ERF1 of Plumbago indica L. receives ethylene signaling and regulates cold tolerance together with the DREB-COR pathway

ERF1 of Plumbago indica L. receives ethylene signaling and regulates cold tolerance together with the DREB-COR pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ERF1 of Plumbago indica L. receives ethylene signaling and regulates cold tolerance together with the DREB-COR pathway Zi-An Zhao, Yi-Rui Li, Ting Lei, Cai-Lei Liu, Qing-Xiao Zeng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4448738/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ethylene is an essential regulatory factor in the signaling pathway of plant responses to abiotic stress, included cold stress, and also plays a regulatory role in cold response. Recent studies have shown that exogenous application of ACC (1-aminocyclopropane-1-carboxylate), an ethylene promoter, affects plant cold hardiness. It is well known that the cold-responsive specific gene DREB (dehydration-responsive element (DRE)-binding protein) plays a crucial role in enhancing cold hardiness in plants by activating some cold-responsive ( CORs ) genes. However, the molecular mechanism of how the ethylene biosynthesis pathway regulated this gene in the cold response of thermophilic plants had yet to be well explained. In this study, using the thermophilic plant P. indica ( Plumbago indica L.) as an example, physiological and transcriptomic analyses revealed that cold stress treatment induced the synthesis of endogenous ACC and regulated the ethylene signaling activator PiERF1 , while cold signaling also activated PiDERB1A . Spraying experiments were also showed that ACC-induced up-regulation of the PiERF1 gene reduced cold tolerance of P. indica , and decreased the expression level of the PiDREB1A gene; reverse experiments have shown that spraying AVG (aminoethoxyvinylglycine) resulted in the down-regulation of the PiERF1 gene, while the expression level of PiDREB1A was increased, and chilled symptoms were alleviated. These results indicated that ethylene signaling directly regulates the downstream gene PiERF1 and initiates the DREB-COR cold-responsive signaling pathway to regulate cold hardiness, exhibiting negative regulation of cold hardiness in thermophilic plants. Ethylene signaling pathway PiERF1 PiDREB1A Cold tolerance 1-aminocyclopropane-1-carboxylate Plumbago indica L. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction With the frequent occurrence of global environmental extremes, low-temperature stress poses a severe test for sequestering organisms, especially thermophilic plants, which must face unfavorable growing conditions [ 1 – 3 ]. When fall arrives, plants with cold hardiness undergo domestication to overwinter successfully [ 4 ]. Some thermophilic plants with higher ornamental value cannot cope with extremely low temperatures, such as such as tropical water lilies ( Nymphaea rubra ), Chrysanthemum morifolium ( Dendranthema grandiflorum var. Jinba), forage ( Medicago sativa L.) [ 5 – 7 ], and P. indica , the material of this paper, where low temperatures have become a key constraint to successful introductions to the north or higher latitudes [ 5 ]. P. indica is an evergreen perennial herb native to the tropics. Because of both ornamental and medicinal functions [ 8 – 10 ], it has excellent prospects for flower commercial development. During the previous introduction process, we found that P. indica was susceptible to wilt and even death in the open field in winter (Fig. 1 ), which was coincided with the flowering period, seriously affected the ornamental value. In order to clarify the mechanism of low-temperature effects on P. indica , members of the ERF subfamily of the Apetala 2/ Ethylene-responsive factor (AP2/ERF) family of transcription factors were found to be most induced in the process as a result of transcriptome sequencing. In past studies, a large number of transcription factors had been identified as significant regulators associated with cold stress [ 11 ]. AP2/ERF, as was one of the most prominent transcription factor families in plants, widely regulates plant biotic and abiotic stresses, such as heavy metals, temperature extremes, and drought [ 12 – 15 ], among others. Meanwhile, it is also a key transcription factor in the significant response to the ethylene signaling pathway in plant stress response [ 2 ]. Among the five subfamilies under AP2/ERF, AP2, ERF, DREB, RAV, and Soloist [ 16 ], the CBF/DREB-COR-mediated cold-responsive pathway, as the classical cold chain, had been much reported in plants [7, 17]; however, ERFs, members of the same family, were still not been reported in cold resistance [ 18 ], and the correlation between the two subfamily during cold response were not known. Ethylene was played a vital role in stress tolerance as a plant growth-regulating hormone that mediates the plant cold response in a species-dependent manner [ 19 ]. ACC was a prerequisite substance for ethylene synthesis, and was often applied in the field instead of gaseous ethylene. Synthesized ethylene was bound to the ETR1 receptor, blocked the triple response CTR1 phosphorylation and inhibited the downstream positive regulator EIN2 . EIN2 was activated to induce the transcription factor EIN3/EIL , which directly induced the ERF transcription factor, initiated the expression of downstream genes in response to ethylene signaling [ 20 ]. In more cold-tolerant plants, such as apples, ERF1B was up-regulated in expression by ethylene regulation. It acted on DRE components to promote downstream CBF1 ( DREB1B ) expression and enhance cold tolerance [ 19 ]. Whereas in some thermophilic plants such as wheat ( Triticum aestivum L.) [ 20 ], bean ( Phaseolus vulgaris L.) [ 21 ] and Bermuda grass ( Cynodon dactylon (L). Pers.) [ 22 ], ACC was thought to regulate their cold tolerance negatively. In 2019, Robison found in soybeans that ACC repressed the expression levels of GmCBF/DREB1s by regulating GmEIN3 and that antagonism of the ethylene pathway may be one of the reasons for cold acclimation in soybeans [ 23 ]. The above two opposite results were shown that the specificity of ACC in regulating plant cold tolerance, further were indicated the complex crosstalk relationship between ethylene and cold signaling pathway signals. Then, for other thermophilic plants, how does the cold stress response crosstalk specifically with ethylene signaling, and does the ethylene signaling pathway contribute to cold sensitivity in thermophilic plants? More evidence supporting which downstream gene expression ACC acts on in response to cold stress is needed. In the present study, we analyzed the transcriptome of P. indica by cold stress treatment. We found that the AP2/ERF-ERF was the most annotated, and one of its members designated PiERF1 ( Unigene_83531 ) in the present study, was detected in a full-length sequence. It was more strongly induced (highest degree induced at 24h, |log 2 FC|=2.46). Meanwhile, ACC content was significantly increased in P. indica leaves after cold stress. It was hypothesized that PiERF1 might mediate the cold response of P. indica by participating in the ethylene signaling pathway and directly or indirectly affecting the expression of the critical cold response gene PiDREB1A ( Unigene_05023 ). Further pretreatment of P. indica leaves were sprayed with 50 µmol/L ACC with the ethylene inhibitor AVG, which was revealed that spraying ACC resulted in reduced cold tolerance and activated downstream up-regulation of PiERF1 expression and down-regulation of PiDREB1A expression; conversely, sprayed AVG enhanced cold tolerance of P. indica , resulted in down-regulation of PiERF1 and up-regulation of PiDREB1A . These results were revealed that the increase of the ethylene precursor ACC in P. indica induced the up-regulated expression of PiERF1 , suppressed the expression level of PiDREB1A , and increased its cold sensitivity under the low-temperature environment in winter. This negative regulation mechanism of cold tolerance explained the senescence or even death of P. indica overwintering leaves in the open field. The results could further understand the crosstalk network relationship between ethylene biosynthesis and the CBF/DREB-COR cold-response pathway, which supported the previous conclusion of negative regulation of cold tolerance in thermophilic plants by ethylene and clarifies the correlation between ACC action on the downstream genes PiERF1 and PiDREB1A , which could provide specific reference for the introduction of thermophilic plants to the north and the development of open-ground over-wintering protection programs. 2. Materials and methods 2.1 Plant material and treatment The experimental material was 2-year-old asexual seedlings of P. indica . The plants were planted in the nursery of Sichuan Agricultural University, Chengdu, China. Before the experimental treatments, 54 pots of healthy potted plants with uniform growth were selected, pruned, and moved into an RXZ-500D-LED type light incubator (Jiangnan instrument, Ningbo, China). Among them, the light time was 16/8h, the temperature was 30/25℃, light intensity was 80%, the humidity was 60%, and the substrate was mixed (peat soil: garden soil: vermiculite: 3:2:1). Uniform routine water and fertilizer management was used to pre-cultivate the plants for 60 d. During this period, to place the error caused by the placement position, the position of the large pots was changed weekly in a clockwise direction to ensure the same environmental conditions. The plants were then divided into two groups of 27 pots each, which were used for two sets of experiments: direct exposure to low temperature at 4℃ or pretreatment with phytohormones followed by 4℃ treatment. The plants were incubated in total darkness to avoid the compound stress of light on them, and they were photoinhibited [ 24 ]. For the low-temperature treatment, plants were exposed to 4℃ for 72h and were sampled at 0h, 1h, 4h, 8h, 12h, 24h, and 72h, respectively. For the hormone group treatments, 27 pots of plants were divided into three groups and the leaves of the experimental group were sprayed with 50 µmol/L ACC and AVG, respectively, and the leaves of the control group were sprayed with RO water (reverse osmosis water) at intervals of 2d. After three consecutive sprays, the plants were exposed to cold stress for 72h at 4℃, cultured for 72h after cold stress, and restored to customary conditions, respectively. Samples were taken at 0h, 1h, 4h, 8h, 12h, 24h, 72h and 72h after recovery from cold stress. Three biological replicates were taken for each group, and each experiment was repeated three times. 2.2 Measurement of physiological and biochemical indicators 2.2.1 Chlorophyll fluorescence parameters and their visualization Whole-leaf fluorescence imaging was scanned using IMAGING-PAM (WALZ, Germany); chlorophyll fluorescence parameters were determined by the Handy PEA Plant Efficiency Analyzer (Hansatech Instruments Ltd, UK), and the key metrics, Fv/Fm, were calculated using the instrument's accompanying PEA Plus 1.12 software package. The top 3–5 leaves of the branches were selected for determination. Three biological replicates were tested for each sample, and each experiment was repeated three times. 2.2.2 Electrolyte leakage, MDA content, and reactive oxygen species scavenging system determination Electrolyte leakage (EL), MDA, SOD, POD, CAT, and soluble sugar were measured concerning the methods of the previous authors [ 25 ] using the relevant kits (Nanjing Jiancheng Bioengineering Institute, China). For sampling, the branches' top 3–5 leaves were taken. Three biological replicates were tested for each sample. 2.3 Determination of ACC content and ACS and ACO enzyme activities Referring to the method of Yuan [ 26 ], plant ethylene precursor (ACC), plant ACC synthase (ACS), and plant ACC oxidase (ACO) were determined using ELISA kits (MM-36135O1 for ACC content; MM-33691O2 for ACS activity; MM-33691O2 for ACO activity; Jiangsu Meimian Industrial Co., Ltd, China) for determination. For sampling, the apical 3–5 leaves were taken. Three biological replicates were tested for each sample. 