Genome-Wide Analysis of the SRPP/REF Gene Family in Taraxacum kok-saghyz Provides Insights into Its Expression Patterns in Response to Ethylene and Methyl Jasmonate Treatments

Taraxacum kok-saghyz (TKS) is a model plant and a potential rubber-producing crop for the study of natural rubber (NR) biosynthesis. The precise analysis of the NR biosynthesis mechanism is an important theoretical basis for improving rubber yield. The small rubber particle protein (SRPP) and rubber elongation factor (REF) are located in the membrane of rubber particles and play crucial roles in rubber biosynthesis. However, the specific functions of the SRPP/REF gene family in the rubber biosynthesis mechanism have not been fully resolved. In this study, we performed a genome-wide identification of the 10 TkSRPP and 2 TkREF genes’ family members of Russian dandelion and a comprehensive investigation on the evolution of the ethylene/methyl jasmonate-induced expression of the SRPP/REF gene family in TKS. Based on phylogenetic analysis, 12 TkSRPP/REFs proteins were divided into five subclades. Our study revealed one functional domain and 10 motifs in these proteins. The SRPP/REF protein sequences all contain typical REF structural domains and belong to the same superfamily. Members of this family are most closely related to the orthologous species T. mongolicum and share the same distribution pattern of SRPP/REF genes in T. mongolicum and L. sativa, both of which belong to the family Asteraceae. Collinearity analysis showed that segmental duplication events played a key role in the expansion of the TkSRPP/REFs gene family. The expression levels of most TkSRPP/REF members were significantly increased in different tissues of T. kok-saghyz after induction with ethylene and methyl jasmonate. These results will provide a theoretical basis for the selection of candidate genes for the molecular breeding of T. kok-saghyz and the precise resolution of the mechanism of natural rubber production.


