Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress

Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress | 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 Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress Minkun Pei, Ping Yang, Jian Li, Yanzhuang Wang, Juan li, Hongjun Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4399432/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2024 Read the published version in BMC Genomics → Version 1 posted 13 You are reading this latest preprint version Abstract Background The RAV gene family in Capsicum annuum remains largely unexplored in response to chilling stress, despite its known significance in plant abiotic stress responses. Results In this study, we identified and characterized six members of the CaRAVs gene subfamily in pepper through genome-wide analysis. Subsequently, the CaRAVs subfamily was classified into four branches based on homology with Arabidopsis thaliana , each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs , whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT-PCR analysis revealed that CaRAV1 , CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAVs gene family, resulting in plants with increased susceptibility to chilling damage and reduced antioxidant enzyme activity, particularly evident under repeated chilling stress. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress. Conclusions Silencing targeting key members of the CaRAVs gene family, results in plants with increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly evident under repeated chilling stress. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in ppepper. RAV gene family Transcription factor Chilling stress VIGS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background The B3 transcription factor (TF) is exert significantly influences over the ability of plants to respond to stresses, growth and development. Typically manifesting as a DNA-protein binding domain in plants [ 1 ]. There are several distinct groups of B3-specific family members, including REM, LAV, RAV and ARF family members. Recent research indicated that there was a fifth member, HSI, Recent research indicated that there was a fifth member, HSI, which expands the B3 superfamily evolutionary footprint, and is particularly evident in cruciferous plants [ 2 ]. This expansion has led to the emergence of more complex structures, such as domain repeats and gene tandem repeats. Notably, the RELATED to ABI3/VP1 (RAV) transcription factor stands out as exclusive to higher plants [ 3 ]. While most B3 superfamily genes feature at least one B3 domain, certain RAV genes subfamily also contain a second domain known as APET-ALA2 (AP2). Akhter et al. classified genes that exhibited double domains and domain replication as members of the RAV subfamily [ 4 ]. On the other hand, the RAV subfamily, which comprises a single B3 domain is commonly referred to as the NGA translation factor subfamily. Thirteen RAV family members have been identified thus far in Arabidopsis. Among these, six belong to the AP2 domain and seven are members of the B3 RAV subfamily ( NGATHE) [ 5 ]. RAV serving as a multifunctional transcription factor, has been implicated in the negative regulation of plant growth under various stress conditions, including salt stress and drought, across multiple plant species such as rice [ 6 ], soybean [ 7 ], barley [ 8 ], cotton [ 9 , 10 ] and cucumber [ 11 ]. Additionally, abscisic acid (ABA), auxin (IAA), and additional stresses regulate RAV expression [ 12 ]. Previous studies have employed Arabidopsis RAV1 as a transcription factor for chilling stress [ 13 ]. More recent reports have further substantiated this claim. RAV expression in pepper [ 14 ], tea plant [ 15 ] and rape [ 16 ] is up-regulated under chilling stress. In addition, aside from its known regulatory effects on ABA and brassinosteroids (BRs) and its role in the abiotic stress response, Song et al. also reported that the celery RAV negatively regulates zeaxanthin cyclooxygenase (ZEP), and ultimately positively regulates carotenoid synthesis [ 17 ]. Although they have been studied in various species, the number and functions of pepper RAV TFs are still unclear. Pepper( Capsicum annuum L.) stands as a pivotal cash crop with a long history of cultivation [ 18 ]. Ranked as the second largest vegetable crop globally, it spans a plantinh area of 1.4–1.6 million hectares. However, its susceptibility to temperature and light renders it vulnerable to damage under conditions of low temperature and inadequate light exposure. The impact of temperature fluctuations, especially during autumn and spring, significantly influences both the productivity and quality of pepper [ 19 ]. Moreover, growth and development are impeded and yields diminish when temperatures drop below 15 o C or light intensity falls below 25 µmol·m − 2 ·s − 1 [ 20 ]. Low temperatures not only have a macroscopic impact on plants but also disrupt the intracellular metabolic equilibrium. Upon initial exposure to low temperature, plants experience an accumulation of reactive oxygen species (ROS) within their cells, resulting in the peroxidation of membrane lipids. When the production of reactive oxygen species (ROS) surpasses the body's ability to remove them, it results in an imbalance of antioxidants and triggers an oxidative stress response, which in turn can induce DNA damage [ 21 ]. Although there are existing reports on the RAV transcription factor subfamily, detailed information on the specific activities of its members, apart from RAV1, remains limited. A comprehensive characterization of the RAV genes family in pepper, especially its response mechanisms to chilling stress, has yet to be established.This study identified revealed six RAVs within the entire pepper genome and analysed them. Conserved domains evolutionary relationships, gene structure, and cis -acting elements in the promoter region. Additionally, virus-induced gene silencing and Quantitative Real-time PCR (qRT-PCR) techniques were employed to investigate the expression patterns of key genes from the CaRAVs gene family in response to low temperatures. These results indicate that RAV proteins may play roles in plant responses to chilling stress. Given the potential importance of RAV genes in plant responses to chilling stress environments. The aim of this study was to examine the impact of the CaRAV1 and CaNGA1 genes, which are characterized by distinct domains, on pepper plants experiencing chilling stress. Materials and Methods Identification of gene family members The acquired genomic materials consisted of the tomato, pepper and B3 superfamily genomes of Arabidopsis. The AtRAVs gene family database ( https://www.arabidopsis.org/browse/genefamily/index.jsp ) was accessed to obtain 87 protein sequences belonging to the Arabidopsis B3 family. Additionally, the database ( http://plants.ensembl.org/index.html ) provides relevant data regarding the Solanum lycopersicum and Capsicum annuum genomes. The NCBI online software BLSAT was utilized to compare the predicted peppers ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The conserved domain of the protein was identified using the second alignment of pepper protein sequences in the InterPro online software ( https://www.ebi.ac.uk/interpro/search/sequence/ ). Subsequently, sequences that were in a disarray state were eliminated, resulting in the acquisition of pepper B3 family genes. Physicochemical properties of amino acids in the CaRAVs subfamily The amino acid length, relative molecule, relative isoelectric point, amino acid stability, fat solubility index, and average hydrophilic coefficient of the pepper CaRAVs were predicted using an online website ( https://web.ExPASy.org/protparam/ ). Subsequently, subcellular localization prediction of the gene was performed using online software ( http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ ). Protein structure prediction for the CaRAVs subfamily Using PSIPRED online software ( http://bioinf.cs.ucl.ac.uk/psipred/ ) and SWISS-MODEL ( https://swisodel.ExPASy.org/ ) provided by the protein expert system ExPASy, the secondary structures of proteins encoded by target CaRAVs were predicted respectively. Analysis of the domains, gene structure, and phylogenetic tree The amino acid sequences of the target CaRAVs were compared with those of Clustal 2.1, and the online tool MEME ( https://meme-suite.org/meme/tools/meme ) was used to identify highly conserved motifs. Highly homologous RAV transcription factors from rice and Arabidopsis that matched each target CaRAVs were acquired from the transcription factor database PlantTFDB ( http://planttfdb.cbi.pku.edu.cn/ ). A phylogenetic tree was then constructed using the neighbor-joining (NJ) method in the software MEGA 11. The relevant parameter settings are as follows: the bootstrap value is set to 500, the partition deletion is set to 50%, and the genetic distance model is selected as JJT + G for calculation, which is the optimal recommendation. The remaining parameter values are set to their default values. Prediction analysis of cis -acting elements in the CaRAVs promoter The promoter region (2000 bp upstream of ATG) of the target RAV was obtained from NCBI ( https://www.ncbi.nlm.nih.gov/ ). The cis -acting elements of the promoter region were screened and analysed using PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and visualized with TBtools. The PlantCARE website (available at .be/web tools/plantcare/html/) was utilized to examine and analyse the cis -acting elements found in the promoter region. These elements were then visualized using TBtools. Collinearity analysis of the CaRAVs gene family The collinearity analysis was conducted using the MCScanX software, and the resulting collinearity data were visualized using TBtools, which can be found at the following link: https://github.com/CJ-Chen/TBtools . Plant materials and chilling stress treatment The test material was Capsicum annuum “Shenghan 740”. The seedlings were grown in a mixed substrate consisting of peat, perlite, and vermiculite at a ratio of 3:1:1(v/v). The plants were cultivated within 25 o C, a photoperiod of 12 h, and a relative humidity of 75%. After the peppers had grown to 5–6 true leaves, they were placed in a 5 o C thermostat for chilling stress treatment. The leaves of the pepper plants were then cut and treated for different durations of 0, 1, 3, 5, 7, 12, 24, 48, and 72 h. These treated leaves were promptly frozen in liquid nitrogen for further experiments. Each treatment was replicated at least three times. For repeated treatments of chilling stressed plants, the seedlings exposed to low temperatures a undergone a 24 h recovery period before they were exposed to chilling stress again. qRT-PCR Leaf samples (100 mg) were pulverized in liquid nitrogen, and total RNA was isolated using a TRIzol kit (Beijing Tsingke Biotech Co., Ltd.) following the provided instructions. The RNA concentration was measured using a NanoDrop microspectrophotometer (Thermo Fisher Scientific, USA), and its purity was assessed using the A260/280 ratio. The A260/280 ratio was used to determine the purity of the sample. The error-free RNA was diluted to a concentration of 400 ng/µL using distilled water and then stored at -80 o C for subsequent reverse transcription. Complementary DNA (cDNA) was synthesized in the reverse direction using Tsingke RT6 reverse transcriptase following the provided instructions. The cDNA was synthesized by designing real-time quantitative primers using Primer-BLAST from the NCBI website. The chimeric fluorescence method was used, with pepper Actin serving as an internal reference and SYBR Green I from Tsingke Biotech was used as the fluorescent dye. Real-time polymerase chain reaction (PCR) was performed using a Bio-Rad system. The primers used can be found as Supplementary Table 1 online. The relative expression of genes was calculated using the 2 −∆∆Ct method. Three replicates were performed for each sample. Vector construction The ligation primer that was designed using CE Design V1.03 software, based on the target gene sequence and intended for use with the Tobacco Rattle Virus (TRV) vector, the length of the primer fragment containing the homology arm of the vector is around 40bp. The process of obtaining RNA has been previously described. However, the acquisition of cDNA differs from that of qRT-PCR because the reverse transcription system utilizes Oligo (dT 17 ) instead of Randomer. Use of Rapid 2 × Taq Master Mix from Nanjing Vazyme Biotech Co., Ltd to amplify the target gene fragment. Next, Saml enzyme digestion was used to release the linear fragment of the TRV vector. The 300 bp purified fragment was connected to TRV using T4 ligase. The monoclonal antibody (kanamycin) was then incubated with the cells overnight on an inverted plate. Positive clones were identified through electrophoresis after a single colony was shaken at 37 o C for 12 hours. To identify false-positive vectors, it is essential to verify whether they align with the target sequence during the sequencing process. Following sequencing, the Escherichia coli solution containing the homologous arm was subsequently transferred to Agrobacterium competent GV3101. The Agrobacterium was then cultured on three different antibiotics: kanamycin, gentamicin, and rifampicin. Following agitation of the bacteria in a solitary colony, electrophoresis was subsequently performed to verify the uniformity of the vectors. Ultimately, the silencing vectors pTRV2- RAV1 , pTRV2- NGA1 , and pTRV2- PDS were generated. Fifty percent glycerin was combined with the bacterial solution at a 1:1 ratio, and the mixture was stored at -80 o C. Silencing efficiency assay for VIGS vectors A single colony was selected after the vector containing the target fragment was coated with three antibodies on luria bertani (LB) solid media and cultured at a temperature at 28 o C for two days. After three cycles of oscillation in LB activated bacterial solution, a single colony was selected. Twelve hours after the initial bacterial