2.4 RNA extraction, RNA-Seq, library construction and gene annotation Young leaves of P. indica were sampled at 4℃ under cold stress (see 2.1 for details) for 0h, 1h, 4h, 8h, 12h, and 24h (label the six-time points as Z1-Z6, respectively). According to the instructions, the total RNA of P. indica was extracted using RNAprep Pure Plant Kit (TIANGEN, China). RNA purity was ensured by 1% agarose gel electrophoresis. Qubit 2.0 and Agilent 2100 systems were used to detect the library and ensure the reliability of the results. cDNA libraries were sequenced using the Illumina NovaSeq high-throughput sequencing platform based on Sequencing By Synthesis (SBS) technology [ 24 ]. The Unigene sequences were compared with public databases NR, Swiss-Prot, GO, COG (Clusters of Orthologous Groups), KOG (euKaryotic Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes) databases were compared using the BLASTx algorithm [ 27 ]. After predicting Unigene's amino acid sequence, Unigene's annotation information was obtained using HMMER software compared with the Pfam database. Metabolic pathways of Unigenes were determined according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [ 28 ]. The Unigene sequences were compared with the databases NR [ 29 ], Swiss-Prot [ 30 ], COG [ 31 ], KOG [ 32 ], eggNOG4.5 [ 33 ], KEGG [ 34 ] comparisons. interProScan [ 35 ] analyzed the GO [ 36 ] Orthology results of the new genes using the InterPro-integrated databases and used KOBAS [ 37 ] to obtain the Unigene's KEGG Orthology results in KEGG, and used HMMER [ 38 ] software to compare the amino acid sequences of the predicted completed Unigene with the Pfam [ 39 ] database to obtain the annotation information of Unigene. 2.5 RT- qPCR validation Quantitative real-time PCR (qRT-PCR) analysis was accomplished using an iCycler iQ5 system (Bio-Rad, Hercules, CA, USA) and SYBR Green PCR Master Mix (transgene), with each reaction containing 5 µL of 2x QuantiFast SYBR Green PCR Each reaction consisted of 5 µL of 2x QuantiFast SYBR Green PCR Master Mix (SYBR Green Pro Taq HS qPCR Kit, Accurate Biotechnology, China), 1 µL of cDNA, 2 µM of gene-specific forward primer, and 2 µM of gene-specific reverse primer, and the final volume was spiked up to 10 µL with sterile water. The PCR reaction was carried out at 95℃ for 3 min, 95℃ for 15 min, and 95℃ for 10 min. for 3 min; 95℃ for 15 s, 60℃ for 20 s, and the above steps were repeated for 40 cycles. The expression of EDGs was calculated after normalization using the 2−ΔΔCT algorithm with PaActin as the internal reference gene. Detailed information about the qRT-PCR primers is provided in Supplementary Table 1( Tab. S1 ). 2.6 Data analysis The results of a representative experiment are shown. The values presented are means ± SE and analyzed via IBM SPSS Statistics statistical software (Version 25.0). Draw and analyze using Origin2020. Statistical differences were compared with a one-way analysis of variance (ANOVA) based on LSD’s multiple range test at significance levels of P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). 3. Results 3.1 Low temperature stress reduces photochemical efficiency of P. indica Continuous exposure to low temperatures can cause some damage to plant leaves and reduce their photosynthetic efficiency. The chlorophyll fluorescence visualization of P. indica leaves (Fig. 2 a) was showed that the chlorophyll fluorescence of P. indica gradually decreased with the increase of low-temperature exposure. Meanwhile, the response of plant photochemical efficiency to low temperatures can be effectively shown by Fv/Fm photosynthetic parameters [ 26 ]. Under 4℃ stress, chlorophyll fluorescence significantly was decreased to 0.64 at 4h, rebounded to 0.76 after 8h, and then decreased to 0.73 after 24h and continued to stabilize in the following 72h (Fig. 2 b), but significantly decreased relative to the control group. These phenomena was indicate that P. indica suffered some degree of leaf damage due to reduced photosynthetic efficiency under cold exposure at 4℃. Although some plants could gradually adapt and recover from cold stress, P. indica leaves continue to be irreversibly damaged. 3.2 P. indica has enhanced membrane permeability under low-temperature stress, possessing stronger oxidase activity and elevated soluble sugar content The larger the EL, the more severe the plant damage. The experimental results were showed that the EL of P. indica gradually increased with the increase in the duration of cold exposure and was significantly higher at 72h (Fig. 3 a).When plants suffer from adversity injury, at this time, the reactive oxygen scavenging mechanism is triggered. Malondialdehyde (MDA) is one of the products of cell membrane lipid peroxidation after excessive accumulation of reactive oxygen radicals in plants. When the antioxidant mechanism in P. indica could not maintain the balance between output and scavenging, MDA increased significantly, significantly elevated, and peaked at 8h. However, with the initiation of the protective enzyme system, the activities of the CAT enzyme (Fig. 3 b), POD enzyme (Fig. 3 c), and SOD enzyme (Fig. 3 d) in the scavenging system of the ROS system were enhanced. Its membrane lipid peroxidation was alleviated to a certain extent after 24h (Fig. 3 e). During low-temperature stress, sugar metabolism undergoes phase changes. In P. indica , the soluble sugar content increased significantly at 8h during the early stage of cold stress, peaked at 12h, decreased gradually, and increased significantly at 72h (Fig. 3 f). Physiological and biochemical data was indicated that P. indica could enhance its cold tolerance through ROS scavenging enzyme activity system and sugar metabolism pathway under low-temperature stress. However, the EL gradually increased with the increase of cold stress time, indicating that the damage to the membrane system was intensified and the P. indica leaves were severely damaged. 3.3 Low-temperature stress increases ACC content and ACS and ACO activities in P. indica leaves As an essential substrate in the ethylene biosynthesis pathway, ACC is commonly used to study cold stress and ethylene signaling pathway models. Therefore, we examined the dynamic changes in ACC content. Under cold stress, the ACC content in P. indica leaves was increased with time, significantly elevated from 94.27 nmol/L at 0 h to 132.93 nmol/L (Fig. 4 a), which was 1.41 times higher than the initial level. Meanwhile, the enzyme activities of ACC synthase ACS and ACC oxidase ACO were also heavily induced (Fig. 4 b,c), with ACS activity significantly was elevated from 5.33 U/L to 8.08 U/L at 0 h, and ACO activity significantly was elevated from 268.68 U/mL to 383.80 U/mL at 0 h. The former was elevated by 1.51 times, while the latter was elevated by 1.42 times. It was indicated that the ethylene biosynthesis pathway was induced to be activated in cold stress, one of the critical pathways for cold response in P. indica . 3.4 RNA-seq sequencing reveals changes in the ethylene pathway of P. indica under cold treatment 3.4.1 Validation of RNA-seq results by quantitative real-time RT-PCR (qPCR) A cDNA library was constructed from equal amounts of RNA extracted from P. indica leaves subjected to 4℃. The transcriptome sequencing of 18 samples was finally completed, and a total of 128.88 Gb Clean Data was obtained, and the Clean Data of each sample reached 5.73 Gb, with the percentage of Q30 bases at 93.42% and above ( Tab. S2 ). A total of 90,725 Unigenes were obtained after assembly, among which 31,073 Unigenes were above 1kb in length. After obtaining high-quality sequencing data, the sequence was assembled using Trinity. A total of 90,725 Unigenes were obtained from the assembly, and the N50 of Unigene was 2963, which showed high assembly integrity. The Clean Data of each sample was compared with the assembled Transcript or Unigene libraries, and the statistics of the comparison results were shown in Tab. S2 . The statistical results indicate that the transcriptome data are sufficient and valid for the following analysis step. Twelve randomly selected DEGs were subjected to qPCR for transcript abundance to verify that the RNA-seq results were accurate. There were 7 Unigene data up-regulated expression and 5 down-regulated (Fig. 5 ). The qPCR results of the tested genes Unigene 30052 , Unigene 69681 and Unigene79700 , Unigene 11281, Unigene40972 were generally consistent with the changes in the RNA-seq data. Unigene08110 , Unigene64040 , Unigene03061 , Unigene82860 , Unigene 43750 , Unigene 44592 , and Unigene 51682 . These seven genes were weakly up- or down-regulated in expression within 4h, which may have led to a slight bias in the q-PCR test results, but the overall expression changes were consistent. This result was validated the reliability of the RNA-seq data and supports further bioinformatics analysis. Note There are 7 up-regulated and 5 down-regulated expressed genes in the graph; the X-axis represents the time (h) of low-temperature treatment, the left Y-axis displays the corresponding RNA-seq expression data (blue histogram), and the right Y-axis displays the relative gene expression level by qPCR (black dash line). Bars represent ± SE (n = 3) and broken lines represent ± SE (n = 9). 3.4.2 GO and KEGG Enrichment Reveals Cold Stress Response is Closely Linked to Transcriptional Regulation and Hormonal Signaling The BLAST parameter E-value ≤ 1e-5 and the HMMER parameter E-value ≤ 1e-10 were selected to obtain 41,111 Unigenes with annotated information in the total database ( Tab. S3 ). The GO database categorized the annotated 33,538 Unigenes into 45 functional groups (Fig. 5 a), which were further enriched into three GO level II functions, including "cellular components," "molecular functions," and "biological processes." The top three "cellular components" were "cellular anatomical entity (19589)", "intracellular (11698)" and "protein-containing complex (3515)"; The top three "molecular functions" were "structural domain binding (16993)", "catalytic activity (15944)" and "transcription regulator activity (1112)"; the top three "biological process" were "cellular process (18945) ", "metabolic process (16477)" and "biological regulation (5812)". The enrichment degree of Pathway was analyzed in KEGG using the Enrichment Factor and the significance of enrichment was calculated using the hypergeometric test. Analysis shows that in the 0h vs. 24h group, the most significant KEGG enrichment pathways are "Plant hormone signal transmission," "Plant pathway interaction," "MAPK signaling pathway - plant," and "Circadian rhythm - plant" (Fig. 5 b). By ranking GO and KEGG, it was not difficult to find that P. indica cold stress response was closely related to transcriptional regulation and hormone signaling, which were also coincided with our previous finding of increased ACC content, ACO and ACS enzyme activities in the phytohormone ethylene synthesis pathway. 3.4.3 Differential expression gene annotation of ethylene response factors PiERF1 and PiDREB1A involved in P. indica cold response Transcription factor prediction analysis by BMKCloud (BMKCloud) was showed that AP2/ERF transcription factors were induced in the most significant number (108) during the cold response of P. indica within 24h, suggested that transcription factors ERFs play an essential role in the molecular mechanism of the cold response of P. indica (Fig. 7 a). Analysis of the differentially expressed ERFs genes in the four treatment groups of 0h vs 4h, 0h vs. 8h, 0h vs 12h and 0h vs 24h revealed that the Unigene_83531 gene (named PiERF1 ) was significantly up-regulated in response to cold stress, with |log 2 FC| reaching 2.46 at 24h. Combined with the correlation matrix analysis, PiERF1 was found to have a common relationship with several colds. In combination with the correlation matrix analysis, PiERF1 was positively correlated with several physiological indicators related to cold stress, including ACC content, ACO and ACS activity. It was hypothesized that PiERF1 might be regulated by the ethylene precursor ACC (Fig. 7 b) and positively regulated by cold stress. In the ethylene signaling pathway, included ETR1 ( Unigene 12465 ), EIN3 ( Unigene 53149 ) with the ACC (1-aminocyclopropane-1-carboxylic acid) synthase gene, ACS6 ( Unigene 81360 ), as well as the upstream genes of ACS6 , EIN3/EIN1 , MPK3 (Unigene 78362 , Unigene 64590 ), both responded positively to cold stress (Fig. 7 c). In the DREB-COR pathway, PiDREB1A , a fast cold-responsive gene, was the highest expression at 8h, followed by a gradual decrease. The COR gene ( Unigene 28684 ) was regulated downstream by PiDREB1A , with a gradual up-regulation of its expression at 24h (Fig. 7 d), which were regulated the genes of the plant group to resist cold stress. The results were indicated that both key pathways, ethylene signaling and DREB-COR, responded to cold stress, but the crosstalk between them needs further exploration. 