4.3 in Arabidopsis was shown to 603 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 19 significantly improve chlorophyll content and drought resistance of plants (Zhao et al., 604 2020). In Paeonia ostia, PoWRKY71 directly binds to the W -box element of the 605 PoCAB151 promoter, thereby activating its transcription . Furthermore, plants 606 overexpressing PoCAB151 exhibited enhanced drought resistance, increased 607 chlorophyll content, and improved photosynthetic activity, compare to those of 608 PoCAB151-silenced plants (Luan et al., 2023) . OsERF19 plays an active role in salt 609 stress and ABA response in rice and that OsERF19 can directly target promoters of 610 OsOTS1 and OsNCED5 and further increase their transcription level (Huang et al., 611 2021). We further confirmed the interaction between HcERF5 and HcCAB in the 612 nucleus using BIFC technology (Fig. 10). Therefore, we hypothesized that HcEFR5 613 may bind to specific cis -acting elements of the HcCAB promoter to regulate HcCAB 614 expression and thus participate in response to drought stress. Further work will focus 615 on the screening and identifying the cis-acting elements of HcEFR5 that specifically 616 bind to HcCAB promoters by experimental means such as Co -IP and EMSA, thereby 617 regulating their expression and further revealing their molecular mechanisms in 618 response to drought stress. 619

620 This study systematically investigates the role and mechanism of HcERF5 in 621 regulating drought stress in kenaf . HcERF5 expression is significantly induced by 622 drought and ABA. Mutation of the aterf5 gene in Arabidopsis significantly increased 623 the sensitivity of seeds and seedlings to drought, whereas overexpression of HcERF5 624 enhanced their drought stress tolerance. Silencing HcERF5 in kenaf resulted in 625 increased ROS levels and decreased ABA content, leading to poor drought tolerance, 626 thus indicating the critical role of HcERF5 in kenaf's drought resistance. Moreover, 627 six stress-responsive genes were identified to interact with HcERF5 via yeast Y2H 628 assays, all of which were down-regulated in HcERF5-silenced kenaf plants under 629 drought conditions compared to WT. RNA-seq revealed 2,489 DEGs in 630 HcERF5-silenced plants, including genes involved in the ABA signaling pathway and 631 antioxidant enzyme activity. A proposed regulatory model suggests that HcERF5 632 enhances drought tolerance in kenaf by improving ROS scavenging abilities through 633 the regulation of ABA synthesis and key stress resistance genes (Fig. 12). This study 634 is of great significance for understanding the mechanism of HcERF5 regulation in 635 kenaf drought stress. 636 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 20

637 Identification, sequence analysis and subcellular localization of HcERF5 638 The amino acid sequence of the HcERF5 gene was predicted using DNAMAN 639 8.0 and Jalview software. The NCBI BLAST tool 640 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to perform 641 conserved domain analysis and homology comparison study. The Phyre2 server 642 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) was utilized to predict 643 the three -dimensional structures and transmembrane regions. The neighbour -joining 644 (NJ) method was employed to construct the phylogenetic tree in MEGA 6.06. The 645 pBI121-HcERF5-EGFP vector, under the control of the CaMV 35S promoter (Fig. 646 S1), was employed to examine the subcellular localization of the HcERF5-GFP fusion 647 construct, following the detailed protocol outline d by Yue et al. (2022). The primers 648 used in this study areprovided in Table S1. 