suspension was shaken, suitable concentrations of acetosyringone (AS) and 2-Morpholinoethanesulfonic acid (MES) were added to the second shaking bacterial suspension, the third polar fungus was added prior to incubation, and the levels of MES and AS in the LB liquid media were elevated. The mixture was then incubated for 12 hours and subsequently subjected to centrifugation at a speed at 4000 rpm for 15 minutes at 4 o C. The infection buffer was prepared, and the bacterial solution was resuspended in MaCl 2 buffer solution. The bacterial suspension (OD 600 ) was diluted to approximately 0.04 using a buffer solution. A 1:1 ratio of infected liquid pTRV1 was combined with pTRV2:GFP (blank vector with label), pTRV2: PDS (positive albino control), pTRV2: RAV1 , and pTRV2: NGA1 to infect the leaves. Virus-infected seedlings were placed in a temperature-controlled room with 75% humidity and 22 o C for 24 hours. After this time, the growth conditions were maintained at a temperature of approximately 22 o C, following a standard photoperiod of 12 hours of light and 12 hours of darkness. After three days of culture, the fluorescence emitted by GFP, which had been labeled with a green dye, was observed using a 254 nm ultraviolet lamp. After approximately two weeks, the pTRV2-PDS plants were albino and subjected to qRT‒PCR analysis to determine their silencing efficiency. Sixty plants with a silencing efficiency above 50% and sixty plants with pTRV2 were chosen for low-temperature treatment. Malondialdehyde (MDA) concentration, membrane permeability, and antioxidant enzyme activity were quantified The MDA concentration was ascertained using a modified version of the thiobarbituric acid colorimetry method as described by Dhindsa [ 22 ]. The relative membrane permeability (REC) was calculated as a percentage according to Yang et al. [ 23 ]. The quantification of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity was conducted using the guaiacol method as described by Jebara et al. [ 24 ] and Almeselmani [ 25 ]. SPSS version 19.0 software was used to analyse the significance of difference by t tests. Results Identification and prediction of the physicochemical properties of CaRAVs family genes BLAST analysis was performed using the Arabidopsis B3 transcription factor family and the complete pepper genome, resulting in the prediction of 12 related pepper CDS sequences. The software TBtools was utilized to acquire the 12 protein sequences. Subsequently, the structural domains were identified and the extraneous sequences were eliminated using InterPro. Ultimately, out of all the identified proteins, only six were classified as typical pepper RAV proteins, as indicated as Supplementary Table 2 online. To summarize, a total of six candidate genes have been verified as members of the pepper RAV family. The CaRAVs of pepper were named CaRAV1, CaRAV2, CaRAV3, CaNGA1, CaNGA2, and CaNGA3 in this study due to their homology and structural domain differences with those of Arabidopsis. According to the results presented in Table 1 , all the CaRAVs were found in the nucleus, while CaNGA3 was also present in the cytoplasm. The amino acid length of the CaRAVs ranged from 130 to 480 aa. Specifically, CaRAV2 had the fewest amino acids with a length of 137 aa, while CaRAV3 had the greatest number of amino acids with a length of 477 aa. The molecular weights of CaRAVs ranged from 14,940 to 52,496 kDa. Among them, CaRAV3 had the highest molecular weight of 52,495.23 kD, while CaRAV1 had the lowest molecular weight of 14,940.15 kDa. The isoelectric points of the CaRAVs amino acids varied between 5.93 and 9.78, with CaRAV2 having the highest isoelectric point of 9.78 and CaNGA2 having the lowest isoelectric point of 5.93. Furthermore, the stability index (> 40) and average total hydrophilicity (< -0.5) suggest that CaRAV1, CaRAV3, and CaNGA3 are amino acids that are both unstable and hydrophilic. On the other hand, CaRAV2, CaNGA1, and CaNGA2 are both stable and hydrophilic amino acids. Table 1 Analysis of important physicochemical properties of amino acids encoded by CaRAVs Amino acid Number of amino acids(aa) Molecular weight (kDa) Theoretical (pI) Instability index Grand average of hydropathicity Aliphatic index Subcellular localization CaRAV1 244 27790.89 9.78 26.65 -0.577 75.82 Nucleus CaRAV2 137 14940.15 6.58 43.65 -0.472 66.28 Nucleus CaRAV3 477 52495.23 6.16 59.1 -0.538 60.27 Nucleus CaNGA1 176 20310.77 6.12 37.02 -0.603 71.93 Nucleus CaNGA2 331 38699.81 5.93 38.23 -0.679 69.15 Nucleus CaNGA3 357 40182.73 8.3 53.62 -0.728 52.91 Cytoplasm/Nucleus Distribution of CaRAVs on chromosomes Using TBtools software in conjunction with the chilli genome annotation files, the location and distribution of CaRAVs was mapped on chromosomes. The graph (Fig. 1 ) shows that among the 12 chromosomes of pepper (see Supplementary Table 3 online), CaRAVs was exclusively found on Chr1, Chr10, Chr11 and Chr12. Chr1 and Chr11 each had two members, while the other two chromosomes had only one RAV . Moreover, all members of this gene family were located at the ends of chromosomes, and no RAV was present in the middle region of any chromosome. A linkage region comprising the genes CaRAV1 and CaRAV2 was established at the beginning of Chr11. The two genes were surrounded by continuous bases extending 100 bp upstream and downstream, based on their location and length. CaRAVs protein structure correlation The PSIPRED software was used to predict the secondary structure of the protein. Based on the results (Table 2 ), it was discovered that CaRAVs protein consisted of three components: β corner, extended main chain, and an irregular curl (see Supplementary Fig. 1 online). However, the relative sizes of each structure varied. Irregular curling accounts for 49.85–78.62% of the total proportion. The β rotation angle and extended main chain components had relatively low percentages, ranging from 11.74–28.98% and 5.60–33.53%, respectively. The findings indicated that the primary secondary structure of CaRAVs is an irregular curl. The extended backbone and β angle, on the other hand, may serve a supplementary and modifying function, thereby aiding in the formation of complex protein conformations at a later stage and performing specific functions. SWISS-MODEL was used to predict the tertiary structure of the CaRAVs protein (Table 3 ). The alignment rate between all members of the CaRAVs and templates exceeded 70%, and the root mean square deviation (RMSD) was less than 2.5 Å. These findings suggest that the homology modeling outcomes are reasonably dependable [ 26 ]. CaNGA1, CaNGA2 and CaNGA3 matched protein templates containing the B3 structural domain, whereas CaRAV1, CaRAV2 and CaRAV3 matched protein templates containing both the B3 and AP2 structural domains. Table 2 Secondary structure analysis of the CaRAVs protein Protein β-Strand amount β-Strand Helix amount Helix Coil amount Coil CaRAV1 59 24.18% 35 14.34% 150 61.48% CaRAV2 17 12.41% 16 11.68% 104 75.91% CaRAV3 56 11.74% 46 9.64% 375 78.62% CaNGA1 51 28.98% 22 12.50% 103 58.52% CaNGA2 55 16.62% 111 33.53% 165 49.85% CaNGA3 65 18.21% 20 5.60% 272 76.19% Phylogenetic tree of CaRAVs, SlRAVs and AtRAVs The amino acid sequences of CaRAVs were subjected to BLAST searches against PlantTFDB. RAV proteins in Arabidopsis and tomato that were found to be highly homologous to CaRAVs (top 10 in terms of score) were screened, repetitive sequences were eliminated, and a phylogenetic tree was constructed using 9 SlRAVs and 13 AtRAVs in conjunction with 6 CaRAVs present in CaRAVs (Fig. 2 ). The findings indicated that all individuals from the three species were classified into four distinct clusters (I - IV), with minimal variation in the population amount across the four groups. A comparison of phylogenetic trees revealed that SlRAVs and CaRAVs are more homologous than are AtRAVs. The phylogenetic tree, gene structure, and domain of AtRAVs, SlRAVs, and CaRAVs Figure 3 shows the Arabidopsis and tomato RAV gene families in the tree, along with the domain and conserved sequence of the RAV gene family. Additionally, we included pepper in the analysis. We utilized the MEME online tool to analyse the amino acid sequences of the CaRAVs. From this analysis, we identified 10 conserved motifs based on their P-values (see Supplementary Table 4 online). The findings indicated variations in the location and abundance of each motif within the amino acid sequence of CaRAVs. Two motifs, motif 1 (LLNFEDENGKVWRFRYSYWNSSQSYVLTKGWSRFVKEKKLDAGDVVSFQR) and motif 2 (ALIEREHLFEKVVTPSDVGKLNRLVIPKQHAEKYFPLDSSN), are found to be highly conserved across various CaRAVs sequences. Thus, motif 1 and motif 2 comprise the conserved region of CaRAVs transcription factors. CaRAV2 lacks motif 1 and motif 2, but it exhibits greater similarity to the conserved region of AtRAVs. Additionally, CaRAV2 is highly homologous to CaRAV2 in Arabidopsis (Fig. 3 A), the cause of this outcome remains uncertain, and it is possible that CaRAVs have undergone a distinct evolutionary process. CaRAV2 has been determined to have a close relationship with the evolution of Arabidopsis, potentially due to the gene linkage previously observed in chromosome location. By examining the domain visualization, it is evident that the three species possess only two domains each, namely, the B3 and AP2 domains. Only domains within a 500 bp range are shown in Fig. 3 B. Based on the exon-intron structure depicted in Fig. 3 C, it is evident that CaRAVs lack introns, with five members consisting of a single exon. In contrast, CaNGA2 contains multiple exons and introns. Collinearity of the three species To elucidate the evolutionary relationship of the RAV across various plants, we used the genomes of Arabidopsis, tomato, and pepper plants to collinearity analysis using MCScanX software (Fig. 4 ). Three pairs of homologous genes were identified in Arabidopsis and pepper ( CaNGA1 / AtRAV-like1 , CaNGA1 / AtRAV-like2 , and CaNGA1 / AtRAV-like3 ), while two pairs of homologous genes were identified in tomato and pepper ( CaNGA3 / SlNGA2 and CaNGA2 / SlRAV3 ). Cis -acting elements in CaRAVs gene family promoters To determine the biological function of the Ca RAVs , an analysis of cis -acting elements was conducted on the 2 kb promoter region located upstream of the initiation codon of the CaRAVs . This analysis was performed using the Plant-CARE database. In the CaRAVs promoter, we discovered 12 regulatory elements that are conserved and have a significant impact on plant stress response (see Supplementary Table 5 online), and growth regulation (Fig. 5 A). The promoter region of the CaRAVs family contains photoresponsive, MeJA-responsive, and anaerobic-inducing elements. In terms of quantity, light responsive elements are the most abundant. However, low-temperature responsive elements are only present in the Ca NGAs subfamily. The results showed that the cis -acting elements amount of CaRAVs members varied to some extent, with CaNGA1 having a maximum of 24 components and CaRAV2 having a minimum of 16 components (Fig. 5 B). Expression pattern of CaRAVs under chilling stress conditions To assess the response of the CaRAVs to natural low temperatures and determine the extent of CaRAVs involvement in chilling stress, we conducted qRT-PCR analysis of the CaRAVs family after pepper plants were subjected to 3 days of treatment at 5 o C. The relative expression levels of the CaRAVs were calculated using Actin as an internal reference gene. The relative expression levels were visualized using a cluster thermograph (Fig. 6 ). The results demonstrated that the relative expression levels of all examined genes increased following exposure to chilling stress. During periods of chilling stress, the expression of CaRAVs is typically elevated at 12 hours and 24 hours after stress occurs. Among the CaRAVs , CaRAV1 , CaRAV2 , and CaNGA1 showed the most significant changes in expression. Response of CaRAV1 and CaNGA1 under repeated chilling stress Based on qRT-PCR heat map data, we selected two genes from the CaRAVs gene family that exhibited the greatest response to chilling stress. These genes are CaRAV1 , which possesses AP2 and B3 domains, and CaNGA1 , which possesses a B3 domain. Low temperatures in nature may occur repeatedly for a period of time. We subjected WT pepper plants to low temperatures of 5 o C for 24 h (see Supplementary Fig. 2 online). The results showed that the expression of CaRAV1 and CaNGA1 was up-regulated after 24 hours of chilling stress in the Str0 group (Fig. 7 ). When plants were again subjected to chilling stress (Str1 group), RAV1 and NGA1 produced higher and faster expression at 24 h. Assessment of the effectiveness of silencing in CaRAV1 and CaNGA1 To study the role of various domains in the CaRAVs gene family under chilling stress, we created virus-induced silencing vectors (pTRV2:GFP, pTRV2: PDS , pTRV2: RAV1 , and pTRV2: NGA1 ) using tobacco crisp virus (TRV). All of the electrophoretic bands that were transferred into Agrobacterium GV3101 showed positive results when analysed using a gel imaging system, and the length of the target gene fragment was determined to be 300 base pairs(Fig. 8 A,C). Furthermore, two groups with two cotyledons each were infected with the vector. After a period of 3–7 days, the presence of green fluorescence, marked by the GFP tag, was detected using a 254 nm ultraviolet lamp. The study revealed that the plants with silenced genes were effectively infected during the initial phase (see Supplementary Fig. 3 online). The silencing efficiency of pTRV2- PDS was confirmed by qRT-PCR after the albino phenotype appeared two weeks later. The findings indicated that the two vectors exhibited substantial suppression of expression in comparison to pTRV2:GFP and the control (Ctl), with a silencing efficiency exceeding 50% (Fig. 8 B,D). Once the plants reached the stage of having six true leaves, the GFP label of the silenced plants ceased to exhibit green fluorescence (see Supplementary Fig. 4 online). MDA content and relative membrane permeability of the silenced peppers To