3.5 Evidence of negative regulation of cold hardiness of P. indica by exogenous spraying of ethylene promoter ACC and inhibitor AVG by ethylene signaling P. indica leaves were sprayed with 50 µmol/L ACC and AVG respectively (control group sprayed with RO water), and it was found that the ACC group was more sensitive to cold relative to the control group, and the foliage crumpling was evident at 72h of 4℃ stress, and the leaves were dry and severely damaged after 72h of recovery incubation (Fig. 8 a). Chlorophyll fluorescence parameters were greatly reduced, and the photochemical efficiency was significantly inhibited (Fig. 8 b); at the same time, ACC increased EL and membrane permeability; ROS scavenging mechanism was weakened, resulting in an increase in MDA, which caused P. indica to exhibit cold sensitivity (Fig. 8 c).In contrast to this, the AVG group was able to increase P. indica cold tolerance, and the degree of damage was lower relative to that of the CONTROL group after 72h of restoration culture (Fig. 8 a) and higher photochemical efficiency (Fig. 8 b); it could reduce EL permeability, maintain cellular homeostasis, enhance ROS enzyme activity, and inhibit MDA overaccumulation (Fig. 8 c). Excess accumulation of ACC in P. indica induced reduced photochemical efficiency, elevated cell membrane permeability, and increased lipid peroxidation, leading to reduced cold tolerance. On the contrary, AVG inhibited ACC accumulation and suppressed ethylene signaling, resulting in a certain protective effect on P. indica ; however, further experiments need to demonstrate how ethylene signaling regulates its downstream genes in response to cold. 3.6 PiDREB1A and PiDREB1A are downstream regulated by crosstalk between ethylene and cold signaling Transcriptome expression analysis of P. indica indicated that cold stress rapidly up-regulated PiETR1 , PiEIN3 , and PiERF1 in the ethylene signaling pathway, as well as the cold-responsive specific gene PiDREB1A (dehydration-responsive element (DRE)-binding protein 1A). However, whether they are co-regulated by ethylene and cold signaling must be further verified. The results of the previous experiments with externally applied ACC and AVG revealed that under 4℃, ACC promoted the up-regulated expression of the PiERF1 gene (Fig. 9 a) but suppressed the expression of the PiDREB1A gene (Fig. 9 b) and the epistatic response was sensitive to cold; on the contrary, spraying of AVG suppressed the expression of the PiERF1 gene (Fig. 9 a), but promoted the expression of the PiDREB1A gene (Fig. 9 b), the epistasis showed better cold tolerance. piEIN3 was also co-regulated by ethylene and cold signaling, and ACC promoted the up-regulated expression of the PiEIN3 gene, while AVG suppressed its expression (Fig. 9 c). ACC and AVG were both inhibited the regular expression of PiETR1 , but the inhibitory effect of AVG was more evident (Fig. 9 d). In the time dimension, ACC treatment rapidly induced the expression of the cold-responsive gene PiERF1 , which was higher than the control at 4h. PiERF1 expression was down-regulated in cold stress within 24h after ethylene signaling inhibition and was lowest at 4h; this was indicated that the gene wa s susceptible to ethylene signaling, responded to ethylene and cold signaling at 4h, and was unable to be expressed in low temperature after ethylene signaling inhibition. After spraying ACC with AVG, PiDREB1A was cold-regulated for overall up-regulated expression. However, gene expression was suppressed in the ACC group relative to the control group after 4h and continued to be lower than the control group for the following 24h. The AVG group was more highly expressed at 8h than the control group and continued to be over-expressed during the following 24h. The expression pattern of this gene was consistent with previous studies, as a fast-response gene was highly expressed at 8h and then gradually decreased. However, after spraying AVG, PiDREB1A continued to be overexpressed during 24h of cold stress without a decreasing trend, suggested that this gene might be regulated by the ethylene signaling pathway in tandem with cold signaling at 8h. When ethylene signaling was interfered with, PiDREB1A lost its upstream inhibitory target for overexpression. It was opposite to the expression pattern of PiERF1 , and PiERF1 was activated by two signaling stimuli at 4h. PiDREB1A is regulated by cold signaling only at 4h and was activated by dual signaling at 8h. The former was activated by dual-signaling stimuli in response to an earlier activation than the latter. ACC treatment increased the expression of PiEIN3 , which reached a maximum and slowly decreased at 8h. As a potential upstream gene of PiERF1 , it responded rapidly to the stimulation of cold and ethylene signals, and the gene was suppressed by AVG treatment, with a continuous negative expression at 24h. The ethylene receptor gene PiETR1 expression increased by cold. However, ACC and AVG negatively regulated it, and the gene expression in the two experimental groups was close to or lower than that of the control group within 24h. The gene might be co-regulated by other factors. Therefore, we suggested that the ethylene signaling pathway is activated by cold upstream, and PiEIN3 was rapidly induced by 8h, which promotes the continuous up-regulation of PiERF1 expression within 24h and suppresses the downstream PiDREB1A expression, and that ethylene acts as a negative regulator during the cold response of P. indica , which might be the reason for the cold-intolerance of P. indica . 4 Discussion 4.1 Physiological responses of P. indica under cold stress and response to ethylene regulators The chlorophyll fluorescence parameter Fv/Fm can assess plant cold tolerance [ 40 – 41 ]. In the present study, cold stress resulted in decreased chlorophyll fluorescence parameters of P. indica , decreased photosynthetic efficiency, and an imbalance in plant energy supply. Exogenous spraying of the ethylene promoter ACC exacerbated photosystem II inhibition (PSII) inhibition, leading to leaf depletion and wilting. The inhibition of the ethylene pathway resulted in PSII recovery after cold stress. The experimental results indicated that inhibition of ethylene signaling would help P. indica to recover photosynthetic efficiency more quickly. It is in general agreement with previous findings in Bermuda grass ( Cynodon dactylon (L). Pers.), where the authors concluded that plants sprayed with ethylene inhibitors could achieve better cold hardiness as the duration of cold stress increased, as found from photosynthetic indicators [ 22 ]. The EL levels are commonly used to analyze the extent of cell membrane damage in plants under abiotic stress [ 3 ], and the greater the EL, the more severe the damage to the cell membrane. MDA is used as a lipid peroxidation parameter and as an indicator of stress mediated by the ROS system [ 42 ]. P. indica suffered cell membrane damage under cold stress, and EL was continuously elevated over 72h. Spraying ACC inhibited the ROS scavenging system and exacerbated the degree of membrane damage, leading to a sustained increase in MDA values during cold stress, which was not recoverable after normoxia. Spraying AVG in advance could maintain cellular homeostasis during cold stress, and the EL and MDA values were lower than those of the control group after room temperature incubation and could be recovered more quickly. At the beginning of low temperature stress, soluble sugar accumulation in plant cells increases with cold tolerance [ 43 ]. Early spraying of AVG resulted in higher levels of soluble sugars and thus higher defense against cold in advance, which was suppressed by ACC. This suggested that thermophilic plants may lack the ability to anticipate the onset of winter in advance and require artificial regulation to help them acquire cold tolerance. And that the ethylene inhibitor AVG positively affected plant cold resistance by increasing cell membrane stability. This may be related to ethylene signaling and plant development, and more evidence in other plants is needed to verify this. 4.2 The cold response mechanism of thermophilic plants P. indica may be negatively regulated by ethylene signaling Plant hormones are critical in various abiotic stresses [ 44 ]. In the present study, transcriptomic data was showed that genes involved in multiple phytohormone transduction signals were differentially expressed within 24h of cold stress. Among them, the most active transcription factor family members, ERFs, which are widely known to play critical roles in plant and abiotic stress [ 3 , 18 ] and downstream in response to ethylene signaling, were targeted. In Arabidopsis , ethylene signaling regulates freezing tolerance by repressing the expression of CBFs [ 45 ]. ERFs are regulated directly or indirectly by binding to the DREB1s promoter, which is positively regulated [ 46 ]. The positive effect of ethylene has also been demonstrated in large trees such as apple [ 19 ] and Poncirus trifoliata [ 18 ]. However, in thermophilic plants such as Bermuda grass [ 22 ], wheat ( Ocimum sanctum ) [ 47 ], and soybean [ 21 , 23 ], exogenous sprays of ethylene promoters negatively affected cold hardiness. In this experiment, after P. indica was sprayed with the ethylene promoter ACC and the inhibitor ACG, the expression of downstream response genes was similarly targeted to PiERF1 and PiDREB1A , and the crosstalk relationship between the two was manifested in the sense that ethylene signaling promoted the elevated expression of PiERF1 and suppressed the expression of PiDREB1A ; on the contrary, suppressed ethylene signaling resulted in the decrease of the expression of PiERF1 and the promotion of the PiDREB1A expression. Combined with the morphological expression of cold tolerance, this result indicated that P. indica might be negatively regulated by ethylene signaling, which generally agrees with previous findings in thermophilic plants such as wheat [ 47 ]. Does this imply that for thermophilic plants, ethylene signaling excites an antagonistic relationship between the downstream classical cold chain CBF/DREB-COR and some members of the ERFs family? Deeper mechanisms need to be supported by further evidence. Regardless, it is clear that P. indica receives ethylene and CBF/DREB-COR signaling pathways to co-regulate cold tolerance in the face of low-temperature stress in PiERF1 . 4.3 Other signaling pathways may simultaneously mediate P. indica cold response processes In the transcriptome KEGG enrichment, we were interested in the MAPK signaling pathway being heavily enriched. According to previous reports, MAPK cascade signaling mediates ethylene signaling under biotic or abiotic stresses in two ways: first, it regulated the downstream ACS6 gene through MAPK6, which was involved in the ethylene synthesis pathway; the second was that after activation of the ethylene signal, MKK9 or MPK3/6 is directly or indirectly regulated through the ETR1/RS1 receptor, which then promotes the involvement of EIN3 in early transcription downstream of ethylene in the nucleus [ 48 ]. Previously, in a cold response study of Arabidopsis thaliana , it was reported that MAPK3/6 negatively regulates Arabidopsis cold tolerance by phosphorylating ICE1 to destabilize it [ 49 ]. During the cold response of P. indica , many genes were induced to be expressed in MAPK signaling and ethylene signaling, including the previously mentioned ACS6 , ETR1 , EIN3 , and MAPK3 . Whether these MAPK signals are involved in the complex response to cold and ethylene in P. indica and how to crosstalk PiERF1 and PiDREB1A was still worthy of in-depth exploration. In addition to transcriptional regulation playing an essential role in cold-responsive stress, we detected that metabolic processes were also involved in cold response in GO enrichment. It coincides with the previous result that spraying the ethylene inhibitor AVG enhances plant defenses against the cold by increasing two metabolic pathways: soluble sugars and soluble proteins. SIMAPK3 was previously reported in tomato [ 50 ] to regulate cold tolerance by regulating the synthesis of its soluble sugars. Surprisingly, two MAPK3 genes were found to be expressed in response to cold in P. indica transcriptome DEGs analysis. From this, we speculate that MAPK signaling may exercise cold tolerance by regulating soluble sugars in plants. AVG was confer this function in advance, but further validation is needed to elucidate this. Meanwhile, in KEGG enrichment, it was found that in addition to MAPK signaling, the circadian rhythms-plant pathway was also significantly up-regulated in expression. However, this may be caused by the transfer of plants to all-day dark conditions during cold stress, and all genes in this pathway were excluded in this study to avoid the results being influenced by circadian rhythms-plant. 5 conclusion In this study, we found that ethylene signaling leads to cold sensitivity in thermophilic plants P. indica , as evidenced by reduced photosynthetic efficiency, enhanced membrane permeability, weakened ROS scavenging mechanism, and inability to maintain cellular homeostasis, suggested that P. indica might be negatively regulated by ethylene signaling. PiERF1 and PiDREB1A , two downstream genes were related to ethylene signaling, and co-regulated by ethylene and cold signals (Fig. 10 ). The former was expressed earlier than the latter by double signaling. The expression of the two genes had a reciprocal relationship, which was explicitly reflected in the fact that the expression of PiDREB1A was up-regulated after the inhibition of ethylene, which gave P. indica cold tolerance, and PiERF1 might be one of the critical genes leading to cold sensitivity. one of them. These results provide some evidence for the effect of ethylene signaling on plants under low-temperature stress and provide a solution for overwintering thermophilic plants in the open field. Declarations Ethics approval and consent to participate Not applicable. Ethical consideration The wild seed collection and the trial conducted in this study were in no violation of any legislation, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The plants were planted in the nursery of Sichuan Agricultural University, Chengdu, China. Consent for publication Not applicable. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials No data was used for the research described in the article. Funding This work is supported by grants from the Sichuan Province Science and Technology Support Program (2021YFYZ0006). Authors' contributions Z-A .Z. and Y-R .L.: conceived and designed the experiments. Z-A .Z., Y-R .L., T .L., C-L .L., Q-X .Z. , X .L., L-J. Y., and J-N .L.: conducted the experiments and performed data analysis, interpretation, and investigation. Z-A .Z. wrote the manuscript. S-P .G.: revised the original manuscript. All authors have read and approved the final manuscript. Acknowledgements We acknowledge the support provided by our labs at Sichuan Agricultural University. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4448738","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309983857,"identity":"b79911e6-ca59-4c15-a945-275bd4aa9d5c","order_by":0,"name":"Zi-An Zhao","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zi-An","middleName":"","lastName":"Zhao","suffix":""},{"id":309983859,"identity":"0b878b12-3d33-41fc-8595-0d954c394201","order_by":1,"name":"Yi-Rui Li","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Rui","middleName":"","lastName":"Li","suffix":""},{"id":309983861,"identity":"5797e8a6-109f-498a-926b-ac7668605b0f","order_by":2,"name":"Ting Lei","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Lei","suffix":""},{"id":309983862,"identity":"5654dc01-bfb2-4119-9812-99ae8d4fdd1e","order_by":3,"name":"Cai-Lei Liu","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Cai-Lei","middleName":"","lastName":"Liu","suffix":""},{"id":309983864,"identity":"f971f54b-f68d-4931-a6ed-4ebd83c74b91","order_by":4,"name":"Qing-Xiao Zeng","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qing-Xiao","middleName":"","lastName":"Zeng","suffix":""},{"id":309983866,"identity":"60cb0c3a-9e42-4888-a513-102aca94bae2","order_by":5,"name":"Xuan Liu","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Liu","suffix":""},{"id":309983868,"identity":"59d98ec9-bb65-478b-a79b-840835952f5a","order_by":6,"name":"Li-Juan Yang","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Li-Juan","middleName":"","lastName":"Yang","suffix":""},{"id":309983869,"identity":"724357af-b6fe-4b81-b348-a03e0ed7f829","order_by":7,"name":"Jia-Ni Li","email":"","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Ni","middleName":"","lastName":"Li","suffix":""},{"id":309983870,"identity":"966771ec-993e-41ca-9373-19b42074e84d","order_by":8,"name":"Su-Ping Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBADHgYG5gMMCRCOAbFa2BJI0wLSBVeJX4u8++EDjD/b7siY86/5/OFBjV1iA3vzNgmGmjs4tRieSUtg5m17xmM54+02iYRjyYkNPMfKJBiOPcOtpSHHgJmx7TCPwY2z2xgSG5gTGyRyzCQYGw7j1tL/xgDoMJCWM48/JDbUJzbIv8GvRV4ix4CBF6TlfA+DRGLDYaAtPPi1GEg8S2DmOfcMaAubGdAvx43beNKKLRKO4bGlP/kA44+yO/YG5w8//vijplq2n/3wxhsfavDYcoCB/QcDwwEGBokEiAgbiEjAqQFoSwOYAmrhP4BH2SgYBaNgFIxoAABQg1d5HMXtiwAAAABJRU5ErkJggg==","orcid":"","institution":"College of Landscape Architecture, Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Su-Ping","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-05-20 11:15:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4448738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4448738/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57824872,"identity":"0bc6b49c-2fb3-4f47-80d2-b6c5ab79718f","added_by":"auto","created_at":"2024-06-06 06:46:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134756,"visible":true,"origin":"","legend":"\u003cp\u003etwo-year-old \u003cem\u003eP. indica \u003c/em\u003ewithered by cold stress during flowering. (a) Localized effect of flowering in November; (b) It is growing normally during the flowering of \u003cem\u003eP. indica\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e; (c) Low temperatures severely affect full petal expansion, resulting in a severely shortened flowering period.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/503f7ca36ba91502639b4b8d.jpeg"},{"id":57826306,"identity":"e395c603-6ea1-4a78-b81b-fea8d4f497b2","added_by":"auto","created_at":"2024-06-06 07:10:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42431,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll fluorescence changes in \u003cem\u003eP. indica\u003c/em\u003e leaves at 4℃ low temperature. (a) Chlorophyll fluorescence imaging; (b) Fv/Fm values of chlorophyll fluorescence parameters. Error bars represent ± SE (n= 9).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/63bf32172a4cc8afba895bf6.jpeg"},{"id":57824873,"identity":"ff5d740a-264c-40a5-8c98-a0842d1236a8","added_by":"auto","created_at":"2024-06-06 06:46:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96883,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of physiological and biochemical indicators of \u003cem\u003eP. indica\u003c/em\u003e leaves treated at 4℃. (a)EL determination of \u003cem\u003eP. indica\u003c/em\u003e leaves' EL; (b) CAT activity; (c) PODnactivity; (d) SOD activity; (e) malondialdehyde content; (f) soluble sugar content. Error bars represent ± SE (n= 3), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/7618c5bf21b43b94fb9127bb.jpeg"},{"id":57825761,"identity":"1487eeb3-5a49-4851-9823-101710a9ba33","added_by":"auto","created_at":"2024-06-06 07:02:09","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49623,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of precursor substance ACC content and two key enzyme activities in the ethylene synthesis pathway of \u003cem\u003eP. indica\u003c/em\u003e under cold stress. (a) Endogenous ACC content; (b) ACS activity; (c) ACO activity. Error bars represent ± SE (n= 3), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/2402696f5066cb643e5771ee.jpeg"},{"id":57825298,"identity":"9aba76d1-5ea1-4bc4-aa2d-dc7d6303f071","added_by":"auto","created_at":"2024-06-06 06:54:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":206634,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of RNA-seq results with qPCR trend of randomly selected DEGs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: There are 7 up-regulated and 5 down-regulated expressed genes in the graph; the X-axis represents the time (h) of low-temperature treatment, the left Y-axis displays the corresponding RNA-seq expression data (blue histogram), and the right Y-axis displays the relative gene expression level by qPCR (black dash line). Bars represent ± SE (n=3) and broken lines represent ± SE (n=9).\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/c8887014eb620a4d87e112bc.jpeg"},{"id":57825297,"identity":"1633f997-d2a2-4f73-98b1-52cf806544b8","added_by":"auto","created_at":"2024-06-06 06:54:09","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90518,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment and KEGG enrichment plots of differentially expressed genes in response to cold in\u003cem\u003e P. indica. \u003c/em\u003e(a) GO pathway enrichment results, the horizontal coordinate was the GO classification, the vertical coordinate was the percentage of the number of genes on the left, and the right was the number of genes; (b) KEGG pathway enrichment results at 0h vs. 24h, each circle in the figure represents a KEGG pathway, the vertical coordinate was indicated the pathway name and the horizontal coordinate was the enrichment factor, indicating the ratio of the proportion of genes annotated to a specific pathway to the proportion of genes annotated to that pathway among all genes in the differentially expressed genes. The horizontal coordinate was the Enrichment Factor, which was indicated the ratio of the proportion of genes annotated to a specific pathway in the differential genes to the proportion of genes annotated to that pathway in all genes.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/4eeee0c40548ab0027d0b419.jpeg"},{"id":57824881,"identity":"6129ebe2-5039-4550-abcb-01eedcdcc483","added_by":"auto","created_at":"2024-06-06 06:46:09","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":131800,"visible":true,"origin":"","legend":"\u003cp\u003eTwo essential pathways of ethylene signaling and DREB-COR in\u003cem\u003e P. indica\u003c/em\u003e in response to cold stress. (a) Statistics on the number of transcription factors during cold response in \u003cem\u003eP. indica\u003c/em\u003e within 24h; (b) correlation analysis between \u003cem\u003ePiERF1\u003c/em\u003e and physiological indicators of cold response; (c) q-PCR results of some essential genes in the ethylene signaling pathway, ± SE (n=9); (d) FPKM values of some essential genes in the DREB-COR pathway by RNA-seq, ± SE (n=3).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/25b4281f984f09463ec724bf.jpeg"},{"id":57824876,"identity":"12d59018-9946-425d-badc-108f1fc41544","added_by":"auto","created_at":"2024-06-06 06:46:09","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":130139,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in physiological and biochemical indicators of \u003cem\u003eP. indica\u003c/em\u003e after exogenous spraying of RO, ACC, and AVG. (a) Leaf phenotypes before, after, and after 72h of recovery from different treatments of cold stress; (b) Chlorophyll fluorescence imaging before, after, and after 72h of recovery from different treatments of cold stress; (c) Leaf Fv/Fm values, EL, MDA content, soluble protein, soluble sugar, ACC content, ACO enzyme activity vs. ACS enzyme activity within 72h of different treatments and after 72h of recovery. ± SE (n=3).\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/5928e70ac65f83c919de3413.jpeg"},{"id":57827109,"identity":"f74f17c8-efe5-4d8d-b429-8f381ab164a0","added_by":"auto","created_at":"2024-06-06 07:18:09","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":80004,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of downstream specific genes after spraying ethylene promoters and inhibitors under cold stress. (a) \u003cem\u003ePiERF1\u003c/em\u003eexpression; (b) \u003cem\u003ePiDREB1A\u003c/em\u003e expression; (c) \u003cem\u003ePiEIN3\u003c/em\u003e expression; (d) \u003cem\u003ePiETR1\u003c/em\u003eexpression. Yellow, orange, and blue histogram bars represent the expression levels of genes after spraying RO, ACC, AVG respectively. ± SE (n=9).\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/16b67381596351e9245fe2ee.jpeg"},{"id":57825300,"identity":"cd1d1121-0d10-4326-80db-0fbe7d964c97","added_by":"auto","created_at":"2024-06-06 06:54:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":48122,"visible":true,"origin":"","legend":"\u003cp\u003eModel diagram of cold stress and ethylene signaling pathway in\u003cem\u003e P. indica. \u003c/em\u003e\u003cstrong\u003eNote:\u003c/strong\u003eUnder 4°C ACC inhibited the expression of\u003cem\u003e PiDREB1A\u003c/em\u003e and promoted the up-regulated expression of \u003cem\u003ePiERF1\u003c/em\u003e, leading to cold sensitivity of plants. AVG increased cold tolerance by inhibiting ACC accumulation. Yellow arrows represent the changes under cold stress, orange arrows represent the changes under the compound effect of ethylene and cold signaling, and blue arrows represent the changes under the compound effect of ethylene inhibition and cold.