649 Expression analysis of the HcERF5 and GUS staining 650 Total RNA was extracted from kenaf leaves treated with 20% PEG6000 and 100 651 μM ABA for various time points (0, 2, 6, 12, 24, and 48 hours) using TRIzol reagent 652 (Vazyme Biotech Co., Ltd), following the manufacturer's protocol. Additionally, total 653 RNA was iso lated from the leaves, petioles, stems, and roots of naturally growing 654 kenaf seedlings. First -strand cDNA synthesis was carried out using a reverse 655 transcription kit from Vazyme Biotech Co., Ltd, and total RNA. Quantitative real-time 656 PCR (qRT-PCR) was caried out in a Bio -Rad CFX-96 RT-PCR amplifier (Bio -Rad, 657 USA) using Cham SYBR qPCR master mix (Vazyme Biotech Co., Ltd, Nanjing, 658 China). The 2 -ΔΔCT method was employed to analyze the relative expression levels, 659 with the Actin3 gene serving as an internal refe rence. Table S1 contains a list of the 660 qRT-PCR primers utilized in this investigation. 661 A 1.5 kilobase region upstream of the start codon ATG of the HcERF5 gene was 662 extracted and utilized to control the expression of the HcERF5 gene in the binary 663 expression vector pCambia1391Z. The constructed vector was transferred into 664 Agrobacterium tumefaciens GV3101 using the freeze -thaw method, and then 665 transgenic strain was introduced into Arabidopsis thaliana via the floral-dip technique. 666 Following stress treatment, the positive seedlings were subjected by GUS stain ing. 667 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 21 Subsequently, the seedlings were decolorized in a 75% (v/v) ethanol solution to 668 remove chlorophyll before being photographed. 669 Abiotic stress assays 670 In this study, wild-type (WT), aterf5 mutants, and HcERF5-overexpressing 671 (HcERF5-OE) Arabidopsis lines were used for abiotic stress analysis. For 672 overexpressing HcERF5-OE lines, Agrobacterium tumefaciens GV3101 strain 673 containing the full -length coding sequence of HcERF5 was introduced into WT 674 Arabidopsis thaliana through the floral -dip method and were grown until T3 675 generation. T-DNA inserted aterf5 mutant (SALK_208574) seed were obtained from 676 the Arabidopsis Biological Resources Centre. For stress analysis, the surface sterilized 677 seeds of WT, aterf5 mutants and HcERF5-OE were sown on the half-strength MS 678 medium or a mixture of peat moss, vermiculite, and perlite in a 3:1:1 ratio. The plants 679 were grown at 23 ± 2°C under long day conditions, with 16 hours of light and 8 680 hours of darkness. After 1 week, Arabidopsis seedlings were initially grown vertically 681 on half -strength MS plates and then transferred to new plates containing mannitol 682 (200 or 400 mM ) and ABA (2 or 4 µM ) for root growth assay . T he primary root 683 lengths were measured seven days after transplantation. For drought stress analysis on 684 HcERF5-OE Arabidopsis plants, seven-day-old seedlings grown on half-strength MS 685 plates were transferred to 7 cm square pots containing a mix of peat moss, organic 686 substrate, and vermiculite in a 2:2:1 ratio. Drought stress was induced by withholding 687 water for seven days, followed by a three -day rehydration period. Measurements of 688 chlorophyll content, fresh weight, survival rate, and relative water content (RWC) 689 were taken for WT, aterf5 mutants, and HcERF5-OE seedlings. RWC was calculated 690 using the formula: RWC (%) = [(FW − DW)/(TW − DW)] × 100, where fresh 691 weight (FW), turgid weight (TW, after a 6 -hour incubation in distilled water at room 692 temperature), and dry weight (DW) were recorded. 