assess the reactions of CaRAV1 and CaNGA1 to chilling stress. We analysed the indices associated with cell membrane damage (Fig. 9 ), including the levels of MDA and REC, which exhibited a consistent upwards trend. The MDA (malondialdehyde) and REC (relative electrical conductivity) levels in the RAV1 -silenced plants were generally greater than those in the NGA1 -silenced plants. In all treatments, plants subjected to low temperature experienced a moderate increase in cell membrane permeability and MDA levels, but their ability to return to the original range was limited. Compared to blank vector damage, after the plants were subjected to the second stress, there was no significant change in the MDA content of the Str0 and Str1 silenced plants. Antioxidant enzyme activity of the silenced peppers Low-temperature treatment resulted in an increase in the activity of antioxidant enzymes. However, antioxidant enzyme activities were significantly lower in silenced plants compared to pTRV2 (Fig. 10 ). After a single day of recovery from the initial chilling stress, the POD and CAT activities returned to their original levels in all the treatments, with the exception of SOD. The levels and rates of activity of antioxidant enzymes in the plants exposed to low temperature were greater and more rapid, while the silenced plants exhibited significantly lower levels than did the pTRV2 plants. Discussion RAV, one of the subfamilies of the B3 transcription factor superfamily, has been demonstrated to have a significant impact on the physiology and biology of plants. The RAV subfamily is involved in plant growth, development, and responses to abiotic stress. Pepper is a significant horticultural crop in the global economy, and its demand is steadily rising each year [ 27 ]. Currently, pepper production often experiences economic and yield losses due to chilling stress. Despite significant advancements in chilling resistance, there is a scarcity of information regarding the response of pepper plants to chilling stress and CaRAVs [ 28 ]. This study discovered six members of the RAV gene subfamily in the entire pepper genome. These members include three members with the complete B3 domain, namely CaNGA1 , CaNGA2 , and CaNGA3 , and two members with both the B3 and AP2 domains, namely CaRAV1 , CaRAV2 , and CaRAV3 . Through the chromosome mapping, we determined that the RAV gene family members in pepper exhibit limited distribution, are distributed at both end positions of the chromosome, and the two closely related genes. Linkage regions was formed at the top of Chr11, comprising two genes, CaRAV1 and CaRAV2 . The 100 bp upstream and downstream of these two genes were considered continuous bases. This positional relationship may be associated with the evolutionary development of the RAV gene family and could also impact the regulatory function of the RAV . Tandem replication is a frequently observed phenomenon in the evolution of plant genomes, particularly in Solanaceae plants [ 29 , 30 ]. These repetitive sequences, known as tandem repeats, have a significant impact on the ability of plants to adapt to environmental changes [ 31 ]. In the present study, these two interlocking genes (CaRAV1 and Ca RAV2) produced higher relative expression in response to chilling stress. Compared with those of other members of the CaRAV gene family, the closely associated genes CaRAV1 and CaRAV2 exhibit higher and more rapid increases in gene expression. It has been demonstrated that genes that are replicated in tandem are more likely to be involved in abiotic stress responses in Arabidopsis [ 32 , 33 ]. Furthermore, based on the modeling of homologous proteins, it has been observed that their structural homology indicates functional similarity. The 28 members of the three species were classified into four branches based on their gene structure and conserved motifs. This classification was used to construct a phylogenetic tree, which represents the homologous relationships among CaRAVs, AtRAVs, and SlRAVs. The findings indicated that the similarity between pepper and tomato was higher. Furthermore, based on the collinearity analysis of Arabidopsis, tomato, and pepper, we discover CaNGA1 exhibited collinearity with three AtRAVs members. This finding suggested that these genes likely have comparable functions and contributed significantly to the evolutionary dynamics of RAV gene families in pepper and Arabidopsis. It is worth noting that CaRAV1, CaRAV2, and CaNGA2 in tomato and Arabidopsis do not share a collinear relationship with the other two species, this suggests that these genes may be exclusive to the evolution of pepper. In addition to their role in gene expression regulation, introns also play a role in gene evolution [ 34 ]. According to exon-intron analysis, CaNGA2 exhibited multiple introns, whereas other members lacked introns. Furthermore, among the 13 members in Arabidopsis, only 5 possess introns. The origin of intron-free genes and whether they lost introns during evolution or were absent from the beginning of development remain unclear. Studies in Arabidopsis have demonstrated that intron-free genes can be significantly activated in response to stress. Additionally, research in rice and Arabidopsis has shown that intron-free genes can play a crucial role in adapting to salt stress and drought [ 35 ]. Exposure to chilling stress can disrupt the equilibrium of reactive oxygen species in plant cells, resulting in the accumulation of reactive oxygen species and subsequent membrane lipid peroxidation. During this process, plants initiate a highly efficient detoxification system and synchronize the activities of antioxidant enzymes and non-enzymatic substances. Currently, the antioxidant enzymes that are widely recognized include SOD, CAT, POD, and ascorbic acid POD (APX). The accumulation of these enzymes can safeguard plants against harm caused by low temperature [ 36 , 37 ]. In pepper production, various antistressors are commonly employed to mitigate abiotic stresses [ 38 – 41 ]. Additionally, chilling acclimation is utilized to enhance pepper tolerance to low temperatures [ 42 ]. This phenomenon, also known as “stress memory” [ 43 ], represents the plasticity mechanism of plants in response to production conditions. Within this study, wild-type pepper plants exhibited a recovery period of one day following exposure to low temperatures for one day. The relative expression levels of CaRAV1 and CaNGA1 also returned to their original baseline levels with increasing recovery time. Under repetitive chilling stress, these genes in plants respond more quickly thereby producing higher relative expressions, it consistent with the typical epigenetic alterations induced by chilling stress in plants. According to qRT-PCR validation of the virus-induced gene-silenced plants, the funny thing is that the CaRAV1 exhibited a greater relative expression level subsequent to albinism in the pTRV2: PDS positive plants. The cause of this phenomenon remains uncertain, possibly due to the suppression of the octahydro lycopene dehydrogenase (PDS) activity, which leads to the inhibiting carotenoid production. As previously stated, RAV acts as a suppressor of zeaxanthin cyclooxygenase [ 44 ], RAV1 and NGA1 may have significant functions in carotenoid synthesis following PDS silencing. MDA is a significant outcome of membrane peroxidation in plants, while the REC represents the extent of electrolyte leakage in cells. These indicators are crucial for assessing the level of damage to the cell membrane [ 45 , 46 ]. Typically, their changes will be positively correlated with the timing of abiotic stresses, which aligns with the pattern of pTRV2 expression observed during a single day of low temperature in this study [ 47 , 48 ]. When the plants in which gene silencing was induced by the virus were subjected to chilling stress, the levels of MDA and cell membrane permeability were difficult to restore to the initial levels of those in the control group (Ctl). and the degree of cell membrane damage in the silenced plants was greater than that in the plants with pTRV2: RAV1 and pTRV2: NGA1 . Furthermore, antioxidants prevent cell membrane damage by specifically targeting reactive oxygen species and nitrogen, as well as lipid peroxidation products and transcription factors that control the expression of genes related to antioxidant response elements. Plants exhibit physiological and biochemical reactions involving cell membrane systems and protective enzyme systems. SOD, POD, and CAT are crucial enzymes with protective functions in plants [ 49 ]. The antioxidant enzyme activities of pepper were notably greater at 5 o C for 12 and 24 h than at 25 o C for 0 h. These findings align with the results reported by Nai et al [ 50 ]. In plants undergoing gene silencing, SOD and CAT did not show significant increase in enzyme activity under repeated stress, while POD enzyme activity was significantly increased. Moreover, the POD and CAT enzyme activities of silenced pepper plants were significantly lower than those of pTRV2 in response to chilling stress. It also shown that RAVs may be involved in the response of chilli plants to chilling stress and increase the tolerance of plants to chilling stress, resulting in epigenetic modifications in the plants. These findings provide a basis for future investigations into the roles of RAV transcription factors in the response of pepper plants to chilling stress. The potential physiological responses identified in this study could be utilized in future molecular studies on enhancing chilling resistance in pepper. Conclusions In summary, our study is the first time to identify and characterize the whole genome of pepper CaRAVs, including chromosome location, protein structure and domain, phylogenetic tree and collinearity information. We further detected the expression of the CaRAV1 and CaNGA1 under repeated low-temperature conditions. Based on qRT-PCR data, CaRAV1 and CaNGA1 with high relative expression and different domains under chilling stress were screened from the CaRAVs gene subfamily, and a gene silencing vector was constructed by virus induction. Therefore, under chilling stress, understanding the low-temperature resistance of the RAV transcription factor response and improving the chilling resistance of pepper plants by using silenced plants is highly important for determining the key role of CaRAVs in the chilling response of pepper plants and chilling stress memory under repeated stress conditions. Declarations Acknowledgements We appreciate the financial support given by the projects. We thank the teacher and all schoolmates in our laboratory for providing useful discussions and technical assistance. We very look forward to the editor and reviewers for critically evaluating the manuscript. Author Contributions Minkun Pei: Writing-original draft, Methodology, Data curation. Ping Yang: Data curation, Formal analysis, Investigation. Jian Li and Yanzhuang Wang: Investigation, Methodology. Juan Li: Investigation. Hongjun Xu: Supervision, Funding acquisition. Jie Li: Supervision, Writing - review & editing. All authors read and approved the final manuscript. Funding This work was supported by the Xinjiang Uyghur Autonomous Region Key Research and Development Plan Project (2022B02032), Yunnan Fundamental Research Projects (202401AT070059), Yunnan Young and Middle Aged Academic and Technical Leaders Reserve Talents (202205AC160056). Availability of data and materials The entire Capsicum annuum genome sequence information was obtained from the Ensembl Genomes website. The plant materials “Shenghan 740” used in the experiment were supplied by the Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences. The datasets supporting the conclusions of this article are included in the article and its Supplementary Files. Ethics approval and consent to participate All plant materials used in the current study complied with the relevant institutional, national, and international guidelines and legislation. This article does not contain any studies with human participants or animals performed by the authors. These methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not Applicable. Competing interests The authors declare that they have no conflicts of interest. Publisher ’ s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Wang Y, Deng D, Zhang R, Wang S, Bian Y, Yin Z. Systematic analysis of plant-specific B3 domain-containing proteins based on the genome resources of 11 sequenced species. Mol Biol Rep. 2012; 39(5): 6267 - 6282. Peng F, Weselake RJ. 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Nai G, Liang G, Ma W, Lu S, Li Y, Gou H, et al. Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme. Bmc Plant Biol. 2022; 22(1): 344. Table Table 3 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1SecondarystructureofCaRAVsprotein.png SupplementaryFigure2PlantperformanceofShenghan740underrepeatedchillingstress.png SupplementaryFigure3ExpressionofGFPgreenfluorescenceinsilencedplantspeciesaftersevendays.png SupplementaryFigure4ExpressionofGFPgreenfluorescenceinsilencedplantspeciesAftersixtrueleaves.png SupplementaryFigure5FulluncroppedGels.jpg SupplementaryTable1Primerofsequenceusedinthisstudy.xlsx SupplementaryTable2Listofthe6CaRAVsgenesidentifiedinthisstudy.xlsx SupplementaryTable3DistributionofCaRAVsonchromosomes.xlsx SupplementaryTable410conservedmotifs.xlsx SupplementaryTable512regulatoryelements.xlsx Table.3AnalysisofthetertiarystructureoftheCaRAVsprotein.xlsx Cite Share Download PDF Status: Published Journal Publication published 29 Jul, 2024 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 10 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviews received at journal 05 Jun, 2024 Reviews received at journal 04 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers agreed at journal 02 Jun, 2024 Reviewers agreed at journal 31 May, 2024 Reviewers agreed at journal 26 May, 2024 Reviewers invited by journal 25 May, 2024 Editor invited by journal 21 May, 2024 Editor assigned by journal 21 May, 2024 Submission checks completed at journal 14 May, 2024 First submitted to journal 10 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4399432","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304884214,"identity":"ee9b1102-dbf3-4641-b48a-a737154c9884","order_by":0,"name":"Minkun Pei","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Minkun","middleName":"","lastName":"Pei","suffix":""},{"id":304884215,"identity":"22da2c02-7776-4d15-9e46-4fdd88940be1","order_by":1,"name":"Ping Yang","email":"","orcid":"","institution":"Honghe University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Yang","suffix":""},{"id":304884216,"identity":"3957aecb-b0dd-4c36-8bcf-3fcfc41f839c","order_by":2,"name":"Jian Li","email":"","orcid":"","institution":"Honghe University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Li","suffix":""},{"id":304884220,"identity":"d354c97e-8ec4-4e6d-aa42-55e38e717379","order_by":3,"name":"Yanzhuang Wang","email":"","orcid":"","institution":"Honghe University","correspondingAuthor":false,"prefix":"","firstName":"Yanzhuang","middleName":"","lastName":"Wang","suffix":""},{"id":304884221,"identity":"96b62de9-3e40-40cd-93fd-b7c8a532d52c","order_by":4,"name":"Juan li","email":"","orcid":"","institution":"Honghe University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"li","suffix":""},{"id":304884222,"identity":"9d5faae5-9789-4bc5-b3e4-1c5ff0c7b901","order_by":5,"name":"Hongjun Xu","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"Xu","suffix":""},{"id":304884223,"identity":"5f194795-2fb9-44a5-b1b8-1bc69ee9fa29","order_by":6,"name":"Jie Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYLCCDz9sePjZG0jQwTizJ01GsucACVqYedgO2xjccCBSubz/4WcSPDzneRhuMDB++JhDhBbDG2lmEhIWt3kYZzcwS87cRoyWGQxmEgY8t3mYZQ6wMfMSpaX/+DeJBLZzPGxAkjgt8gw5ZhIH2A7w8BCtxUAip9iysSeZR4LnYDNxfpHvP77x9p8fdvb2x5sPfvhIlC0H4EzGBiLUg2whUt0oGAWjYBSMZAAAonEywavHT8IAAAAASUVORK5CYII=","orcid":"","institution":"Honghe University","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-05-10 08:51:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4399432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4399432/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-024-10639-x","type":"published","date":"2024-07-29T15:58:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56984188,"identity":"8c5e3ae5-78a6-4b51-b0ec-fe0e54b8b280","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":564622,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram illustrating the arrangement of pepper \u003cem\u003eRAVs\u003c/em\u003e on the chromosomes. The chromosomes of the pepper plants are represented by vertical bars. The chromosome number is displayed adjacent to each chromosome. The vertical axis on the left side of the diagram represents the length of the chromosomes.\u003c/p\u003e","description":"","filename":"Figure1DistributionofCaRAVsonchromosomes.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/0a27b7e37eef6c931474750b.jpg"},{"id":56984631,"identity":"a67b2893-f202-4539-ae04-f325c9687806","added_by":"auto","created_at":"2024-05-23 04:43:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":710881,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree without a root, illustrating the connections between the \u003cem\u003eRAV\u003c/em\u003e of Arabidopsis, tomato, and pepper. The organisms are categorized into four clades based on their homologous evolutionary relationships. Clades I, II, III, and IV are represented by lilac, carnation, olive-green, and smalt branch lines, respectively. The \u003cem\u003eRAV\u003c/em\u003e obtained from pepper is designated with an asterisk prefix.\u003c/p\u003e","description":"","filename":"FIgure2PhylogenetictreeofCaRAVsSlRAVsandAtRAVs.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/f34013282101690915e92060.jpg"},{"id":56984196,"identity":"cabd3a98-42ac-4071-a4d1-1e303776b912","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":811874,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motif, structural domain, and gene structure analysis of AtRAVs, SlRAVs, and CaRAVs gene families. (A) The ten conserved motifs are represented by rectangles of different colors. (B) The domains are shown in three colored rectangles green for B3, yellow for AP2, and gray for non-domain areas. (C) Exons and introns are replaced by green matrices and gray lines, respectively, and can be judged by the number of corresponding colors and rectangles. The black lines indicate relative lengths. The \u003cem\u003eRAV\u003c/em\u003e from pepper is labeled with an asterisk suffix.\u003c/p\u003e","description":"","filename":"Figure3ThephylogenetictreegenestructureanddomainofAtRAVsSlRAVsandCaRAVs.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/6eb3994c8ab844eede5c87fb.jpg"},{"id":56984194,"identity":"2a355281-d99b-4105-8a29-c0d08db6a32c","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":935187,"visible":true,"origin":"","legend":"\u003cp\u003eHomology analysis of the \u003cem\u003eRAV\u003c/em\u003e between pepper\u003cem\u003e \u003c/em\u003eand the representative plants Arabidopsis and tomato. The colored circular rectangles denote the chromosomes of the three plants. The grey curves indicate collinear blocks within the genomes, and the blue curves represent collinear gene pairs. The red triangle represents the location of the \u003cem\u003eRAV\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4Collinearityofthethreespecies.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/dde5b3d2186522dfe4047299.jpg"},{"id":56984206,"identity":"8b3ca8c0-4da5-4bdd-8075-4a731d58f603","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1222141,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of \u003cem\u003ecis\u003c/em\u003e-elements in \u003cem\u003eRAV\u003c/em\u003e promoters, which are conserved regulatory elements associated with plant hormone response, stress response, and growth regulation. (A) Distribution of the 12 main \u003cem\u003ecis\u003c/em\u003e-elements in \u003cem\u003eCaRAVs\u003c/em\u003e, with different colored shapes indicating the positions of the different elements in the promoter. (B) Clustering heatmap of the number of promoter \u003cem\u003ecis\u003c/em\u003e-elements. Black numbers are numbers, red means ten or more, and gray means zero.\u003c/p\u003e","description":"","filename":"Figure5CisactingelementsinCaRAVsgenefamilypromoters.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/7b6cac6e024cf3b079ec2003.jpg"},{"id":56984205,"identity":"e663f1b0-651a-42e5-8915-c4c231f98e2b","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":315479,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression profiles of the \u003cem\u003eCaRAVs\u003c/em\u003e gene subfamily were analysed after the plants were subjected to three days of chilling stress. The expression amount is converted to a 2-based log function and then normalized by the row using the normalization method. The color scale to the right of the heatmap indicates the relative expression level, with an elevated expression level shown by the color gradient ranging from dodger blue to red. Pepper \u003cem\u003eActin\u003c/em\u003e primers were used as internal reference primers for qRT-PCR (see Supplementary Table 1 online). The following figure is the same as.\u003c/p\u003e","description":"","filename":"Figure6ExpressionpatternofCaRAVsunderchillingstressconditions.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/5f89507069aa9813c9b4d7cc.jpg"},{"id":56984198,"identity":"c165bf01-9415-42bb-a987-d01197458cee","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1065191,"visible":true,"origin":"","legend":"\u003cp\u003eChilling stress induces the expression of \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e. The experiment consisted of four treatments: the control group (Ctl); the direct stress group (Stro), which was subjected to chilling stress at 5\u003csup\u003eo\u003c/sup\u003eC for 24 hours; the recovery group (Rec), which experienced chilling stress for 24 hours at 25\u003csup\u003eo\u003c/sup\u003eC followed by 1 day of recovery; and the restress group (Str1) which underwent an additional 24 hours of chilling stress after the recovery period. (A) The relative expression of \u003cem\u003eCaRAV1\u003c/em\u003e was measured in the four treatment groups. (B) The relative expression of \u003cem\u003eCaNGA1\u003c/em\u003e. The experiments were replicated three times. The provided data represent the mean values of three repeated measurements, with the standard deviation (SD) indicated by the error bars. The presence of distinct letters above or below the error bars indicates significant differences at a significance level of \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, the same below.\u003c/p\u003e","description":"","filename":"Figure7ResponseofCaRAV1andCaNGA1underrepeatedchillingstress.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/c5dedfe77133a68949d54d30.jpg"},{"id":56984201,"identity":"fcae3658-791f-4c88-93e3-6cd2db159ddd","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":858043,"visible":true,"origin":"","legend":"\u003cp\u003eSemi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to analyse the expression levels of \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaRAV2\u003c/em\u003e in pepper plants subjected to virus-induced gene silencing, full uncropped Gels are shown as Supplementary Fig. 5 online. (A) Maker (2000 bp) is shown on the left side of the Figure. The white band on the right side of Maker, a positive control, is between 250 bp and 500 bp. It can be inferred that \u003cem\u003eCaRAV1\u003c/em\u003e contains three groups of repetitions of approximately 300 bp. (B) Four groups of different plants were used: blank control (Ctl), positive control (pTRV2:\u003cem\u003ePDS\u003c/em\u003e), blank vector (pTRV2:GFP), and pTRV2:\u003cem\u003eRAV1\u003c/em\u003e silenced plants. (C) Semi-quantitative electrophoresis (EP) of\u003cem\u003e CaNGA1\u003c/em\u003e, which was repeated four times, revealed that the band position was approximately 300 bp. (D) The relative expression of \u003cem\u003eCaNGA1\u003c/em\u003e was measured in four different groups of pepper plants.\u003c/p\u003e","description":"","filename":"Figure8AssessmentoftheeffectivenessofsilencinginCaRAV1andCaNGA1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/043b365c22e7437a5211b884.jpg"},{"id":56984191,"identity":"203ac595-bc33-43b8-b6c8-60ae4f96fdda","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1746774,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of the alterations in the MDA content and REC in pepper leaves subjected to four different groups of chilling stress treatments. (A) MDA content. (B) REC change trend. The treatment of chilling stress is the same as above.\u003c/p\u003e","description":"","filename":"Figure9MDAcontentandrelativemembranepermeabilityofthesilencedpeppers.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/857772c126b73aeaf2bc370f.jpg"},{"id":56984203,"identity":"20daf8ea-abbe-47f7-8f3c-4ef6b4332e3a","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":992959,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of alterations in SOD, POD, and CAT enzyme activities in pepper leaves subjected to four different groups of chilling stress treatments.(A)SOD, (B)POD, (C)CAT.\u003c/p\u003e","description":"","filename":"Figure10Antioxidantenzymeactivityofthesilencedpeppers.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/1386588036ef56eab98e48df.jpg"},{"id":61793785,"identity":"892bfc7d-0a47-4ede-8346-8b098024f24d","added_by":"auto","created_at":"2024-08-05 16:15:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10225716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/61e09830-842d-4400-ba7a-53882c91c104.pdf"},{"id":56984626,"identity":"256b70a9-6df7-4623-a2db-1ad832dbbf9f","added_by":"auto","created_at":"2024-05-23 04:43:00","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4541559,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1SecondarystructureofCaRAVsprotein.png","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/35f04425cd62171876037476.png"},{"id":56984630,"identity":"97939bf8-ed9a-4617-a740-b09cd5334bc4","added_by":"auto","created_at":"2024-05-23 04:43:00","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14156694,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2PlantperformanceofShenghan740underrepeatedchillingstress.png","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/f5197ef3b76654ce1a4e1196.png"},{"id":56984193,"identity":"28467965-4db2-46f4-ac52-d0f52d877a81","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8822261,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3ExpressionofGFPgreenfluorescenceinsilencedplantspeciesaftersevendays.png","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/89358416b03e97cb9fd2e05a.png"},{"id":56984208,"identity":"066e9a5d-4caf-46ab-b6f1-7926080bca13","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21786594,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4ExpressionofGFPgreenfluorescenceinsilencedplantspeciesAftersixtrueleaves.png","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/a5df7cce75a10e366c4c218c.png"},{"id":56984199,"identity":"5172bbdf-d684-4ae3-b114-0203e5d498ea","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":199054,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure5FulluncroppedGels.