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/ac6b32fde41a819cf5b8bbe6.png"},{"id":63349088,"identity":"e91fbc6e-7a93-4015-b255-b7f649df2b63","added_by":"auto","created_at":"2024-08-27 08:01:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2050885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/06d0e452-3ce2-4353-8c68-7adc11eb64d5.pdf"},{"id":57824875,"identity":"77a979ff-b790-490c-8a50-ed2aaa81ab0c","added_by":"auto","created_at":"2024-06-06 06:46:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20658,"visible":true,"origin":"","legend":"","description":"","filename":"5Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4448738/v1/0f24e92a391f9bc4a5eb50ec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ERF1 of Plumbago indica L. receives ethylene signaling and regulates cold tolerance together with the DREB-COR pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWith the frequent occurrence of global environmental extremes, low-temperature stress poses a severe test for sequestering organisms, especially thermophilic plants, which must face unfavorable growing conditions [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. When fall arrives, plants with cold hardiness undergo domestication to overwinter successfully [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Some thermophilic plants with higher ornamental value cannot cope with extremely low temperatures, such as such as tropical water lilies (\u003cem\u003eNymphaea rubra\u003c/em\u003e), Chrysanthemum morifolium (\u003cem\u003eDendranthema grandiflorum\u003c/em\u003e var. Jinba), forage ( \u003cem\u003eMedicago sativa\u003c/em\u003e L.) [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and \u003cem\u003eP. indica\u003c/em\u003e, the material of this paper, where low temperatures have become a key constraint to successful introductions to the north or higher latitudes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. indica\u003c/em\u003e is an evergreen perennial herb native to the tropics. Because of both ornamental and medicinal functions [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], it has excellent prospects for flower commercial development. During the previous introduction process, we found that \u003cem\u003eP. indica\u003c/em\u003e was susceptible to wilt and even death in the open field in winter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was coincided with the flowering period, seriously affected the ornamental value. In order to clarify the mechanism of low-temperature effects on \u003cem\u003eP. indica\u003c/em\u003e, members of the ERF subfamily of the Apetala 2/ Ethylene-responsive factor (AP2/ERF) family of transcription factors were found to be most induced in the process as a result of transcriptome sequencing. In past studies, a large number of transcription factors had been identified as significant regulators associated with cold stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. AP2/ERF, as was one of the most prominent transcription factor families in plants, widely regulates plant biotic and abiotic stresses, such as heavy metals, temperature extremes, and drought [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], among others. Meanwhile, it is also a key transcription factor in the significant response to the ethylene signaling pathway in plant stress response [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among the five subfamilies under AP2/ERF, AP2, ERF, DREB, RAV, and Soloist [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the CBF/DREB-COR-mediated cold-responsive pathway, as the classical cold chain, had been much reported in plants [7, 17]; however, ERFs, members of the same family, were still not been reported in cold resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the correlation between the two subfamily during cold response were not known.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEthylene was played a vital role in stress tolerance as a plant growth-regulating hormone that mediates the plant cold response in a species-dependent manner [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ACC was a prerequisite substance for ethylene synthesis, and was often applied in the field instead of gaseous ethylene. Synthesized ethylene was bound to the ETR1 receptor, blocked the triple response CTR1 phosphorylation and inhibited the downstream positive regulator \u003cem\u003eEIN2\u003c/em\u003e. \u003cem\u003eEIN2\u003c/em\u003e was activated to induce the transcription factor \u003cem\u003eEIN3/EIL\u003c/em\u003e, which directly induced the \u003cem\u003eERF\u003c/em\u003e transcription factor, initiated the expression of downstream genes in response to ethylene signaling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In more cold-tolerant plants, such as apples, \u003cem\u003eERF1B\u003c/em\u003e was up-regulated in expression by ethylene regulation. It acted on DRE components to promote downstream \u003cem\u003eCBF1\u003c/em\u003e (\u003cem\u003eDREB1B\u003c/em\u003e) expression and enhance cold tolerance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Whereas in some thermophilic plants such as wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Bermuda grass (\u003cem\u003eCynodon dactylon\u003c/em\u003e (L). Pers.) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], ACC was thought to regulate their cold tolerance negatively. In 2019, Robison found in soybeans that ACC repressed the expression levels of \u003cem\u003eGmCBF/DREB1s\u003c/em\u003e by regulating \u003cem\u003eGmEIN3\u003c/em\u003e and that antagonism of the ethylene pathway may be one of the reasons for cold acclimation in soybeans [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The above two opposite results were shown that the specificity of ACC in regulating plant cold tolerance, further were indicated the complex crosstalk relationship between ethylene and cold signaling pathway signals. Then, for other thermophilic plants, how does the cold stress response crosstalk specifically with ethylene signaling, and does the ethylene signaling pathway contribute to cold sensitivity in thermophilic plants? More evidence supporting which downstream gene expression ACC acts on in response to cold stress is needed.\u003c/p\u003e \u003cp\u003eIn the present study, we analyzed the transcriptome of \u003cem\u003eP. indica\u003c/em\u003e by cold stress treatment. We found that the AP2/ERF-ERF was the most annotated, and one of its members designated \u003cem\u003ePiERF1\u003c/em\u003e (\u003cem\u003eUnigene_83531\u003c/em\u003e) in the present study, was detected in a full-length sequence. It was more strongly induced (highest degree induced at 24h, |log\u003csub\u003e2\u003c/sub\u003eFC|=2.46). Meanwhile, ACC content was significantly increased in \u003cem\u003eP. indica\u003c/em\u003e leaves after cold stress. It was hypothesized that PiERF1 might mediate the cold response of \u003cem\u003eP. indica\u003c/em\u003e by participating in the ethylene signaling pathway and directly or indirectly affecting the expression of the critical cold response gene \u003cem\u003ePiDREB1A\u003c/em\u003e (\u003cem\u003eUnigene_05023\u003c/em\u003e). Further pretreatment of \u003cem\u003eP. indica\u003c/em\u003e leaves were sprayed with 50 \u0026micro;mol/L ACC with the ethylene inhibitor AVG, which was revealed that spraying ACC resulted in reduced cold tolerance and activated downstream up-regulation of \u003cem\u003ePiERF1\u003c/em\u003e expression and down-regulation of \u003cem\u003ePiDREB1A\u003c/em\u003e expression; conversely, sprayed AVG enhanced cold tolerance of \u003cem\u003eP. indica\u003c/em\u003e, resulted in down-regulation of \u003cem\u003ePiERF1\u003c/em\u003e and up-regulation of \u003cem\u003ePiDREB1A\u003c/em\u003e. These results were revealed that the increase of the ethylene precursor ACC in \u003cem\u003eP. indica\u003c/em\u003e induced the up-regulated expression of \u003cem\u003ePiERF1\u003c/em\u003e, suppressed the expression level of \u003cem\u003ePiDREB1A\u003c/em\u003e, and increased its cold sensitivity under the low-temperature environment in winter. This negative regulation mechanism of cold tolerance explained the senescence or even death of \u003cem\u003eP. indica\u003c/em\u003e overwintering leaves in the open field. The results could further understand the crosstalk network relationship between ethylene biosynthesis and the CBF/DREB-COR cold-response pathway, which supported the previous conclusion of negative regulation of cold tolerance in thermophilic plants by ethylene and clarifies the correlation between ACC action on the downstream genes \u003cem\u003ePiERF1\u003c/em\u003e and \u003cem\u003ePiDREB1A\u003c/em\u003e, which could provide specific reference for the introduction of thermophilic plants to the north and the development of open-ground over-wintering protection programs.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material and treatment\u003c/h2\u003e \u003cp\u003eThe experimental material was 2-year-old asexual seedlings of \u003cem\u003eP. indica\u003c/em\u003e. The plants were planted in the nursery of Sichuan Agricultural University, Chengdu, China. Before the experimental treatments, 54 pots of healthy potted plants with uniform growth were selected, pruned, and moved into an RXZ-500D-LED type light incubator (Jiangnan instrument, Ningbo, China). Among them, the light time was 16/8h, the temperature was 30/25℃, light intensity was 80%, the humidity was 60%, and the substrate was mixed (peat soil: garden soil: vermiculite: 3:2:1). Uniform routine water and fertilizer management was used to pre-cultivate the plants for 60 d. During this period, to place the error caused by the placement position, the position of the large pots was changed weekly in a clockwise direction to ensure the same environmental conditions.\u003c/p\u003e \u003cp\u003eThe plants were then divided into two groups of 27 pots each, which were used for two sets of experiments: direct exposure to low temperature at 4℃ or pretreatment with phytohormones followed by 4℃ treatment. The plants were incubated in total darkness to avoid the compound stress of light on them, and they were photoinhibited [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For the low-temperature treatment, plants were exposed to 4℃ for 72h and were sampled at 0h, 1h, 4h, 8h, 12h, 24h, and 72h, respectively. For the hormone group treatments, 27 pots of plants were divided into three groups and the leaves of the experimental group were sprayed with 50 \u0026micro;mol/L ACC and AVG, respectively, and the leaves of the control group were sprayed with RO water (reverse osmosis water) at intervals of 2d. After three consecutive sprays, the plants were exposed to cold stress for 72h at 4℃, cultured for 72h after cold stress, and restored to customary conditions, respectively. Samples were taken at 0h, 1h, 4h, 8h, 12h, 24h, 72h and 72h after recovery from cold stress. Three biological replicates were taken for each group, and each experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Measurement of physiological and biochemical indicators\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Chlorophyll fluorescence parameters and their visualization\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eWhole-leaf fluorescence imaging was scanned using IMAGING-PAM (WALZ, Germany); chlorophyll fluorescence parameters were determined by the Handy PEA Plant Efficiency Analyzer (Hansatech Instruments Ltd, UK), and the key metrics, Fv/Fm, were calculated using the instrument's accompanying PEA Plus 1.12 software package. The top 3\u0026ndash;5 leaves of the branches were selected for determination. Three biological replicates were tested for each sample, and each experiment was repeated three times.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Electrolyte leakage, MDA content, and reactive oxygen species scavenging system determination\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eElectrolyte leakage (EL), MDA, SOD, POD, CAT, and soluble sugar were measured concerning the methods of the previous authors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] using the relevant kits (Nanjing Jiancheng Bioengineering Institute, China). For sampling, the branches' top 3\u0026ndash;5 leaves were taken. Three biological replicates were tested for each sample.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of ACC content and ACS and ACO enzyme activities\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eReferring to the method of Yuan [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], plant ethylene precursor (ACC), plant ACC synthase (ACS), and plant ACC oxidase (ACO) were determined using ELISA kits (MM-36135O1 for ACC content; MM-33691O2 for ACS activity; MM-33691O2 for ACO activity; Jiangsu Meimian Industrial Co., Ltd, China) for determination. For sampling, the apical 3\u0026ndash;5 leaves were taken. Three biological replicates were tested for each sample.