693 VIGS 694 The kenaf cultivar 'Fuhong 992' (FH992) was used to explore the role of the 695 HcERF5 gene in response to drought stress using virus-induced gene silencing 696 (VIGS). The SGN-VIGS web tool (https://vigs.solgenomics.net/) was used to identify 697 the optimal target and design primers. The primer sequences can be obtained in Table 698 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 22 S1, whereas the precise positions for amplification are marked in Fig. S6 . VIGS 699 derived tobacco rattle virus (TRV) vectors, pTRV1 and pTRV2 were used for fur ther 700 analysis. To create pTRV2-HcERF5, a partial fragment of the HcERF5 gene was 701 cloned into the pTRV2 vector. Then the auxiliary plasmid pTRV1, the empty plasmid 702 pTRV2, and the positive recombinant plasmid pTRV2-HcERF5 were successfully 703 transformed into the Agrobacterium strain GV3101. At the first true leaf stage, the 704 leaves of FH992 were injected with a cell suspension of the Agrobacterium (Luo et al., 705 2023). After being exposed to darkness for twenty -four hours, kenaf seedlings were 706 then grown normally for 10 d ays. To assess the efficacy of gene silencing, random 707 samples of kenaf plants were taken at the third true leaf stage and subjected to 708 qRT-PCR analysis. The VIGS seedlings that tested positive were then exposed to a 709 10-day natural drought stress in the soil and the growth and physiological indicators 710 were examined accordingly. 711 The leaves of kenaf plants were examined with an inverted NICON microscope 712 with a 40X objective lens, and structural analysis was conducted with ProgRes. The 713 guard cell located on the lower epidermis of the leaves was captured using ProgRes® 714 CapturePro 2.8.8 software. The stomatal aperture was measured using ImageJ 715 software. The experiment was replicated thrice, with 50 -60 stomata measured in each 716 treatment group. The density of stomata was obtained by calculating the number of 717 pores in each photo/the area of the photo (204.8×153.6 μm2), with 10 photos used for 718 statistical analysis 719 The content of hydrogen peroxide (H 2O2), superoxide anion radical (O 2 -) and 720 proline content were measured according to the method described by Luo et al. (2023) 721 (Luo et al., 2023) . Malondialdehyde (MDA) conten t and the activities of superoxide 722 dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR) 723 were measured according to the method described in our previous stu dy (Chen et al., 724 2020). The concentrations of hydrogen peroxide (H 2O2) and superoxide radicals (O 2 -) 725 were determined by staining with 3, 30 -diaminobenzidine (DAB) and nitroblue 726 tetrazolium (NBT), respectively. The ABA content was determined by LC -MS 727 (Agilent 1290 infinity-SCIEX B5000trap, https://www.agilent.com/). 728 For the water loss rate assay, t he true leaves of pTRV2 and pTRV2 -HcERF5 729 plants were used as material. The intact leaves were cut off to measure fresh weight 730 (FW) immediately. When measuring the water loss rate of leaves, the isolated leaves 731 were placed in a greenhouse to ensure that the environment of the isolated leaves was 732 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 23 the same as that of the experimental and control plants at intervals of 0.5 h, 1 h, 1.5 h, 733 2 h, 3 h and 5 h. The weight of the isolated blade W0, and calc ulate the final result 734 according to the formula of water loss rate (%) = (W0-FW)/W0. 735 Transcriptome sequencing 736 The leaves of kenaf seedlings in pTRV2 and pTRV2 -HcERF5 plants under 737 drought stress were collected for transcriptome sequencing, and each treatment 738 contained three biological replicates. RNA extraction, library construction, 739 sequencing, and data processing were conducted by Majorbio (Shanghai, China) 740 based on Illumina Novaseq 6,000 sequencing platform. The clean data was then 741 mapped to the reference genome sequence 742 (https://bigd.big.ac.cn/gwh/Assembly/1033/show). The expression levels of 743 genes/transcripts were quantitatively analyzed usimg Fragments Per Kiloba ses per 744 Millionreads (FPKM). DEseq2 software was employed to search for differentially 745 expressed genes (DEGs) between two different treatment groups. The p -value＜0.05 746 and |log 2 fold change| ≥1 was considered as the threshold of DEGs. Functional 747 analysis of DEGs was performed via Gene Ontology (GO), and Kyoto Encyclopedia 748 of Genes and Genomes (KEGG) on the free online platform of Majorbio 749 (www.majorbio.com). 