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/d84143f452654cd47ba64de9.jpg"},{"id":56984204,"identity":"7f5a8e98-1d53-461f-a510-9eebd3304405","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10180,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1Primerofsequenceusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/40115a122ccbff3300dca869.xlsx"},{"id":56984632,"identity":"fac22994-4635-4d68-84ce-18293dbc600f","added_by":"auto","created_at":"2024-05-23 04:43:01","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12982,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2Listofthe6CaRAVsgenesidentifiedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/52c1fbe20d39b8e02c52441f.xlsx"},{"id":56984209,"identity":"2604e219-cff1-4cbe-a656-085719eb48ae","added_by":"auto","created_at":"2024-05-23 04:35:02","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":9518,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3DistributionofCaRAVsonchromosomes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/942b9d82d4c95c8b8a1c9bd3.xlsx"},{"id":56984200,"identity":"2dd65fe5-339e-4872-af1b-16b791075d01","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":9715,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable410conservedmotifs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/5a5dde4864e3b36f0ee80de2.xlsx"},{"id":56984195,"identity":"31c185d1-1990-49fe-8288-e799b0ad9384","added_by":"auto","created_at":"2024-05-23 04:35:00","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":12787,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable512regulatoryelements.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/d8628016539d8869f614cb96.xlsx"},{"id":56984202,"identity":"d5ac0cb3-9b6a-4ec4-9bf6-b23d8ace18fe","added_by":"auto","created_at":"2024-05-23 04:35:01","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":33979,"visible":true,"origin":"","legend":"","description":"","filename":"Table.3AnalysisofthetertiarystructureoftheCaRAVsprotein.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4399432/v1/8f09f3299be79f81788910f2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress","fulltext":[{"header":"Background","content":"\u003cp\u003eThe B3 transcription factor (TF) is exert significantly influences over the ability of plants to respond to stresses, growth and development. Typically manifesting as a DNA-protein binding domain in plants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There are several distinct groups of B3-specific family members, including REM, LAV, RAV and ARF family members. Recent research indicated that there was a fifth member, HSI, Recent research indicated that there was a fifth member, HSI, which expands the B3 superfamily evolutionary footprint, and is particularly evident in cruciferous plants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This expansion has led to the emergence of more complex structures, such as domain repeats and gene tandem repeats. Notably, the RELATED to ABI3/VP1 (RAV) transcription factor stands out as exclusive to higher plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While most B3 superfamily genes feature at least one B3 domain, certain RAV genes subfamily also contain a second domain known as APET-ALA2 (AP2). Akhter et al. classified genes that exhibited double domains and domain replication as members of the RAV subfamily [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On the other hand, the RAV subfamily, which comprises a single B3 domain is commonly referred to as the NGA translation factor subfamily. Thirteen \u003cem\u003eRAV\u003c/em\u003e family members have been identified thus far in Arabidopsis. Among these, six belong to the AP2 domain and seven are members of the B3 RAV subfamily (\u003cem\u003eNGATHE)\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. RAV serving as a multifunctional transcription factor, has been implicated in the negative regulation of plant growth under various stress conditions, including salt stress and drought, across multiple plant species such as rice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], soybean [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], barley [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], cotton [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and cucumber [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, abscisic acid (ABA), auxin (IAA), and additional stresses regulate RAV expression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous studies have employed Arabidopsis RAV1 as a transcription factor for chilling stress [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. More recent reports have further substantiated this claim. \u003cem\u003eRAV\u003c/em\u003e expression in pepper [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], tea plant [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and rape [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] is up-regulated under chilling stress. In addition, aside from its known regulatory effects on ABA and brassinosteroids (BRs) and its role in the abiotic stress response, Song et al. also reported that the celery \u003cem\u003eRAV\u003c/em\u003e negatively regulates zeaxanthin cyclooxygenase (ZEP), and ultimately positively regulates carotenoid synthesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although they have been studied in various species, the number and functions of pepper RAV TFs are still unclear.\u003c/p\u003e \u003cp\u003ePepper(\u003cem\u003eCapsicum annuum\u003c/em\u003e L.) stands as a pivotal cash crop with a long history of cultivation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Ranked as the second largest vegetable crop globally, it spans a plantinh area of 1.4\u0026ndash;1.6\u0026nbsp;million hectares. However, its susceptibility to temperature and light renders it vulnerable to damage under conditions of low temperature and inadequate light exposure. The impact of temperature fluctuations, especially during autumn and spring, significantly influences both the productivity and quality of pepper [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, growth and development are impeded and yields diminish when temperatures drop below 15\u003csup\u003eo\u003c/sup\u003eC or light intensity falls below 25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Low temperatures not only have a macroscopic impact on plants but also disrupt the intracellular metabolic equilibrium. Upon initial exposure to low temperature, plants experience an accumulation of reactive oxygen species (ROS) within their cells, resulting in the peroxidation of membrane lipids. When the production of reactive oxygen species (ROS) surpasses the body's ability to remove them, it results in an imbalance of antioxidants and triggers an oxidative stress response, which in turn can induce DNA damage [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough there are existing reports on the RAV transcription factor subfamily, detailed information on the specific activities of its members, apart from RAV1, remains limited. A comprehensive characterization of the RAV genes family in pepper, especially its response mechanisms to chilling stress, has yet to be established.This study identified revealed six \u003cem\u003eRAVs\u003c/em\u003e within the entire pepper genome and analysed them. Conserved domains evolutionary relationships, gene structure, and \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter region. Additionally, virus-induced gene silencing and Quantitative Real-time PCR (qRT-PCR) techniques were employed to investigate the expression patterns of key genes from the CaRAVs gene family in response to low temperatures. These results indicate that RAV proteins may play roles in plant responses to chilling stress. Given the potential importance of \u003cem\u003eRAV\u003c/em\u003e genes in plant responses to chilling stress environments. The aim of this study was to examine the impact of the \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e genes, which are characterized by distinct domains, on pepper plants experiencing chilling stress.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of gene family members\u003c/h2\u003e \u003cp\u003eThe acquired genomic materials consisted of the tomato, pepper and B3 superfamily genomes of Arabidopsis. The \u003cem\u003eAtRAVs\u003c/em\u003e gene family database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/browse/genefamily/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/browse/genefamily/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was accessed to obtain 87 protein sequences belonging to the Arabidopsis B3 family. Additionally, the database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) provides relevant data regarding the \u003cem\u003eSolanum lycopersicum\u003c/em\u003e and \u003cem\u003eCapsicum annuum\u003c/em\u003e genomes. The NCBI online software BLSAT was utilized to compare the predicted peppers (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conserved domain of the protein was identified using the second alignment of pepper protein sequences in the InterPro online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/search/sequence/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/search/sequence/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, sequences that were in a disarray state were eliminated, resulting in the acquisition of pepper B3 family genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical properties of amino acids in the CaRAVs subfamily\u003c/h2\u003e \u003cp\u003eThe amino acid length, relative molecule, relative isoelectric point, amino acid stability, fat solubility index, and average hydrophilic coefficient of the pepper \u003cem\u003eCaRAVs\u003c/em\u003e were predicted using an online website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.ExPASy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.ExPASy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, subcellular localization prediction of the gene was performed using online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein structure prediction for the CaRAVs subfamily\u003c/h2\u003e \u003cp\u003eUsing PSIPRED online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"http://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swisodel.ExPASy.org/\u003c/span\u003e\u003cspan address=\"https://swisodel.ExPASy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) provided by the protein expert system ExPASy, the secondary structures of proteins encoded by target CaRAVs were predicted respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the domains, gene structure, and phylogenetic tree\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of the target CaRAVs were compared with those of Clustal 2.1, and the online tool MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify highly conserved motifs. Highly homologous RAV transcription factors from rice and Arabidopsis that matched each target CaRAVs were acquired from the transcription factor database PlantTFDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://planttfdb.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A phylogenetic tree was then constructed using the neighbor-joining (NJ) method in the software MEGA 11. The relevant parameter settings are as follows: the bootstrap value is set to 500, the partition deletion is set to 50%, and the genetic distance model is selected as JJT\u0026thinsp;+\u0026thinsp;G for calculation, which is the optimal recommendation. The remaining parameter values are set to their default values.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction analysis of\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-acting elements in the CaRAVs promoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe promoter region (2000 bp upstream of ATG) of the target \u003cem\u003eRAV\u003c/em\u003e was obtained from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003ecis\u003c/em\u003e-acting elements of the promoter region were screened and analysed using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized with TBtools. The PlantCARE website (available at .be/web tools/plantcare/html/) was utilized to examine and analyse the \u003cem\u003ecis\u003c/em\u003e-acting elements found in the promoter region. These elements were then visualized using TBtools.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity analysis of the\u003c/b\u003e \u003cb\u003eCaRAVs\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe collinearity analysis was conducted using the MCScanX software, and the resulting collinearity data were visualized using TBtools, which can be found at the following link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/CJ-Chen/TBtools\u003c/span\u003e\u003cspan address=\"https://github.com/CJ-Chen/TBtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and chilling stress treatment\u003c/h2\u003e \u003cp\u003eThe test material was \u003cem\u003eCapsicum annuum\u003c/em\u003e \u0026ldquo;Shenghan 740\u0026rdquo;. The seedlings were grown in a mixed substrate consisting of peat, perlite, and vermiculite at a ratio of 3:1:1(v/v). The plants were cultivated within 25\u003csup\u003eo\u003c/sup\u003eC, a photoperiod of 12 h, and a relative humidity of 75%. After the peppers had grown to 5\u0026ndash;6 true leaves, they were placed in a 5\u003csup\u003eo\u003c/sup\u003eC thermostat for chilling stress treatment. The leaves of the pepper plants were then cut and treated for different durations of 0, 1, 3, 5, 7, 12, 24, 48, and 72 h. These treated leaves were promptly frozen in liquid nitrogen for further experiments. Each treatment was replicated at least three times. For repeated treatments of chilling stressed plants, the seedlings exposed to low temperatures a undergone a 24 h recovery period before they were exposed to chilling stress again.