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA extraction, RNA-Seq, library construction and gene annotation\u003c/h2\u003e \u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eYoung leaves of \u003cem\u003eP. indica\u003c/em\u003e were sampled at 4℃ under cold stress (see 2.1 for details) for 0h, 1h, 4h, 8h, 12h, and 24h (label the six-time points as Z1-Z6, respectively). According to the instructions, the total RNA of \u003cem\u003eP. indica\u003c/em\u003e was extracted using RNAprep Pure Plant Kit (TIANGEN, China). RNA purity was ensured by 1% agarose gel electrophoresis. Qubit 2.0 and Agilent 2100 systems were used to detect the library and ensure the reliability of the results. cDNA libraries were sequenced using the Illumina NovaSeq high-throughput sequencing platform based on Sequencing By Synthesis (SBS) technology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The Unigene sequences were compared with public databases NR, Swiss-Prot, GO, COG (Clusters of Orthologous Groups), KOG (euKaryotic Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes) databases were compared using the BLASTx algorithm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After predicting Unigene's amino acid sequence, Unigene's annotation information was obtained using HMMER software compared with the Pfam database. Metabolic pathways of Unigenes were determined according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe Unigene sequences were compared with the databases NR [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Swiss-Prot [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], COG [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], KOG [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], eggNOG4.5 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], KEGG [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] comparisons. interProScan [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] analyzed the GO [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] Orthology results of the new genes using the InterPro-integrated databases and used KOBAS [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] to obtain the Unigene's KEGG Orthology results in KEGG, and used HMMER [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] software to compare the amino acid sequences of the predicted completed Unigene with the Pfam [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] database to obtain the annotation information of Unigene.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RT- qPCR validation\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eQuantitative real-time PCR (qRT-PCR) analysis was accomplished using an iCycler iQ5 system (Bio-Rad, Hercules, CA, USA) and SYBR Green PCR Master Mix (transgene), with each reaction containing 5 \u0026micro;L of 2x QuantiFast SYBR Green PCR Each reaction consisted of 5 \u0026micro;L of 2x QuantiFast SYBR Green PCR Master Mix (SYBR Green Pro Taq HS qPCR Kit, Accurate Biotechnology, China), 1 \u0026micro;L of cDNA, 2 \u0026micro;M of gene-specific forward primer, and 2 \u0026micro;M of gene-specific reverse primer, and the final volume was spiked up to 10 \u0026micro;L with sterile water. The PCR reaction was carried out at 95℃ for 3 min, 95℃ for 15 min, and 95℃ for 10 min. for 3 min; 95℃ for 15 s, 60℃ for 20 s, and the above steps were repeated for 40 cycles. The expression of EDGs was calculated after normalization using the \u003csup\u003e2\u0026minus;ΔΔCT\u003c/sup\u003e algorithm with \u003cem\u003ePaActin\u003c/em\u003e as the internal reference gene. Detailed information about the qRT-PCR primers is provided in Supplementary Table\u0026nbsp;1(\u003cb\u003eTab. S1\u003c/b\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data analysis\u003c/h2\u003e \u003cp\u003eThe results of a representative experiment are shown. The values presented are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE and analyzed via IBM SPSS Statistics statistical software (Version 25.0). Draw and analyze using Origin2020. Statistical differences were compared with a one-way analysis of variance (ANOVA) based on LSD\u0026rsquo;s multiple range test at significance levels of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Low temperature stress reduces photochemical efficiency of \u003cem\u003eP. indica\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eContinuous exposure to low temperatures can cause some damage to plant leaves and reduce their photosynthetic efficiency. The chlorophyll fluorescence visualization of \u003cem\u003eP. indica\u003c/em\u003e leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) was showed that the chlorophyll fluorescence of \u003cem\u003eP. indica\u003c/em\u003e gradually decreased with the increase of low-temperature exposure. Meanwhile, the response of plant photochemical efficiency to low temperatures can be effectively shown by Fv/Fm photosynthetic parameters [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Under 4℃ stress, chlorophyll fluorescence significantly was decreased to 0.64 at 4h, rebounded to 0.76 after 8h, and then decreased to 0.73 after 24h and continued to stabilize in the following 72h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), but significantly decreased relative to the control group. These phenomena was indicate that \u003cem\u003eP. indica\u003c/em\u003e suffered some degree of leaf damage due to reduced photosynthetic efficiency under cold exposure at 4℃. Although some plants could gradually adapt and recover from cold stress, \u003cem\u003eP. indica\u003c/em\u003e leaves continue to be irreversibly damaged.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003ehas enhanced membrane permeability under low-temperature stress, possessing stronger oxidase activity and elevated soluble sugar content\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe larger the EL, the more severe the plant damage. The experimental results were showed that the EL of \u003cem\u003eP. indica\u003c/em\u003e gradually increased with the increase in the duration of cold exposure and was significantly higher at 72h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).When plants suffer from adversity injury, at this time, the reactive oxygen scavenging mechanism is triggered. Malondialdehyde (MDA) is one of the products of cell membrane lipid peroxidation after excessive accumulation of reactive oxygen radicals in plants. When the antioxidant mechanism in \u003cem\u003eP. indica\u003c/em\u003e could not maintain the balance between output and scavenging, MDA increased significantly, significantly elevated, and peaked at 8h. However, with the initiation of the protective enzyme system, the activities of the CAT enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), POD enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and SOD enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) in the scavenging system of the ROS system were enhanced. Its membrane lipid peroxidation was alleviated to a certain extent after 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). During low-temperature stress, sugar metabolism undergoes phase changes. In \u003cem\u003eP. indica\u003c/em\u003e, the soluble sugar content increased significantly at 8h during the early stage of cold stress, peaked at 12h, decreased gradually, and increased significantly at 72h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003ePhysiological and biochemical data was indicated that \u003cem\u003eP. indica\u003c/em\u003e could enhance its cold tolerance through ROS scavenging enzyme activity system and sugar metabolism pathway under low-temperature stress. However, the EL gradually increased with the increase of cold stress time, indicating that the damage to the membrane system was intensified and the \u003cem\u003eP. indica\u003c/em\u003e leaves were severely damaged.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Low-temperature stress increases ACC content and ACS and ACO activities in \u003cem\u003eP. indica\u003c/em\u003e leaves\u003c/h2\u003e \u003cp\u003eAs an essential substrate in the ethylene biosynthesis pathway, ACC is commonly used to study cold stress and ethylene signaling pathway models. Therefore, we examined the dynamic changes in ACC content. Under cold stress, the ACC content in \u003cem\u003eP. indica\u003c/em\u003e leaves was increased with time, significantly elevated from 94.27 nmol/L at 0 h to 132.93 nmol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which was 1.41 times higher than the initial level. Meanwhile, the enzyme activities of ACC synthase ACS and ACC oxidase ACO were also heavily induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c), with ACS activity significantly was elevated from 5.33 U/L to 8.08 U/L at 0 h, and ACO activity significantly was elevated from 268.68 U/mL to 383.80 U/mL at 0 h. The former was elevated by 1.51 times, while the latter was elevated by 1.42 times. It was indicated that the ethylene biosynthesis pathway was induced to be activated in cold stress, one of the critical pathways for cold response in \u003cem\u003eP. indica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RNA-seq sequencing reveals changes in the ethylene pathway of \u003cem\u003eP. indica\u003c/em\u003e under cold treatment\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Validation of RNA-seq results by quantitative real-time RT-PCR (qPCR)\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eA cDNA library was constructed from equal amounts of RNA extracted from \u003cem\u003eP. indica\u003c/em\u003e leaves subjected to 4℃. The transcriptome sequencing of 18 samples was finally completed, and a total of 128.88 Gb Clean Data was obtained, and the Clean Data of each sample reached 5.73 Gb, with the percentage of Q30 bases at 93.42% and above (\u003cb\u003eTab. S2\u003c/b\u003e). A total of 90,725 Unigenes were obtained after assembly, among which 31,073 Unigenes were above 1kb in length. After obtaining high-quality sequencing data, the sequence was assembled using Trinity. A total of 90,725 Unigenes were obtained from the assembly, and the N50 of Unigene was 2963, which showed high assembly integrity. The Clean Data of each sample was compared with the assembled Transcript or Unigene libraries, and the statistics of the comparison results were shown in \u003cb\u003eTab. S2\u003c/b\u003e. The statistical results indicate that the transcriptome data are sufficient and valid for the following analysis step.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTwelve randomly selected DEGs were subjected to qPCR for transcript abundance to verify that the RNA-seq results were accurate. There were 7 Unigene data up-regulated expression and 5 down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The qPCR results of the tested genes \u003cem\u003eUnigene 30052\u003c/em\u003e, \u003cem\u003eUnigene 69681\u003c/em\u003e and \u003cem\u003eUnigene79700\u003c/em\u003e, \u003cem\u003eUnigene 11281, Unigene40972\u003c/em\u003e were generally consistent with the changes in the RNA-seq data. \u003cem\u003eUnigene08110\u003c/em\u003e, \u003cem\u003eUnigene64040\u003c/em\u003e, \u003cem\u003eUnigene03061\u003c/em\u003e, \u003cem\u003eUnigene82860\u003c/em\u003e, \u003cem\u003eUnigene 43750\u003c/em\u003e, \u003cem\u003eUnigene 44592\u003c/em\u003e, and \u003cem\u003eUnigene 51682\u003c/em\u003e. These seven genes were weakly up- or down-regulated in expression within 4h, which may have led to a slight bias in the q-PCR test results, but the overall expression changes were consistent. This result was validated the reliability of the RNA-seq data and supports further bioinformatics analysis.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eThere are 7 up-regulated and 5 down-regulated expressed genes in the graph; the X-axis represents the time (h) of low-temperature treatment, the left Y-axis displays the corresponding RNA-seq expression data (blue histogram), and the right Y-axis displays the relative gene expression level by qPCR (black dash line). Bars represent\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;3) and broken lines represent\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;9).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4.2 GO and KEGG Enrichment Reveals Cold Stress Response is Closely Linked to Transcriptional Regulation and Hormonal Signaling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe BLAST parameter E-value\u0026thinsp;\u0026le;\u0026thinsp;1e-5 and the HMMER parameter E-value\u0026thinsp;\u0026le;\u0026thinsp;1e-10 were selected to obtain 41,111 Unigenes with annotated information in the total database (\u003cb\u003eTab. S3\u003c/b\u003e). The GO database categorized the annotated 33,538 Unigenes into 45 functional groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), which were further enriched into three GO level II functions, including \"cellular components,\" \"molecular functions,\" and \"biological processes.