750 Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BIFC) 751 assay 752 Specific cloning primers were designed based on the sequence of the CDS of the 753 HcERF5 gene (Table S1), and seamlessly cloned into the bait vector pGBKT7. The 754 recombinant plasmids pGBKT7 -HcERF5, empty PGBKT7 plasmids (negative 755 control), and pGBKT7 -53+pGADT7-T (positive control) were transformed into the 756 Y2H Gold strain using the LiTE/PEG method. The monoclonal yeast was identified 757 by PCR and then the bacterial solution was diluted 1, 10, 100, and 1000 folds. the 758 diluted sample (2 μL ) was spread on SD/ -Trp board and SD/ -Trp-Leu-His+X-α-gal 759 (SD/-TDO+X-α-gal) plate and cultured at 30 ℃ for 3-5 days for validation of toxicity 760 and self-activation. 761 For initial screening o f HcERF5 protein, the bait vector pGBKT7 -HcERF5 with 762 library mixture (matching) was used on SD/ -Trp-Leu-His medium, and selected 763 monoclonal clones were spread on SD/ -Trp-Leu+X-α-Gal (SD/ -DDO+X-α-Gal) 764 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 24 medium. The blue spot colony was amplified and sequenc ed with T7 and ADF/B 765 primers (Table S1) and the sequencing results were aligned against the kenaf genome 766 (https://bigd.big.ac.cn/gwh) (Zhang et al., 2020) and NCBI BLAST 767 (https://blast.ncbi.nlm. nih.gov). Finally, the bacterial cultures of positive clones, 768 pGBKT7-53+pGADT7-T (positive control), and pGBKT7 - HcERF5+pGADT7 769 (negative control) were cultured to OD=0.5, and spread 3 μL onto SD/ -Trp-Leu and 770 SD/-Trp-Leu-His-Ade+X-α-Gal (SD/ -QDO+X-α-Gal) medium plates. The plates 771 were then incubateed at 30 ℃ for 3 days and photographed. 772 The coding sequences of HcERF5 and HcCAB were cloned into the vectors 773 pCAMBIA1301-nYFP and pCAMBIA1301 -cYFP, and transferred into 774 Agrobacterium GV3101, which can be immediately expressed in tobacco. The 775 fluorescence of YFP was analyzed by confocal laser scanning microscope 776 (TCS-SP8MP; Leica, Germany). The flesh cell on tobacco leaf were observed by 777 adjusting the excitation wavelength at 488 nm and emission wavelength at 507 nm. 778 Statistical analysis 779 Excel 2019 was used to organize the data and GraphPad Prism 8 software was 780 used for plotting . SPSS v24.0 software was used for analysis of variance, and 781 Duncan's new complex range method was used to compare the significance of 782 differences between treatments (* for p < 0.05 and ** for p < 0.01). 783 Supplemental Data 784 Supplemental Table S1 Primers for vector construction, qRT-PCR, and VIGS 785 Supplemental Table S2 The proteins of interaction with HcERF5 786 Supplemental Table S3 List of DEGs 787 Supplemental Table S4 GO analysis of DEGs 788 Supplemental Table S5 KEGG analysis of DEGs 789 Fundings 790 This research work was supported by the National Natural Science Foundatio n of 791 China (Grant No. 31960368), and the National Natural Science Foundation of 792 Guangxi Province (No. 2024GXNSFBA010446). 793 Figure legends 794 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 25 Fig. 1 Expression analysis and sequence analysis of HcERF5. (A) Multiple sequence alignment 795 of HcERF5 with its homologous proteins from other plant species. The conservative AP2 domains 796 are overlined in black. (B) Predicted 3D structure of HcERF5 generated using the Phyre2 server. 797 (C) Phylogenetic tree of HcERF5 wi th its homologous proteins from other plant species. A 798 phylogenetic tree of HcERF5 and its homologous sequences constructed by using the 799 neighbor-joining method using the MEGA 6.0 software. (D) Subcellular localization of HcERF5. 