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eLeaf samples (100 mg) were pulverized in liquid nitrogen, and total RNA was isolated using a TRIzol kit (Beijing Tsingke Biotech Co., Ltd.) following the provided instructions. The RNA concentration was measured using a NanoDrop microspectrophotometer (Thermo Fisher Scientific, USA), and its purity was assessed using the A260/280 ratio. The A260/280 ratio was used to determine the purity of the sample. The error-free RNA was diluted to a concentration of 400 ng/\u0026micro;L using distilled water and then stored at -80\u003csup\u003eo\u003c/sup\u003eC for subsequent reverse transcription. Complementary DNA (cDNA) was synthesized in the reverse direction using Tsingke RT6 reverse transcriptase following the provided instructions. The cDNA was synthesized by designing real-time quantitative primers using Primer-BLAST from the NCBI website. The chimeric fluorescence method was used, with pepper \u003cem\u003eActin\u003c/em\u003e serving as an internal reference and SYBR Green I from Tsingke Biotech was used as the fluorescent dye. Real-time polymerase chain reaction (PCR) was performed using a Bio-Rad system. The primers used can be found as Supplementary Table\u0026nbsp;1 online. The relative expression of genes was calculated using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method. Three replicates were performed for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVector construction\u003c/h2\u003e \u003cp\u003eThe ligation primer that was designed using CE Design V1.03 software, based on the target gene sequence and intended for use with the Tobacco Rattle Virus (TRV) vector, the length of the primer fragment containing the homology arm of the vector is around 40bp. The process of obtaining RNA has been previously described. However, the acquisition of cDNA differs from that of qRT-PCR because the reverse transcription system utilizes Oligo (dT\u003csub\u003e17\u003c/sub\u003e) instead of Randomer. Use of Rapid 2 \u0026times; Taq Master Mix from Nanjing Vazyme Biotech Co., Ltd to amplify the target gene fragment. Next, \u003cem\u003eSaml\u003c/em\u003e enzyme digestion was used to release the linear fragment of the TRV vector. The 300 bp purified fragment was connected to TRV using T4 ligase. The monoclonal antibody (kanamycin) was then incubated with the cells overnight on an inverted plate. Positive clones were identified through electrophoresis after a single colony was shaken at 37\u003csup\u003eo\u003c/sup\u003eC for 12 hours. To identify false-positive vectors, it is essential to verify whether they align with the target sequence during the sequencing process. Following sequencing, the \u003cem\u003eEscherichia coli\u003c/em\u003e solution containing the homologous arm was subsequently transferred to Agrobacterium competent GV3101. The Agrobacterium was then cultured on three different antibiotics: kanamycin, gentamicin, and rifampicin. Following agitation of the bacteria in a solitary colony, electrophoresis was subsequently performed to verify the uniformity of the vectors. Ultimately, the silencing vectors pTRV2-\u003cem\u003eRAV1\u003c/em\u003e, pTRV2-\u003cem\u003eNGA1\u003c/em\u003e, and pTRV2-\u003cem\u003ePDS\u003c/em\u003e were generated. Fifty percent glycerin was combined with the bacterial solution at a 1:1 ratio, and the mixture was stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSilencing efficiency assay for VIGS vectors\u003c/h2\u003e \u003cp\u003eA single colony was selected after the vector containing the target fragment was coated with three antibodies on luria bertani (LB) solid media and cultured at a temperature at 28\u003csup\u003eo\u003c/sup\u003eC for two days. After three cycles of oscillation in LB activated bacterial solution, a single colony was selected. Twelve hours after the initial bacterial suspension was shaken, suitable concentrations of acetosyringone (AS) and 2-Morpholinoethanesulfonic acid (MES) were added to the second shaking bacterial suspension, the third polar fungus was added prior to incubation, and the levels of MES and AS in the LB liquid media were elevated. The mixture was then incubated for 12 hours and subsequently subjected to centrifugation at a speed at 4000 rpm for 15 minutes at 4\u003csup\u003eo\u003c/sup\u003eC. The infection buffer was prepared, and the bacterial solution was resuspended in MaCl\u003csub\u003e2\u003c/sub\u003e buffer solution. The bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e) was diluted to approximately 0.04 using a buffer solution. A 1:1 ratio of infected liquid pTRV1 was combined with pTRV2:GFP (blank vector with label), pTRV2:\u003cem\u003ePDS\u003c/em\u003e (positive albino control), pTRV2:\u003cem\u003eRAV1\u003c/em\u003e, and pTRV2:\u003cem\u003eNGA1\u003c/em\u003e to infect the leaves. Virus-infected seedlings were placed in a temperature-controlled room with 75% humidity and 22\u003csup\u003eo\u003c/sup\u003eC for 24 hours. After this time, the growth conditions were maintained at a temperature of approximately 22\u003csup\u003eo\u003c/sup\u003eC, following a standard photoperiod of 12 hours of light and 12 hours of darkness. After three days of culture, the fluorescence emitted by GFP, which had been labeled with a green dye, was observed using a 254 nm ultraviolet lamp. After approximately two weeks, the pTRV2-PDS plants were albino and subjected to qRT‒PCR analysis to determine their silencing efficiency. Sixty plants with a silencing efficiency above 50% and sixty plants with pTRV2 were chosen for low-temperature treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA) concentration, membrane permeability, and antioxidant enzyme activity were quantified\u003c/h2\u003e \u003cp\u003eThe MDA concentration was ascertained using a modified version of the thiobarbituric acid colorimetry method as described by Dhindsa [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The relative membrane permeability (REC) was calculated as a percentage according to Yang et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The quantification of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity was conducted using the guaiacol method as described by Jebara et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and Almeselmani [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. SPSS version 19.0 software was used to analyse the significance of difference by \u003cem\u003et\u003c/em\u003e tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification and prediction of the physicochemical properties of CaRAVs family genes\u003c/h2\u003e\n \u003cp\u003eBLAST analysis was performed using the Arabidopsis B3 transcription factor family and the complete pepper genome, resulting in the prediction of 12 related pepper CDS sequences. The software TBtools was utilized to acquire the 12 protein sequences. Subsequently, the structural domains were identified and the extraneous sequences were eliminated using InterPro. Ultimately, out of all the identified proteins, only six were classified as typical pepper RAV proteins, as indicated as Supplementary Table\u0026nbsp;2 online. To summarize, a total of six candidate genes have been verified as members of the pepper RAV family. The CaRAVs of pepper were named CaRAV1, CaRAV2, CaRAV3, CaNGA1, CaNGA2, and CaNGA3 in this study due to their homology and structural domain differences with those of Arabidopsis.\u003c/p\u003e\n \u003cp\u003eAccording to the results presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, all the CaRAVs were found in the nucleus, while CaNGA3 was also present in the cytoplasm. The amino acid length of the CaRAVs ranged from 130 to 480 aa. Specifically, CaRAV2 had the fewest amino acids with a length of 137 aa, while CaRAV3 had the greatest number of amino acids with a length of 477 aa. The molecular weights of CaRAVs ranged from 14,940 to 52,496 kDa. Among them, CaRAV3 had the highest molecular weight of 52,495.23 kD, while CaRAV1 had the lowest molecular weight of 14,940.15 kDa. The isoelectric points of the CaRAVs amino acids varied between 5.93 and 9.78, with CaRAV2 having the highest isoelectric point of 9.78 and CaNGA2 having the lowest isoelectric point of 5.93. Furthermore, the stability index (\u0026gt;\u0026thinsp;40) and average total hydrophilicity (\u0026lt; -0.5) suggest that CaRAV1, CaRAV3, and CaNGA3 are amino acids that are both unstable and hydrophilic. On the other hand, CaRAV2, CaNGA1, and CaNGA2 are both stable and hydrophilic amino acids.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnalysis of important physicochemical properties of amino acids encoded by CaRAVs\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmino acid\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of amino acids(aa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular weight (kDa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTheoretical\u003c/p\u003e\n \u003cp\u003e(pI)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstability\u003c/p\u003e\n \u003cp\u003eindex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrand\u003c/p\u003e\n \u003cp\u003eaverage of\u003c/p\u003e\n \u003cp\u003ehydropathicity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAliphatic\u003c/p\u003e\n \u003cp\u003eindex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubcellular\u003c/p\u003e\n \u003cp\u003elocalization\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27790.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14940.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52495.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e176\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20310.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38699.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40182.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.728\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCytoplasm/Nucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eDistribution of CaRAVs on chromosomes\u003c/h2\u003e\n \u003cp\u003eUsing TBtools software in conjunction with the chilli genome annotation files, the location and distribution of CaRAVs was mapped on chromosomes. The graph (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) shows that among the 12 chromosomes of pepper (see Supplementary Table 3 online), \u003cem\u003eCaRAVs\u003c/em\u003e was exclusively found on Chr1, Chr10, Chr11 and Chr12. Chr1 and Chr11 each had two members, while the other two chromosomes had only one \u003cem\u003eRAV\u003c/em\u003e. Moreover, all members of this gene family were located at the ends of chromosomes, and no \u003cem\u003eRAV\u003c/em\u003e was present in the middle region of any chromosome. A linkage region comprising the genes \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaRAV2\u003c/em\u003e was established at the beginning of Chr11. The two genes were surrounded by continuous bases extending 100 bp upstream and downstream, based on their location and length.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCaRAVs protein structure correlation\u003c/h2\u003e\n \u003cp\u003eThe PSIPRED software was used to predict the secondary structure of the protein. Based on the results (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), it was discovered that CaRAVs protein consisted of three components: \u0026beta; corner, extended main chain, and an irregular curl (see Supplementary Fig.\u0026nbsp;1 online). However, the relative sizes of each structure varied. Irregular curling accounts for 49.85\u0026ndash;78.62% of the total proportion. The \u0026beta; rotation angle and extended main chain components had relatively low percentages, ranging from 11.74\u0026ndash;28.98% and 5.60\u0026ndash;33.53%, respectively. The findings indicated that the primary secondary structure of CaRAVs is an irregular curl. The extended backbone and \u0026beta; angle, on the other hand, may serve a supplementary and modifying function, thereby aiding in the formation of complex protein conformations at a later stage and performing specific functions.\u003c/p\u003e\n \u003cp\u003eSWISS-MODEL was used to predict the tertiary structure of the CaRAVs protein (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The alignment rate between all members of the CaRAVs and templates exceeded 70%, and the root mean square deviation (RMSD) was less than 2.5 \u0026Aring;. These findings suggest that the homology modeling outcomes are reasonably dependable [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. CaNGA1, CaNGA2 and CaNGA3 matched protein templates containing the B3 structural domain, whereas CaRAV1, CaRAV2 and CaRAV3 matched protein templates containing both the B3 and AP2 structural domains.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSecondary structure analysis of the CaRAVs protein\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026beta;-Strand amount\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026beta;-Strand\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHelix amount\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHelix\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCoil amount\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCoil\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.18%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.34%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.48%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.41%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.68%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaRAV3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.74%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e78.62%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.98%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58.52%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.62%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.53%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaNGA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003ePhylogenetic tree of CaRAVs, SlRAVs and AtRAVs\u003c/h2\u003e\n \u003cp\u003eThe amino acid sequences of CaRAVs were subjected to BLAST searches against PlantTFDB. RAV proteins in Arabidopsis and tomato that were found to be highly homologous to CaRAVs (top 10 in terms of score) were screened, repetitive sequences were eliminated, and a phylogenetic tree was constructed using 9 SlRAVs and 13 AtRAVs in conjunction with 6 CaRAVs present in CaRAVs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The findings indicated that all individuals from the three species were classified into four distinct clusters (I - IV), with minimal variation in the population amount across the four groups. A comparison of phylogenetic trees revealed that SlRAVs and CaRAVs are more homologous than are AtRAVs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eThe phylogenetic tree, gene structure, and domain of AtRAVs, SlRAVs, and CaRAVs\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the Arabidopsis and tomato RAV gene families in the tree, along with the domain and conserved sequence of the RAV gene family. Additionally, we included pepper in the analysis. We utilized the MEME online tool to analyse the amino acid sequences of the CaRAVs. From this analysis, we identified 10 conserved motifs based on their P-values (see Supplementary Table 4 online). The findings indicated variations in the location and abundance of each motif within the amino acid sequence of CaRAVs. Two motifs, motif 1 (LLNFEDENGKVWRFRYSYWNSSQSYVLTKGWSRFVKEKKLDAGDVVSFQR) and motif 2 (ALIEREHLFEKVVTPSDVGKLNRLVIPKQHAEKYFPLDSSN), are found to be highly conserved across various CaRAVs sequences. Thus, motif 1 and motif 2 comprise the conserved region of CaRAVs transcription factors. CaRAV2 lacks motif 1 and motif 2, but it exhibits greater similarity to the conserved region of AtRAVs. Additionally, CaRAV2 is highly homologous to CaRAV2 in Arabidopsis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), the cause of this outcome remains uncertain, and it is possible that CaRAVs have undergone a distinct evolutionary process. CaRAV2 has been determined to have a close relationship with the evolution of Arabidopsis, potentially due to the gene linkage previously observed in chromosome location.