\" The top three \"cellular components\" were \"cellular anatomical entity (19589)\", \"intracellular (11698)\" and \"protein-containing complex (3515)\"; The top three \"molecular functions\" were \"structural domain binding (16993)\", \"catalytic activity (15944)\" and \"transcription regulator activity (1112)\"; the top three \"biological process\" were \"cellular process (18945) \", \"metabolic process (16477)\" and \"biological regulation (5812)\". The enrichment degree of Pathway was analyzed in KEGG using the Enrichment Factor and the significance of enrichment was calculated using the hypergeometric test. Analysis shows that in the 0h vs. 24h group, the most significant KEGG enrichment pathways are \"Plant hormone signal transmission,\" \"Plant pathway interaction,\" \"MAPK signaling pathway - plant,\" and \"Circadian rhythm - plant\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). By ranking GO and KEGG, it was not difficult to find that \u003cem\u003eP. indica\u003c/em\u003e cold stress response was closely related to transcriptional regulation and hormone signaling, which were also coincided with our previous finding of increased ACC content, ACO and ACS enzyme activities in the phytohormone ethylene synthesis pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4.3 Differential expression gene annotation of ethylene response factors PiERF1 and PiDREB1A involved in\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003ecold response\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTranscription factor prediction analysis by BMKCloud (BMKCloud) was showed that AP2/ERF transcription factors were induced in the most significant number (108) during the cold response of \u003cem\u003eP. indica\u003c/em\u003e within 24h, suggested that transcription factors ERFs play an essential role in the molecular mechanism of the cold response of \u003cem\u003eP. indica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Analysis of the differentially expressed ERFs genes in the four treatment groups of 0h vs 4h, 0h vs. 8h, 0h vs 12h and 0h vs 24h revealed that the \u003cem\u003eUnigene_83531\u003c/em\u003e gene (named \u003cem\u003ePiERF1\u003c/em\u003e) was significantly up-regulated in response to cold stress, with |log\u003csub\u003e2\u003c/sub\u003eFC| reaching 2.46 at 24h. Combined with the correlation matrix analysis, \u003cem\u003ePiERF1\u003c/em\u003e was found to have a common relationship with several colds. In combination with the correlation matrix analysis, \u003cem\u003ePiERF1\u003c/em\u003e was positively correlated with several physiological indicators related to cold stress, including ACC content, ACO and ACS activity. It was hypothesized that \u003cem\u003ePiERF1\u003c/em\u003e might be regulated by the ethylene precursor ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) and positively regulated by cold stress.\u003c/p\u003e \u003cp\u003eIn the ethylene signaling pathway, included \u003cem\u003eETR1\u003c/em\u003e (\u003cem\u003eUnigene 12465\u003c/em\u003e), \u003cem\u003eEIN3\u003c/em\u003e (\u003cem\u003eUnigene 53149\u003c/em\u003e) with the ACC (1-aminocyclopropane-1-carboxylic acid) synthase gene, \u003cem\u003eACS6\u003c/em\u003e (\u003cem\u003eUnigene 81360\u003c/em\u003e), as well as the upstream genes of \u003cem\u003eACS6\u003c/em\u003e, \u003cem\u003eEIN3/EIN1\u003c/em\u003e, \u003cem\u003eMPK3 (Unigene 78362\u003c/em\u003e, \u003cem\u003eUnigene 64590\u003c/em\u003e), both responded positively to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). In the DREB-COR pathway, \u003cem\u003ePiDREB1A\u003c/em\u003e, a fast cold-responsive gene, was the highest expression at 8h, followed by a gradual decrease. The \u003cem\u003eCOR\u003c/em\u003e gene (\u003cem\u003eUnigene 28684\u003c/em\u003e) was regulated downstream by \u003cem\u003ePiDREB1A\u003c/em\u003e, with a gradual up-regulation of its expression at 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), which were regulated the genes of the plant group to resist cold stress. The results were indicated that both key pathways, ethylene signaling and DREB-COR, responded to cold stress, but the crosstalk between them needs further exploration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e3.5 Evidence of negative regulation of cold hardiness of\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003eby exogenous spraying of ethylene promoter ACC and inhibitor AVG by ethylene signaling\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eP. indica\u003c/em\u003e leaves were sprayed with 50 \u0026micro;mol/L ACC and AVG respectively (control group sprayed with RO water), and it was found that the ACC group was more sensitive to cold relative to the control group, and the foliage crumpling was evident at 72h of 4℃ stress, and the leaves were dry and severely damaged after 72h of recovery incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Chlorophyll fluorescence parameters were greatly reduced, and the photochemical efficiency was significantly inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eb); at the same time, ACC increased EL and membrane permeability; ROS scavenging mechanism was weakened, resulting in an increase in MDA, which caused \u003cem\u003eP. indica\u003c/em\u003e to exhibit cold sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).In contrast to this, the AVG group was able to increase \u003cem\u003eP. indica\u003c/em\u003e cold tolerance, and the degree of damage was lower relative to that of the CONTROL group after 72h of restoration culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003ea) and higher photochemical efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eb); it could reduce EL permeability, maintain cellular homeostasis, enhance ROS enzyme activity, and inhibit MDA overaccumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Excess accumulation of ACC in \u003cem\u003eP. indica\u003c/em\u003e induced reduced photochemical efficiency, elevated cell membrane permeability, and increased lipid peroxidation, leading to reduced cold tolerance. On the contrary, AVG inhibited ACC accumulation and suppressed ethylene signaling, resulting in a certain protective effect on \u003cem\u003eP. indica\u003c/em\u003e; however, further experiments need to demonstrate how ethylene signaling regulates its downstream genes in response to cold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cem\u003ePiDREB1A\u003c/em\u003e and \u003cem\u003ePiDREB1A\u003c/em\u003e are downstream regulated by crosstalk between ethylene and cold signaling\u003c/h2\u003e \u003cp\u003eTranscriptome expression analysis of \u003cem\u003eP. indica\u003c/em\u003e indicated that cold stress rapidly up-regulated \u003cem\u003ePiETR1\u003c/em\u003e, \u003cem\u003ePiEIN3\u003c/em\u003e, and \u003cem\u003ePiERF1\u003c/em\u003e in the ethylene signaling pathway, as well as the cold-responsive specific gene \u003cem\u003ePiDREB1A\u003c/em\u003e (dehydration-responsive element (DRE)-binding protein 1A). However, whether they are co-regulated by ethylene and cold signaling must be further verified. The results of the previous experiments with externally applied ACC and AVG revealed that under 4℃, ACC promoted the up-regulated expression of the \u003cem\u003ePiERF1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ea) but suppressed the expression of the \u003cem\u003ePiDREB1A\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eb) and the epistatic response was sensitive to cold; on the contrary, spraying of AVG suppressed the expression of the \u003cem\u003ePiERF1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), but promoted the expression of the \u003cem\u003ePiDREB1A\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), the epistasis showed better cold tolerance. \u003cem\u003epiEIN3\u003c/em\u003e was also co-regulated by ethylene and cold signaling, and ACC promoted the up-regulated expression of the \u003cem\u003ePiEIN3\u003c/em\u003e gene, while AVG suppressed its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). ACC and AVG were both inhibited the regular expression of \u003cem\u003ePiETR1\u003c/em\u003e, but the inhibitory effect of AVG was more evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eIn the time dimension, ACC treatment rapidly induced the expression of the cold-responsive gene \u003cem\u003ePiERF1\u003c/em\u003e, which was higher than the control at 4h. \u003cem\u003ePiERF1\u003c/em\u003e expression was down-regulated in cold stress within 24h after ethylene signaling inhibition and was lowest at 4h; this was indicated that the gene wa s susceptible to ethylene signaling, responded to ethylene and cold signaling at 4h, and was unable to be expressed in low temperature after ethylene signaling inhibition. After spraying ACC with AVG, \u003cem\u003ePiDREB1A\u003c/em\u003e was cold-regulated for overall up-regulated expression. However, gene expression was suppressed in the ACC group relative to the control group after 4h and continued to be lower than the control group for the following 24h. The AVG group was more highly expressed at 8h than the control group and continued to be over-expressed during the following 24h. The expression pattern of this gene was consistent with previous studies, as a fast-response gene was highly expressed at 8h and then gradually decreased. However, after spraying AVG, \u003cem\u003ePiDREB1A\u003c/em\u003e continued to be overexpressed during 24h of cold stress without a decreasing trend, suggested that this gene might be regulated by the ethylene signaling pathway in tandem with cold signaling at 8h. When ethylene signaling was interfered with, \u003cem\u003ePiDREB1A\u003c/em\u003e lost its upstream inhibitory target for overexpression. It was opposite to the expression pattern of \u003cem\u003ePiERF1\u003c/em\u003e, and \u003cem\u003ePiERF1\u003c/em\u003e was activated by two signaling stimuli at 4h. \u003cem\u003ePiDREB1A\u003c/em\u003e is regulated by cold signaling only at 4h and was activated by dual signaling at 8h. The former was activated by dual-signaling stimuli in response to an earlier activation than the latter. ACC treatment increased the expression of \u003cem\u003ePiEIN3\u003c/em\u003e, which reached a maximum and slowly decreased at 8h. As a potential upstream gene of \u003cem\u003ePiERF1\u003c/em\u003e, it responded rapidly to the stimulation of cold and ethylene signals, and the gene was suppressed by AVG treatment, with a continuous negative expression at 24h. The ethylene receptor gene \u003cem\u003ePiETR1\u003c/em\u003e expression increased by cold. However, ACC and AVG negatively regulated it, and the gene expression in the two experimental groups was close to or lower than that of the control group within 24h. The gene might be co-regulated by other factors. Therefore, we suggested that the ethylene signaling pathway is activated by cold upstream, and \u003cem\u003ePiEIN3\u003c/em\u003e was rapidly induced by 8h, which promotes the continuous up-regulation of \u003cem\u003ePiERF1\u003c/em\u003e expression within 24h and suppresses the downstream \u003cem\u003ePiDREB1A\u003c/em\u003e expression, and that ethylene acts as a negative regulator during the cold response of \u003cem\u003eP. indica\u003c/em\u003e, which might be the reason for the cold-intolerance of \u003cem\u003eP. indica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Physiological responses of \u003cem\u003eP. indica\u003c/em\u003e under cold stress and response to ethylene regulators\u003c/h2\u003e \u003cp\u003eThe chlorophyll fluorescence parameter Fv/Fm can assess plant cold tolerance [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the present study, cold stress resulted in decreased chlorophyll fluorescence parameters of \u003cem\u003eP. indica\u003c/em\u003e, decreased photosynthetic efficiency, and an imbalance in plant energy supply. Exogenous spraying of the ethylene promoter ACC exacerbated photosystem II inhibition (PSII) inhibition, leading to leaf depletion and wilting. The inhibition of the ethylene pathway resulted in PSII recovery after cold stress. The experimental results indicated that inhibition of ethylene signaling would help \u003cem\u003eP. indica\u003c/em\u003e to recover photosynthetic efficiency more quickly. It is in general agreement with previous findings in Bermuda grass (\u003cem\u003eCynodon dactylon\u003c/em\u003e (L). Pers.), where the authors concluded that plants sprayed with ethylene inhibitors could achieve better cold hardiness as the duration of cold stress increased, as found from photosynthetic indicators [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe EL levels are commonly used to analyze the extent of cell membrane damage in plants under abiotic stress [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the greater the EL, the more severe the damage to the cell membrane. MDA is used as a lipid peroxidation parameter and as an indicator of stress mediated by the ROS system [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eP. indica\u003c/em\u003e suffered cell membrane damage under cold stress, and EL was continuously elevated over 72h. Spraying ACC inhibited the ROS scavenging system and exacerbated the degree of membrane damage, leading to a sustained increase in MDA values during cold stress, which was not recoverable after normoxia. Spraying AVG in advance could maintain cellular homeostasis during cold stress, and the EL and MDA values were lower than those of the control group after room temperature incubation and could be recovered more quickly. At the beginning of low temperature stress, soluble sugar accumulation in plant cells increases with cold tolerance [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Early spraying of AVG resulted in higher levels of soluble sugars and thus higher defense against cold in advance, which was suppressed by ACC. This suggested that thermophilic plants may lack the ability to anticipate the onset of winter in advance and require artificial regulation to help them acquire cold tolerance. And that the ethylene inhibitor AVG positively affected plant cold resistance by increasing cell membrane stability. This may be related to ethylene signaling and plant development, and more evidence in other plants is needed to verify this.