800 Bar: 10μm. 801 Fig. 2 Stress -induced expression assay of HcERF5. Expression levels of the HcERF5 in kenaf 802 leaves under (A) PEG and (B) ABA treatment. The time points of 0, 2, 6, 12, 24 and 48 h were 803 used to observe changes in expression trends with the untreated group at 0 hours serving as the 804 control. Mean and SD were calculated from more than three biological replicates. Asterisks 805 indicate significant differences from control ( * for p < 0.05 and ** for p < 0.01). (C) Analysis of 806 HcERF5 promoter activity by examining GUS expression in Arabidopsis under ABA and drought 807 treatments 808 Fig. 3 Arabidopsis seeds growth on 1/2 MS medium supplemented with different 809 concentrations of mannitol or ABA, and their germination rate. (A) The phenotype of WT, 810 aterf5 mutant and HcERF5-OE lines in different concentrations of mannitol or ABA. (B) Seed 811 germination rate of WT, aterf5 mutants and HcERF5-OE lines on 1/2 MS medium. (C -D) Seed 812 germination rate of WT, aterf5 mutants and HcERF5-OE lines in response to different 813 concentrations of mannitol. (E -F) Seed germination rate of WT, aterf5 mutants and HcERF5-OE 814 lines in response to different concentrations of ABA. Mean and SD were obtained from three 815 biological replicates. 816 Fig. 4 Respons e of WT, aterf5 mutants and HcERF5-OE Arabidopsis plants to drought and 817 ABA treatment. (A) Visualization of root length of WT, aterf5 mutants, and overexpressed lines 818 under normal, drought and ABA settings. (B -C) Measurement of r oot length under normal, 819 drought, and ABA conditions . (D) Drought stress and rehydration phenotype. (E) Chlorophyll 820 content. (F) Total fresh weight. (G) Relative water content, and (H) survival rate. Data are shown 821 as the means ± SEs of three biological replicates. Different lowercase letters indicate a significant 822 difference (P < 0.05) based on Duncan’s test. 823 Fig. 5 ROS accumulation and activities of antioxidant enzyme s under drought stress. (A) 824 SOD activity. (B) POD activity. (C) CAT activity. (D) MDA content. (E) H 2O2 content. (F) O 2 - 825 content. Data are expressed as the means ± SEs of three biological replicates. Different lowercase 826 letters indicate a significant difference (P < 0.05) based on Duncan’s test. 827 Fig. 6 Silencing of HcERF5 in kenaf reduces tolerance to drought stress. (A) Albino 828 phenotype upon silencing of HcTrx. (B) Phenotypes of mock ( pRTV2) and VIGS plants 829 (pRTV2-HcERF5) under drought stress . (C) H2O2 and O 2 - accumulation was detected by 830 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 26 histochemical staining with DAB and NBT, respectively. (D) Phenotypic analysis of stomata of 831 pRTV2 and pRTV2 -HcERF5 plants. Scale bar = 3 μm. (E) Stomatal density of pRTV2 a nd 832 pRTV2-HcERF5 plants photographed under the microscope. Scale bar = 40 μm. (F) Rate of water 833 loss in detached lea ves. (G) Measurements of stomatal aperture. (H) Measurements of stomatal 834 density. Data are shown as the means ± SEs of three biological replicates. Different lowercase 835 letters indicate a significant difference (P < 0.05) based on Duncan’s test. 836 Fig. 7 Functional analysis of HcERF5 under drought stress using VIGS. (A) Plant height. (B) 837 Total fresh weight. (C) ABA content. (E) SOD activity. (F) POD activity. (G) CAT activity. (H) 838 GR activity. (I) MDA content. (J) H 2O2 content. (K) O 2 - content. (L) Proline content. Data are 839 expressed as means ± SEs of three biological replicates. Different lowercase letters indicate a 840 significant difference (P < 0.05) based on Duncan’s test. 841 Fig. 8 Transcriptome analysis of pTRV2 and pTRV2 -HcERF5 plants under drought 842 treatment. 