\u003c/p\u003e\n \u003cp\u003eBy examining the domain visualization, it is evident that the three species possess only two domains each, namely, the B3 and AP2 domains. Only domains within a 500 bp range are shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB. Based on the exon-intron structure depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, it is evident that \u003cem\u003eCaRAVs\u003c/em\u003e lack introns, with five members consisting of a single exon. In contrast, \u003cem\u003eCaNGA2\u003c/em\u003e contains multiple exons and introns.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eCollinearity of the three species\u003c/h2\u003e\n \u003cp\u003eTo elucidate the evolutionary relationship of the RAV across various plants, we used the genomes of Arabidopsis, tomato, and pepper plants to collinearity analysis using MCScanX software (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Three pairs of homologous genes were identified in Arabidopsis and pepper (\u003cem\u003eCaNGA1\u003c/em\u003e/\u003cem\u003eAtRAV-like1\u003c/em\u003e, \u003cem\u003eCaNGA1\u003c/em\u003e/\u003cem\u003eAtRAV-like2\u003c/em\u003e, and \u003cem\u003eCaNGA1\u003c/em\u003e/\u003cem\u003eAtRAV-like3\u003c/em\u003e), while two pairs of homologous genes were identified in tomato and pepper (\u003cem\u003eCaNGA3\u003c/em\u003e/\u003cem\u003eSlNGA2\u003c/em\u003e and \u003cem\u003eCaNGA2\u003c/em\u003e/\u003cem\u003eSlRAV3\u003c/em\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCis\u003c/strong\u003e \u003cstrong\u003e-acting elements in\u003c/strong\u003e \u003cstrong\u003eCaRAVs\u003c/strong\u003e \u003cstrong\u003egene family promoters\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo determine the biological function of the Ca\u003cem\u003eRAVs\u003c/em\u003e, an analysis of \u003cem\u003ecis\u003c/em\u003e-acting elements was conducted on the 2 kb promoter region located upstream of the initiation codon of the \u003cem\u003eCaRAVs\u003c/em\u003e. This analysis was performed using the Plant-CARE database. In the \u003cem\u003eCaRAVs\u003c/em\u003e promoter, we discovered 12 regulatory elements that are conserved and have a significant impact on plant stress response (see Supplementary Table 5 online), and growth regulation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The promoter region of the \u003cem\u003eCaRAVs\u003c/em\u003e family contains photoresponsive, MeJA-responsive, and anaerobic-inducing elements. In terms of quantity, light responsive elements are the most abundant. However, low-temperature responsive elements are only present in the Ca\u003cem\u003eNGAs\u003c/em\u003e subfamily. The results showed that the \u003cem\u003ecis\u003c/em\u003e-acting elements amount of \u003cem\u003eCaRAVs\u003c/em\u003e members varied to some extent, with \u003cem\u003eCaNGA1\u003c/em\u003e having a maximum of 24 components and \u003cem\u003eCaRAV2\u003c/em\u003e having a minimum of 16 components (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eExpression pattern of\u003c/strong\u003e \u003cstrong\u003eCaRAVs\u003c/strong\u003e \u003cstrong\u003eunder chilling stress conditions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo assess the response of the \u003cem\u003eCaRAVs\u003c/em\u003e to natural low temperatures and determine the extent of \u003cem\u003eCaRAVs\u003c/em\u003e involvement in chilling stress, we conducted qRT-PCR analysis of the \u003cem\u003eCaRAVs\u003c/em\u003e family after pepper plants were subjected to 3 days of treatment at 5\u003csup\u003eo\u003c/sup\u003eC. The relative expression levels of the \u003cem\u003eCaRAVs\u003c/em\u003e were calculated using \u003cem\u003eActin\u003c/em\u003e as an internal reference gene. The relative expression levels were visualized using a cluster thermograph (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The results demonstrated that the relative expression levels of all examined genes increased following exposure to chilling stress. During periods of chilling stress, the expression of \u003cem\u003eCaRAVs\u003c/em\u003e is typically elevated at 12 hours and 24 hours after stress occurs. Among the \u003cem\u003eCaRAVs\u003c/em\u003e, \u003cem\u003eCaRAV1\u003c/em\u003e, \u003cem\u003eCaRAV2\u003c/em\u003e, and \u003cem\u003eCaNGA1\u003c/em\u003e showed the most significant changes in expression.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eResponse of\u003c/strong\u003e \u003cstrong\u003eCaRAV1\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eCaNGA1\u003c/strong\u003e \u003cstrong\u003eunder repeated chilling stress\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBased on qRT-PCR heat map data, we selected two genes from the \u003cem\u003eCaRAVs\u003c/em\u003e gene family that exhibited the greatest response to chilling stress. These genes are \u003cem\u003eCaRAV1\u003c/em\u003e, which possesses AP2 and B3 domains, and \u003cem\u003eCaNGA1\u003c/em\u003e, which possesses a B3 domain. Low temperatures in nature may occur repeatedly for a period of time. We subjected \u003cem\u003eWT\u003c/em\u003e pepper plants to low temperatures of 5\u003csup\u003eo\u003c/sup\u003eC for 24 h (see Supplementary Fig. 2 online). The results showed that the expression of \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e was up-regulated after 24 hours of chilling stress in the Str0 group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). When plants were again subjected to chilling stress (Str1 group), \u003cem\u003eRAV1\u003c/em\u003e and \u003cem\u003eNGA1\u003c/em\u003e produced higher and faster expression at 24 h.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAssessment of the effectiveness of silencing in\u003c/strong\u003e \u003cstrong\u003eCaRAV1\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eCaNGA1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo study the role of various domains in the CaRAVs gene family under chilling stress, we created virus-induced silencing vectors (pTRV2:GFP, pTRV2:\u003cem\u003ePDS\u003c/em\u003e, pTRV2:\u003cem\u003eRAV1\u003c/em\u003e, and pTRV2:\u003cem\u003eNGA1\u003c/em\u003e) using tobacco crisp virus (TRV). All of the electrophoretic bands that were transferred into Agrobacterium GV3101 showed positive results when analysed using a gel imaging system, and the length of the target gene fragment was determined to be 300 base pairs(Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA,C). Furthermore, two groups with two cotyledons each were infected with the vector. After a period of 3\u0026ndash;7 days, the presence of green fluorescence, marked by the GFP tag, was detected using a 254 nm ultraviolet lamp. The study revealed that the plants with silenced genes were effectively infected during the initial phase (see Supplementary Fig.\u0026nbsp;3 online). The silencing efficiency of pTRV2-\u003cem\u003ePDS\u003c/em\u003e was confirmed by qRT-PCR after the albino phenotype appeared two weeks later. The findings indicated that the two vectors exhibited substantial suppression of expression in comparison to pTRV2:GFP and the control (Ctl), with a silencing efficiency exceeding 50% (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB,D). Once the plants reached the stage of having six true leaves, the GFP label of the silenced plants ceased to exhibit green fluorescence (see Supplementary Fig. 4 online).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eMDA content and relative membrane permeability of the silenced peppers\u003c/h2\u003e\n \u003cp\u003eTo assess the reactions of \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e to chilling stress. We analysed the indices associated with cell membrane damage (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), including the levels of MDA and REC, which exhibited a consistent upwards trend. The MDA (malondialdehyde) and REC (relative electrical conductivity) levels in the \u003cem\u003eRAV1\u003c/em\u003e-silenced plants were generally greater than those in the \u003cem\u003eNGA1\u003c/em\u003e-silenced plants. In all treatments, plants subjected to low temperature experienced a moderate increase in cell membrane permeability and MDA levels, but their ability to return to the original range was limited. Compared to blank vector damage, after the plants were subjected to the second stress, there was no significant change in the MDA content of the Str0 and Str1 silenced plants.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eAntioxidant enzyme activity of the silenced peppers\u003c/h2\u003e\n \u003cp\u003eLow-temperature treatment resulted in an increase in the activity of antioxidant enzymes. However, antioxidant enzyme activities were significantly lower in silenced plants compared to pTRV2 (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). After a single day of recovery from the initial chilling stress, the POD and CAT activities returned to their original levels in all the treatments, with the exception of SOD. The levels and rates of activity of antioxidant enzymes in the plants exposed to low temperature were greater and more rapid, while the silenced plants exhibited significantly lower levels than did the pTRV2 plants.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRAV, one of the subfamilies of the B3 transcription factor superfamily, has been demonstrated to have a significant impact on the physiology and biology of plants. The RAV subfamily is involved in plant growth, development, and responses to abiotic stress. Pepper is a significant horticultural crop in the global economy, and its demand is steadily rising each year [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Currently, pepper production often experiences economic and yield losses due to chilling stress. Despite significant advancements in chilling resistance, there is a scarcity of information regarding the response of pepper plants to chilling stress and CaRAVs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study discovered six members of the RAV gene subfamily in the entire pepper genome. These members include three members with the complete B3 domain, namely \u003cem\u003eCaNGA1\u003c/em\u003e, \u003cem\u003eCaNGA2\u003c/em\u003e, and \u003cem\u003eCaNGA3\u003c/em\u003e, and two members with both the B3 and AP2 domains, namely \u003cem\u003eCaRAV1\u003c/em\u003e, \u003cem\u003eCaRAV2\u003c/em\u003e, and \u003cem\u003eCaRAV3\u003c/em\u003e. Through the chromosome mapping, we determined that the RAV gene family members in pepper exhibit limited distribution, are distributed at both end positions of the chromosome, and the two closely related genes. Linkage regions was formed at the top of Chr11, comprising two genes, \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaRAV2\u003c/em\u003e. The 100 bp upstream and downstream of these two genes were considered continuous bases. This positional relationship may be associated with the evolutionary development of the RAV gene family and could also impact the regulatory function of the \u003cem\u003eRAV\u003c/em\u003e. Tandem replication is a frequently observed phenomenon in the evolution of plant genomes, particularly in \u003cem\u003eSolanaceae\u003c/em\u003e plants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These repetitive sequences, known as tandem repeats, have a significant impact on the ability of plants to adapt to environmental changes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present study, these two interlocking genes (CaRAV1 and Ca RAV2) produced higher relative expression in response to chilling stress.