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 The cold response mechanism of thermophilic plants\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003emay be negatively regulated by ethylene signaling\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePlant hormones are critical in various abiotic stresses [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In the present study, transcriptomic data was showed that genes involved in multiple phytohormone transduction signals were differentially expressed within 24h of cold stress. Among them, the most active transcription factor family members, ERFs, which are widely known to play critical roles in plant and abiotic stress [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and downstream in response to ethylene signaling, were targeted. In \u003cem\u003eArabidopsis\u003c/em\u003e, ethylene signaling regulates freezing tolerance by repressing the expression of \u003cem\u003eCBFs\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eERFs\u003c/em\u003e are regulated directly or indirectly by binding to the \u003cem\u003eDREB1s\u003c/em\u003e promoter, which is positively regulated [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The positive effect of ethylene has also been demonstrated in large trees such as apple [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and \u003cem\u003ePoncirus trifoliata\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, in thermophilic plants such as Bermuda grass [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], wheat (\u003cem\u003eOcimum sanctum\u003c/em\u003e) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and soybean [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], exogenous sprays of ethylene promoters negatively affected cold hardiness. In this experiment, after \u003cem\u003eP. indica\u003c/em\u003e was sprayed with the ethylene promoter ACC and the inhibitor ACG, the expression of downstream response genes was similarly targeted to \u003cem\u003ePiERF1\u003c/em\u003e and \u003cem\u003ePiDREB1A\u003c/em\u003e, and the crosstalk relationship between the two was manifested in the sense that ethylene signaling promoted the elevated expression of \u003cem\u003ePiERF1\u003c/em\u003e and suppressed the expression of \u003cem\u003ePiDREB1A\u003c/em\u003e; on the contrary, suppressed ethylene signaling resulted in the decrease of the expression of \u003cem\u003ePiERF1\u003c/em\u003e and the promotion of the \u003cem\u003ePiDREB1A\u003c/em\u003e expression. Combined with the morphological expression of cold tolerance, this result indicated that \u003cem\u003eP. indica\u003c/em\u003e might be negatively regulated by ethylene signaling, which generally agrees with previous findings in thermophilic plants such as wheat [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Does this imply that for thermophilic plants, ethylene signaling excites an antagonistic relationship between the downstream classical cold chain CBF/DREB-COR and some members of the ERFs family? Deeper mechanisms need to be supported by further evidence. Regardless, it is clear that \u003cem\u003eP. indica\u003c/em\u003e receives ethylene and CBF/DREB-COR signaling pathways to co-regulate cold tolerance in the face of low-temperature stress in \u003cem\u003ePiERF1\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Other signaling pathways may simultaneously mediate \u003cem\u003eP. indica\u003c/em\u003e cold response processes\u003c/h2\u003e \u003cp\u003eIn the transcriptome KEGG enrichment, we were interested in the MAPK signaling pathway being heavily enriched. According to previous reports, MAPK cascade signaling mediates ethylene signaling under biotic or abiotic stresses in two ways: first, it regulated the downstream \u003cem\u003eACS6\u003c/em\u003e gene through MAPK6, which was involved in the ethylene synthesis pathway; the second was that after activation of the ethylene signal, MKK9 or MPK3/6 is directly or indirectly regulated through the ETR1/RS1 receptor, which then promotes the involvement of EIN3 in early transcription downstream of ethylene in the nucleus [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Previously, in a cold response study of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, it was reported that MAPK3/6 negatively regulates Arabidopsis cold tolerance by phosphorylating ICE1 to destabilize it [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. During the cold response of \u003cem\u003eP. indica\u003c/em\u003e, many genes were induced to be expressed in MAPK signaling and ethylene signaling, including the previously mentioned \u003cem\u003eACS6\u003c/em\u003e, \u003cem\u003eETR1\u003c/em\u003e, \u003cem\u003eEIN3\u003c/em\u003e, and \u003cem\u003eMAPK3\u003c/em\u003e. Whether these MAPK signals are involved in the complex response to cold and ethylene in \u003cem\u003eP. indica\u003c/em\u003e and how to crosstalk \u003cem\u003ePiERF1\u003c/em\u003e and \u003cem\u003ePiDREB1A\u003c/em\u003e was still worthy of in-depth exploration.\u003c/p\u003e \u003cp\u003eIn addition to transcriptional regulation playing an essential role in cold-responsive stress, we detected that metabolic processes were also involved in cold response in GO enrichment. It coincides with the previous result that spraying the ethylene inhibitor AVG enhances plant defenses against the cold by increasing two metabolic pathways: soluble sugars and soluble proteins. \u003cem\u003eSIMAPK3\u003c/em\u003e was previously reported in tomato [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] to regulate cold tolerance by regulating the synthesis of its soluble sugars. Surprisingly, two MAPK3 genes were found to be expressed in response to cold in \u003cem\u003eP. indica\u003c/em\u003e transcriptome DEGs analysis. From this, we speculate that MAPK signaling may exercise cold tolerance by regulating soluble sugars in plants. AVG was confer this function in advance, but further validation is needed to elucidate this.\u003c/p\u003e \u003cp\u003eMeanwhile, in KEGG enrichment, it was found that in addition to MAPK signaling, the circadian rhythms-plant pathway was also significantly up-regulated in expression. However, this may be caused by the transfer of plants to all-day dark conditions during cold stress, and all genes in this pathway were excluded in this study to avoid the results being influenced by circadian rhythms-plant.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 conclusion","content":"\u003cp\u003eIn this study, we found that ethylene signaling leads to cold sensitivity in thermophilic plants \u003cem\u003eP. indica\u003c/em\u003e, as evidenced by reduced photosynthetic efficiency, enhanced membrane permeability, weakened ROS scavenging mechanism, and inability to maintain cellular homeostasis, suggested that \u003cem\u003eP. indica\u003c/em\u003e might be negatively regulated by ethylene signaling. \u003cem\u003ePiERF1\u003c/em\u003e and \u003cem\u003ePiDREB1A\u003c/em\u003e, two downstream genes were related to ethylene signaling, and co-regulated by ethylene and cold signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The former was expressed earlier than the latter by double signaling. The expression of the two genes had a reciprocal relationship, which was explicitly reflected in the fact that the expression of \u003cem\u003ePiDREB1A\u003c/em\u003e was up-regulated after the inhibition of ethylene, which gave \u003cem\u003eP. indica\u003c/em\u003e cold tolerance, and \u003cem\u003ePiERF1\u003c/em\u003e might be one of the critical genes leading to cold sensitivity. one of them. These results provide some evidence for the effect of ethylene signaling on plants under low-temperature stress and provide a solution for overwintering thermophilic plants in the open field.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eEthical consideration\u003c/h3\u003e\n\u003cp\u003eThe wild seed collection and the trial conducted in this study were in no violation of any legislation, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The plants were planted in the nursery of Sichuan Agricultural University, Chengdu, China.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eNo data was used for the research described in the article.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work is supported by grants from the Sichuan Province Science and Technology Support Program (2021YFYZ0006).\u003c/p\u003e\n\u003ch3\u003eAuthors' contributions\u003c/h3\u003e\n\u003cp\u003eZ-A .Z. and Y-R .L.: conceived and designed the experiments. Z-A .Z., Y-R .L., T .L., C-L .L., Q-X .Z. , X .L., L-J. Y., and J-N .L.: conducted the experiments and performed data analysis, interpretation, and investigation. Z-A .Z. wrote the manuscript. S-P .G.: revised the original manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe acknowledge the support provided by our labs at Sichuan Agricultural University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHong YB, Wang H, Gao YZ, Bi Y, Xiong XH, Yan YQ, Wang JJ, Li DY, Song FM. ERF Transcription factor \u003cem\u003eOsBIERF3\u003c/em\u003e positively contributes to immunity against fungal and bacterial diseases but negatively regulates cold tolerance in rice. 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J Agric Food Chem. 2024;72(10):5185\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.3c09066\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.3c09066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ethylene signaling pathway, PiERF1, PiDREB1A, Cold tolerance, 1-aminocyclopropane-1-carboxylate, Plumbago indica L.","lastPublishedDoi":"10.21203/rs.3.rs-4448738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4448738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEthylene is an essential regulatory factor in the signaling pathway of plant responses to abiotic stress, included cold stress, and also plays a regulatory role in cold response. Recent studies have shown that exogenous application of ACC (1-aminocyclopropane-1-carboxylate), an ethylene promoter, affects plant cold hardiness. It is well known that the cold-responsive specific gene \u003cem\u003eDREB\u003c/em\u003e (dehydration-responsive element (DRE)-binding protein) plays a crucial role in enhancing cold hardiness in plants by activating some cold-responsive (\u003cem\u003eCORs\u003c/em\u003e) genes. However, the molecular mechanism of how the ethylene biosynthesis pathway regulated this gene in the cold response of thermophilic plants had yet to be well explained. In this study, using the thermophilic plant \u003cem\u003eP. indica\u003c/em\u003e ( \u003cem\u003ePlumbago indica\u003c/em\u003e L.) as an example, physiological and transcriptomic analyses revealed that cold stress treatment induced the synthesis of endogenous ACC and regulated the ethylene signaling activator \u003cem\u003ePiERF1\u003c/em\u003e, while cold signaling also activated \u003cem\u003ePiDERB1A\u003c/em\u003e. Spraying experiments were also showed that ACC-induced up-regulation of the \u003cem\u003ePiERF1\u003c/em\u003e gene reduced cold tolerance of \u003cem\u003eP. indica\u003c/em\u003e, and decreased the expression level of the \u003cem\u003ePiDREB1A\u003c/em\u003e gene; reverse experiments have shown that spraying AVG (aminoethoxyvinylglycine) resulted in the down-regulation of the \u003cem\u003ePiERF1\u003c/em\u003e gene, while the expression level of \u003cem\u003ePiDREB1A\u003c/em\u003e was increased, and chilled symptoms were alleviated. These results indicated that ethylene signaling directly regulates the downstream gene \u003cem\u003ePiERF1\u003c/em\u003e and initiates the DREB-COR cold-responsive signaling pathway to regulate cold hardiness, exhibiting negative regulation of cold hardiness in thermophilic plants.\u003c/p\u003e","manuscriptTitle":"ERF1 of Plumbago indica L. receives ethylene signaling and regulates cold tolerance together with the DREB-COR pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 06:46:04","doi":"10.21203/rs.3.rs-4448738/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c454dabf-899c-4d78-bbbf-041c26634f1b","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-27T07:53:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-06 06:46:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4448738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4448738","identity":"rs-4448738","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}