843 (A) Number of DEGs from HcERF5-silenced and pTRV2 plants that are significantly up-regulated 844 and significantly down -regulated. (B) DEGs GO enrichment analysis. (C) DEGs KEGG 845 enrichment analysis. (D) Analysis of gene expression associated with the ABA signaling pathway 846 in pTRV2 and HcERF5-silenced plants under drought treatment. 847 Fig. 9 Validation of the interaction proteins for HcERF5. ( A) Transactivation activity and 848 toxicity assay of HcERF5 in yeast cells. (B) Validation of interaction proteins for HcERF5. ‘+’ 849 represent pGADT7-T+pGBKT7-53，‘-’ represent pGADT7-T+pGBKT7-Lam; 1-29 represent 850 interacting colonies with HcERF5. The transformed yeast cells w ere plated on SD/ -DDO, 851 SD/-TDO+ X -α-gal and SD/ -QDO+X-α-gal. pGADT7 -T+pGBKT7-53 and 852 pGADT7-T+pGBKT7-Lam combinations served as positive and negative controls, respectively. 853 Fig. 10 Interaction verification assay of HcERF5 and HcCAB proteins. (A) Validation of 854 HcERF5 and HcCAB proteins using yeast two-hybrid assay. (B) Interaction between HcERF5 and 855 HcCAB verified by BIFC system. Bar=20 μm 856 Fig. 11 Expression profile of s tress-responsive genes (A -H: HcPRK, HcRD22, HcMAPK2, 857 HcCAB, HcCS, and HcCCoAOMT3) in HcERF5-silenced kenaf plants. Asterisks indicate 858 statistical significance (* for p < 0.05 and ** for p < 0.01). 859 Fig. 12 A proposed model of HcERF5 regulating drought tolerance in kenaf. 860

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The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 31 ethylene-responsive factor 1 (TaERF1) that increases multiple stress tolerance. 65: 719-732 1041 Yang C-Y, Hsu F -C, Li J -P, Wang N -N, Shih M -CJPp (2011) The AP2/ERF transcription factor 1042 AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis. 156: 202-212 1043 Yang Q, Wang Y, Zhang J, Shi W, Qian C, Peng XJP (2007) Identification of aluminum‐responsive 1044 proteins in rice roots by a proteomic approach: Cys teine synthase as a key player in Al 1045 response. 7: 737-749 1046 Yang Z, Dai Z, Lu R, Wu B, Tang Q, Xu Y, Cheng C, Su JJSr (2017) Transcriptome analysis of two 1047 species of jute in response to polyethylene glycol (PEG)-induced drought stress. 7: 16565 1048 Yoshida T, Christmann A, Yamaguchi-Shinozaki K, Grill E, Fernie ARJTiPS (2019) Revisiting the 1049 basal role of ABA–roles outside of stress. 24: 625-635 1050 Yue J, Tang M, Zhang H, Luo D, Cao S, Hu Y, Huang Z, Wu Q, Wu X, Pan JJPC, Tissue, Culture 1051 O (2022) The transcription fa ctor HcERF4 confers salt and drought tolerance in kenaf 1052 (Hibiscus cannabinus L.). 150: 207-221 1053 Zhang B, Su L, Hu B, Li LJIjoms (2018) Expression of AhDREB1, an AP2/ERF transcription factor 1054 gene from peanut, is affected by histone acetylation and increases abscisic acid sensitivity and 1055 tolerance to osmotic stress in Arabidopsis. 19: 1441 1056 Zhang G, Chen M, Chen X, Xu Z, Guan S, Li L -C, Li A, Guo J, Mao L, Ma YJJoeb (2008) 1057 Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean 1058 (Glycine max L.). 59: 4095-4107 1059 Zhang G, Chen M, Chen X, Xu Z, Li L, Guo J, Ma YJMBR (2010) Isolation and characterization of 1060 a novel EAR-motif-containing gene GmERF4 from soybean (Glycine max L.). 37: 809-818 1061 Zhang L, Xu Y, Zhang X, Ma X, Zhang L, Liao Z, Zh ang Q, Wan X, Cheng Y, Zhang JJPBJ 1062 (2020) The genome of kenaf (Hibiscus cannabinus L.) provides insights into bast fibre and 1063 leaf shape biogenesis. 18: 1796-1809 1064 Zhao S, Gao H, Luo J, Wang H, Dong Q, Wang Y, Yang K, Mao K, Ma FJPP, Biochemistry (2020) 1065 Genome-wide analysis of the light -harvesting chlorophyll a/b -binding gene family in apple 1066 (Malus domestica) and functional characterization of MdLhcb4. 3, which confers tolerance to 1067 drought and osmotic stress. 