\u003c/p\u003e \u003cp\u003eCompared with those of other members of the CaRAV gene family, the closely associated genes CaRAV1 and CaRAV2 exhibit higher and more rapid increases in gene expression. It has been demonstrated that genes that are replicated in tandem are more likely to be involved in abiotic stress responses in Arabidopsis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, based on the modeling of homologous proteins, it has been observed that their structural homology indicates functional similarity. The 28 members of the three species were classified into four branches based on their gene structure and conserved motifs. This classification was used to construct a phylogenetic tree, which represents the homologous relationships among CaRAVs, AtRAVs, and SlRAVs. The findings indicated that the similarity between pepper and tomato was higher. Furthermore, based on the collinearity analysis of Arabidopsis, tomato, and pepper, we discover CaNGA1 exhibited collinearity with three AtRAVs members. This finding suggested that these genes likely have comparable functions and contributed significantly to the evolutionary dynamics of RAV gene families in pepper and Arabidopsis. It is worth noting that CaRAV1, CaRAV2, and CaNGA2 in tomato and Arabidopsis do not share a collinear relationship with the other two species, this suggests that these genes may be exclusive to the evolution of pepper. In addition to their role in gene expression regulation, introns also play a role in gene evolution [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. According to exon-intron analysis, CaNGA2 exhibited multiple introns, whereas other members lacked introns. Furthermore, among the 13 members in Arabidopsis, only 5 possess introns. The origin of intron-free genes and whether they lost introns during evolution or were absent from the beginning of development remain unclear. Studies in Arabidopsis have demonstrated that intron-free genes can be significantly activated in response to stress. Additionally, research in \u003cem\u003erice\u003c/em\u003e and Arabidopsis has shown that intron-free genes can play a crucial role in adapting to salt stress and drought [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExposure to chilling stress can disrupt the equilibrium of reactive oxygen species in plant cells, resulting in the accumulation of reactive oxygen species and subsequent membrane lipid peroxidation. During this process, plants initiate a highly efficient detoxification system and synchronize the activities of antioxidant enzymes and non-enzymatic substances. Currently, the antioxidant enzymes that are widely recognized include SOD, CAT, POD, and ascorbic acid POD (APX). The accumulation of these enzymes can safeguard plants against harm caused by low temperature [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In pepper production, various antistressors are commonly employed to mitigate abiotic stresses [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, chilling acclimation is utilized to enhance pepper tolerance to low temperatures [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This phenomenon, also known as \u0026ldquo;stress memory\u0026rdquo; [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], represents the plasticity mechanism of plants in response to production conditions. Within this study, wild-type pepper plants exhibited a recovery period of one day following exposure to low temperatures for one day. The relative expression levels of \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e also returned to their original baseline levels with increasing recovery time. Under repetitive chilling stress, these genes in plants respond more quickly thereby producing higher relative expressions, it consistent with the typical epigenetic alterations induced by chilling stress in plants. According to qRT-PCR validation of the virus-induced gene-silenced plants, the funny thing is that the \u003cem\u003eCaRAV1\u003c/em\u003e exhibited a greater relative expression level subsequent to albinism in the pTRV2:\u003cem\u003ePDS\u003c/em\u003e positive plants. The cause of this phenomenon remains uncertain, possibly due to the suppression of the octahydro lycopene dehydrogenase (PDS) activity, which leads to the inhibiting carotenoid production. As previously stated, \u003cem\u003eRAV\u003c/em\u003e acts as a suppressor of zeaxanthin cyclooxygenase [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], \u003cem\u003eRAV1\u003c/em\u003e and \u003cem\u003eNGA1\u003c/em\u003e may have significant functions in carotenoid synthesis following PDS silencing.\u003c/p\u003e \u003cp\u003eMDA is a significant outcome of membrane peroxidation in plants, while the REC represents the extent of electrolyte leakage in cells. These indicators are crucial for assessing the level of damage to the cell membrane [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Typically, their changes will be positively correlated with the timing of abiotic stresses, which aligns with the pattern of pTRV2 expression observed during a single day of low temperature in this study [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. When the plants in which gene silencing was induced by the virus were subjected to chilling stress, the levels of MDA and cell membrane permeability were difficult to restore to the initial levels of those in the control group (Ctl). and the degree of cell membrane damage in the silenced plants was greater than that in the plants with pTRV2:\u003cem\u003eRAV1\u003c/em\u003e and pTRV2:\u003cem\u003eNGA1\u003c/em\u003e. Furthermore, antioxidants prevent cell membrane damage by specifically targeting reactive oxygen species and nitrogen, as well as lipid peroxidation products and transcription factors that control the expression of genes related to antioxidant response elements. Plants exhibit physiological and biochemical reactions involving cell membrane systems and protective enzyme systems. SOD, POD, and CAT are crucial enzymes with protective functions in plants [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The antioxidant enzyme activities of pepper were notably greater at 5\u003csup\u003eo\u003c/sup\u003eC for 12 and 24 h than at 25\u003csup\u003eo\u003c/sup\u003eC for 0 h. These findings align with the results reported by Nai et al [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In plants undergoing gene silencing, SOD and CAT did not show significant increase in enzyme activity under repeated stress, while POD enzyme activity was significantly increased. Moreover, the POD and CAT enzyme activities of silenced pepper plants were significantly lower than those of pTRV2 in response to chilling stress. It also shown that \u003cem\u003eRAVs\u003c/em\u003e may be involved in the response of chilli plants to chilling stress and increase the tolerance of plants to chilling stress, resulting in epigenetic modifications in the plants. These findings provide a basis for future investigations into the roles of RAV transcription factors in the response of pepper plants to chilling stress. The potential physiological responses identified in this study could be utilized in future molecular studies on enhancing chilling resistance in pepper.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study is the first time to identify and characterize the whole genome of pepper CaRAVs, including chromosome location, protein structure and domain, phylogenetic tree and collinearity information. We further detected the expression of the \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e under repeated low-temperature conditions. Based on qRT-PCR data, \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e with high relative expression and different domains under chilling stress were screened from the \u003cem\u003eCaRAVs\u003c/em\u003e gene subfamily, and a gene silencing vector was constructed by virus induction. Therefore, under chilling stress, understanding the low-temperature resistance of the RAV transcription factor response and improving the chilling resistance of pepper plants by using silenced plants is highly important for determining the key role of \u003cem\u003eCaRAVs\u003c/em\u003e in the chilling response of pepper plants and chilling stress memory under repeated stress conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the financial support given by the projects. We thank the teacher and all schoolmates in our laboratory for providing useful discussions and technical assistance. We very look forward to the editor and reviewers for critically evaluating the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMinkun Pei: Writing-original draft, Methodology, Data curation. Ping Yang: Data curation, Formal analysis, Investigation. Jian Li and Yanzhuang Wang: Investigation, Methodology. Juan Li: Investigation. Hongjun Xu: Supervision, Funding acquisition. Jie Li: Supervision, Writing - review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Xinjiang Uyghur Autonomous Region\u0026nbsp;Key\u0026nbsp;Research and\u0026nbsp;Development Plan\u0026nbsp;Project (2022B02032),\u0026nbsp;Yunnan Fundamental Research Projects (202401AT070059), Yunnan Young and Middle Aged Academic and Technical Leaders Reserve Talents (202205AC160056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe entire \u003cem\u003eCapsicum\u003c/em\u003e\u003cem\u003e\u0026nbsp;annuum\u003c/em\u003e genome sequence information was obtained from the Ensembl Genomes website. The plant materials \u0026ldquo;Shenghan 740\u0026rdquo; used in the experiment were supplied by the Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences. The datasets supporting the conclusions of this article are included in the article and its Supplementary Files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plant materials used in the current study complied with the relevant institutional, national, and international guidelines and legislation.\u0026nbsp;This article does not contain any studies with human participants or animals\u0026nbsp;performed by the authors. These methods were carried out in accordance\u0026nbsp;with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePublisher\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003es Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Y, Deng D, Zhang R, Wang S, Bian Y, Yin Z. Systematic analysis of plant-specific B3 domain-containing proteins based on the genome resources of 11 sequenced species. 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Alleviating damage of photosystem and oxidative stress from chilling stress with exogenous zeaxanthin in pepper (Capsicum annuum L.) seedlings. Plant Physiol Bioch.\u003cem\u003e \u003c/em\u003e2021; 162: 395 - 409.\u003c/li\u003e\n\u003cli\u003eNandagopal JGT, Shanmugam G. Screening and differential oxidative stress responses of hot pepper (Capsicum annuum L.) genotypes under cold stress. S Afr J Bot.\u003cem\u003e \u003c/em\u003e2022; 151, 591 - 599.\u003c/li\u003e\n\u003cli\u003eRamakrishnan M, Zhang ZJ, Mullasseri S, Kalendar R, Ahmad Z, Sharma A, et al. Epigenetic stress memory: A new approach to study cold and heat stress responses in plants. Front Plant Sci.\u003cem\u003e \u003c/em\u003e2022; 13.\u003c/li\u003e\n\u003cli\u003eLi C, Ji J, Wang G, Li Z, Wang Y, Fan Y. Over-Expression of LcPDS, LcZDS, and LcCRTISO, Genes From Wolfberry for Carotenoid Biosynthesis, Enhanced Carotenoid Accumulation, and Salt Tolerance in Tobacco. 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Bmc Plant Biol.\u003cem\u003e \u003c/em\u003e2022; 22(1): 344.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 3 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RAV gene family, Transcription factor, Chilling stress, VIGS","lastPublishedDoi":"10.21203/rs.3.rs-4399432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4399432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe RAV gene family in \u003cem\u003eCapsicum annuum\u003c/em\u003e remains largely unexplored in response to chilling stress, despite its known significance in plant abiotic stress responses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we identified and characterized six members of the CaRAVs gene subfamily in pepper through genome-wide analysis. Subsequently, the \u003cem\u003eCaRAVs\u003c/em\u003e subfamily was classified into four branches based on homology with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of \u003cem\u003eCaRAVs\u003c/em\u003e, whereas low-temperature response elements were specific to the \u003cem\u003eNGA\u003c/em\u003e gene subfamily. After pepper plants were subjected to chilling stress, qRT-PCR analysis revealed that \u003cem\u003eCaRAV1\u003c/em\u003e, \u003cem\u003eCaRAV2\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e were significantly induced in response to chilling stress, indicating that \u003cem\u003eCaRAVs\u003c/em\u003e play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAVs gene family, resulting in plants with increased susceptibility to chilling damage and reduced antioxidant enzyme activity, particularly evident under repeated chilling stress. These findings suggest that \u003cem\u003eCaRAV1\u003c/em\u003e and \u003cem\u003eCaNGA1\u003c/em\u003e positively regulate the response to chilling stress.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSilencing targeting key members of the CaRAVs gene family, results in plants with increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly evident under repeated chilling stress. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in ppepper.\u003c/p\u003e","manuscriptTitle":"Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 04:34:55","doi":"10.21203/rs.3.rs-4399432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-10T05:28:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-07T05:44:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-05T17:41:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-04T10:46:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280779661296331513102116044746125335636","date":"2024-06-03T04:22:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22973479685715134024846179253665764095","date":"2024-06-03T00:36:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86390271261532545009669150718876570062","date":"2024-05-31T10:52:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76968061691757333130764646246439718682","date":"2024-05-26T15:11:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-25T23:39:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-21T07:41:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T07:30:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-14T09:01:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-05-10T08:48:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e273ae43-22b2-4942-bafb-74a40c2f092a","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:07:15+00:00","versionOfRecord":{"articleIdentity":"rs-4399432","link":"https://doi.org/10.1186/s12864-024-10639-x","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2024-07-29 15:58:16","publishedOnDateReadable":"July 29th, 2024"},"versionCreatedAt":"2024-05-23 04:34:55","video":"","vorDoi":"10.1186/s12864-024-10639-x","vorDoiUrl":"https://doi.org/10.1186/s12864-024-10639-x","workflowStages":[]},"version":"v1","identity":"rs-4399432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4399432","identity":"rs-4399432","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}