154: 517-529 1068 1069 Figure 1 1070 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 32 1071 Fig. 1 Expression analysis and sequence analysis of HcERF5. (A) Multiple sequence alignment 1072 of HcERF5 with its homologous proteins from other plant species. The conservative AP2 domains 1073 are overlined in black. (B) Predicted 3D structure of HcERF5 generated using the Phyre2 server. 1074 (C) Phylogenetic tree of HcERF5 with its homologous proteins from other plant species. A 1075 phylogenetic tree of HcERF5 and its homologous sequences constructed by using the 1076 neighbor-joining method using the MEGA 6.0 software. (D) Su bcellular localization of HcERF5. 1077 Bar: 10μm. 1078 1079 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 33 Figure 2 1080 1081 Fig. 2 Stress -induced expression assay of HcERF5. Expression levels of the HcERF5 in kenaf 1082 leaves under (A) PEG and (B) ABA treatment. The time points of 0, 2, 6, 12, 24 and 48 h were 1083 used to observe changes in expression trends with the untreated group at 0 hours serving as the 1084 control. Mean and SD were calculated from more than three biological replicates. Asterisks 1085 indicate significant differences from control ( * for p < 0.05 and ** for p < 0.01). (C) Analysis of 1086 HcERF5 promoter activity by examining GUS expression in Arabidopsis under ABA and drought 1087 treatments. 1088 1089 1090 1091 1092 1093 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 34 1094 1095 1096 1097 1098 1099 Figure 3 1100 1101 Fig. 3 Arabidopsis seeds growth on 1/2 MS medium supplemented with different 1102 concentrations of mannitol or ABA, and their germination rate. (A) The phenotype of WT, 1103 aterf5 mutant and HcERF5-OE lines in different concentrations of mannitol or ABA. (B) Seed 1104 germination rate of WT, aterf5 mutants and HcERF5-OE lines on 1/2 MS medium. (C -D) Seed 1105 germination rate of WT, aterf5 mutants and HcERF5-OE lines in response to different 1106 concentrations of mannitol. (E -F) Seed germination rate of WT, aterf5 mutants and HcERF5-OE 1107 lines in response to different concentrations of ABA. Mean and SD were obtained from three 1108 biological replicates. 1109 1110 1111 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 35 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 Figure 4 1126 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 36 1127 Fig. 4 Response of WT, aterf5 mutants and HcERF5-OE Arabidopsis plants to drought and 1128 ABA treatment. (A) Visualization of root length of WT, aterf5 mutants, and overexpressed lines 1129 under normal, drought and ABA settings. (B -C) Measurement of r oot length under normal, 1130 drought, and ABA conditions . (D) Drought stress and rehydration phenotype. (E) Chlorophyll 1131 content. (F) Total fresh weight. (G) Relative water content, and (H) survival rate. Data are shown 1132 as the means ± SEs of three biological replicates. Different lowercase letters indicate a significant 1133 difference (P < 0.05) based on Duncan’s test. 1134 1135 1136 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 37 Figure 5 1137 1138 Fig. 5 ROS accumulation and activities of antioxidant enzyme s under drought stress. (A) 1139 SOD activity. (B) POD activity. (C) CAT activity. (D) MDA content. (E) H 2O2 content. (F) O 2 - 1140 content. Data are expressed as the means ± SEs of three biological replicat es. Different lowercase 1141 letters indicate a significant difference (P < 0.05) based on Duncan’s test. 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 38 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 Figure 6 1167 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 39 1168 Fig. 6 Silencing of HcERF5 in kenaf reduces tolerance to drought stress. (A) Albino 1169 phenotype upon silencing of HcTrx. (B) Phenotypes of mock ( pRTV2) and VIGS plants 1170 (pRTV2-HcERF5) under drought stress . (C) H2O2 and O 2 - accumulation was detected by 1171 histochemical staining with DAB and NBT, respectively. (D) Phenotypic analysis of stomata of 1172 pRTV2 and pRTV2 -HcERF5 plants. Scale bar = 3 μm. (E) Stomatal density of pRTV2 a nd 1173 pRTV2-HcERF5 plants photographed under the microscope. Scale bar = 40 μm. (F) Rate of water 1174 loss in detached leaves. (G) Measurements of stomatal aperture. (H) Measurements of stomatal 1175 density. Data are shown as the means ± SEs of three biological replicates. Different lowercase 1176 letters indicate a significant difference (P < 0.05) based on Duncan’s test. 1177 1178 1179 1180 1181 1182 1183 1184 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 40 1185 1186 1187 1188 Figure 7 1189 1190 Fig. 7 Functional analysis of HcERF5 under drought stress using VIGS. (A) Plant height. (B) 1191 Total fresh weight. (C) ABA content. (E) SOD activity. (F) POD activity. (G) CAT activity. (H) 1192 GR activity. (I) MDA content. (J) H 2O2 content. (K) O 2 - content. (L) Proline content. Data are 1193 expressed as means ± SEs of three biological replicates. Different lowercase letters indicate a 1194 significant difference (P < 0.05) based on Duncan’s test. 1195 1196 1197 1198 1199 1200 1201 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 41 1202 1203 1204 1205 1206 1207 Figure 8 1208 1209 1210 Fig. 8 Transcriptome analysis of pTRV2 and pTRV2 -HcERF5 plants under drought 1211 treatment. 1212 (A) Number of DEGs from HcERF5-silenced and pTRV2 plants that are significantly up-regulated 1213 and significantly down -regulated. (B) DEGs GO enrichment analysis. (C) DEGs KEGG 1214 enrichment analysis. (D) Analysis of gene expression associated with the ABA signaling pathway 1215 in pTRV2 and HcERF5-silenced plants under drought treatment. 1216 1217 1218 1219 1220 1221 1222 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 42 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 Figure 9 1236 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 43 1237 Fig. 9 Validation of the interaction proteins for HcERF5. ( A) Transactivation activity and 1238 toxicity assay of HcERF5 in yeast cells. (B) Validation of interaction proteins for HcERF5. ‘+’ 1239 represent pGADT7-T+pGBKT7-53，‘-’ represent pGADT7-T+pGBKT7-Lam; 1-29 represent 1240 interacting colonies with HcERF5. The transformed yeast cells w ere plated on SD/ -DDO, 1241 SD/-TDO+ X -α-gal and SD/ -QDO+X-α-gal. pGADT7 -T+pGBKT7-53 and 1242 pGADT7-T+pGBKT7-Lam combinations served as positive and negative controls, respectively. 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 44 1253 1254 1255 1256 1257 1258 Figure 10 1259 1260 Fig. 10 Interaction verification assay of HcERF5 and HcCAB proteins. (A) Validation of 1261 HcERF5 and HcCAB proteins using yeast two-hybrid assay. (B) Interaction between HcERF5 and 1262 HcCAB verified by BIFC system. Bar=20 μm. 1263 1264 1265 1266 1267 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 45 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 Figure 11 1280 1281 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 46 Fig. 11 Expression profile of s tress-responsive genes (A -H: HcPRK, HcRD22, HcMAPK2, 1282 HcCAB, HcCS, and HcCCoAOMT3) in HcERF5-silenced kenaf plants. Asterisks indicate 1283 statistical significance (* for p < 0.05 and ** for p < 0.01). 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 Figure 12 1300 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint 47 1301 Fig. 12 A proposed model of HcERF5 regulating drought tolerance in kenaf. 1302 1303 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 4, 2024. ; https://doi.org/10.1101/2024.08.01.606231doi: bioRxiv preprint Parsed Citations A uriga A , Wrobel JJFEB (2018) Effect of effective m icro-organism s on the proline and m da contents in herb plant m aterial of Ocim um basilicum L. var. 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