Introduction
Natural rubber (NR) is mainly composed of cis-1,4-polyisoprene polymers, which are indispensable and strategically valuable biologically scarce materials in the fields of healthcare and construction due to their strong impact resistance, abrasion resistance, high flexibility, and superior resilience properties [1,2].More than 2500 species of plants can produce NR, but only some can produce high molecular-weight rubber as a high-performance raw material for commercial needs [3][4][5][6].Among these, the Hevea brasiliensis [7], Parthenium argentatum [8], Lactuca sativa, and the genus Russian dandelion produce natural rubber with an average molecular weight of up to 1000 kg/mol [2].Currently, H. brasiliensis is the only source of NR for the manufacturing industry and cannot be replaced with synthetic materials.However, due to its special growing conditions, limited output, increasing demand, pathogens, and allergic reactions [9,10], it is urgent to explore alternative plant sources of natural rubber.
Taraxacum kok-saghyz L. Rodin (TKS) is a perennial herbaceous plant with high NR yield, good quality, low cost, and it can be used as a candidate alternative crop to natural rubber [11,12].Compared to other rubber-producing plants such as the annual herbaceous plant P. argentatum [8], TKS produces NR with higher molecular weights, increased cis-1,4polyisoprene content, and valuable by-products including inulin and bioethanol [13].T. kok-saghyz had five times the NR yield compared to dandelion (Taraxacum brevicorniculatum, TB) [1].However, the molecular mechanism of natural rubber biosynthesis (NRB) in TKS remains incomplete and elusive.
The precise analysis of the molecular regulation mechanism of natural rubber biosynthesis is important for the development of the natural rubber industry.From previous studies, it is known that T. kok-saghyz use both the Mevalonate pathway (MVA) and Methylerythritol pathway (MEP) to synthesize natural rubber, and 102 genes were identified in the roots of TKS during the developmental period that are involved in the pathways [14].Proteome and transcriptome analysis showed that MVA is the key pathway for rubber synthesis, while the genes and proteins involved in the MEP pathway were expressed in low abundance.The rubber particle membrane is a continuous, monolayer membrane structure that provides a compatible interface between the rubber particles and the surrounding environment [15].The small rubber particle protein (SRPP) and rubber elongation factor (REF) are the most important latex proteins in the rubber particle membrane [7].SRPP/REF, Nogo-B receptor, and HRT1-REF bridging protein (HRBP), three rubber particles (RP) are membrane-binding proteins, are involved in the final elongation process of rubber synthesis in the TKS root [9,16].SRPP covers the membrane surface and has a lower affinity for the membrane than REF, which is embedded in the membrane and attached to the rubber particles [17].Both SRPP and REF interact with rubber particles and jointly regulate NRB.SRPP promotes polyisoprene synthesis, affects the molecular weight of rubber, and alters the length of rubber chains [18,19].SRPP also plays a role in stabilizing rubber particles during rubber biosynthesis and can act as a latex coagulation factor in latex coagulation [20,21].REF and SRPP proteins are highly homologous, and both share a common REF-conserved domain [10,22].Compared to SRPP, the REF protein is smaller and has a partial sequence missing at the C-terminus [17].It has been demonstrated that the overexpression of the REF gene is positively correlated with latex yield [23], and RNAi experiments on the REF gene resulted in a significant reduction in rubber yield, but did not affect the molecular weight of natural rubber, and the silencing of REF does not affect the regulation of rubber particle stability by SRPP [18].
Ethylene is an important phytohormone involved in the regulation of plant secondary metabolism, accelerating plant maturation, regulating photosynthesis and carbohydrate metabolism, and promoting chlorophyll degradation [24].Ethylene is also involved in regulating the expression of genes related to biosynthesis such as flavonoid metabolism and anthocyanin glycosides [25].Ethylene activates the laticifers regeneration pathway in H. brasiliensis [26] and inhibits the expression of rubber particle aggregase, which in turn affects latex yield [16,27,28]. Methyl jasmonate (MeJA), as well as other derivatives, can increase crop yields by stimulating secondary plant metabolism [29,30], and is involved in the latex regeneration pathway [7,31].The HbMADS4 gene of H. brasiliensis is induced by ethylene and MeJA significantly and the overexpression of HbMADS4 in tobacco plants significantly represses the promoter activity of the HbSRPP gene [7].The rubber tree is a woody plant; consequently, gene function studies have been slow to progress.Therefore, it is more appropriate to study the regulation mechanism of NRB in T. kok-saghyz, a model plant for rubber production.Because of the recent discovery that ethylene can effectively stimulate H. brasiliensis to enhance latex production, jasmonic acid (JA) and its volatile MeJA can be widely used as inducers of rubber laticifer differentiation [32].However, the study of these two hormones on the same rubber-producing plant T. kok-saghyz has not yet been revealed and needs to be studied systematically and in-depth.
To systematically investigate the effect of TkSRPP/REF genes on NRB in T. kok-saghyz, in this paper, genome-wide identification of the TkSRPP/REF family members was carried out by using the available genome data.The physical and chemical properties, basic structure and evolutionary relationships of the family members were analyzed.In the qRT-PCR analysis of gene expression patterns in different tissues after ethylene and MeJA treatment, we observed the subcellular localization of part of the family genes.Furthermore, we carried out a preliminary identification of the main members of TkSRPP/REF involved in the regulation of natural rubber biosynthesis.These results provide new ideas for further research on exogenous phytohormones affecting natural rubber biosynthesis.

Identification and Classification of SRPP/REF Gene Family in T. kok-saghyz
We obtained 8, 17, and 16 candidate SRPP/REF genes using three methods: keyword search, HMMER search, and BLASTP search, respectively.The motif, domain, repetitive sequences and transcripts of the same genes were screened for de-duplication.After primary screening and secondary identification, we finally identified a total of 12 SRPP/REF family members in the TKS genome.It contained 10 SRPP family members and 2 REF family members (Table 1).The results of the SRPP/REF sequence analyses showed that all the family genes contain typical REF structural domains (Pfam: PF05755), which belong to the REF family.Based on the SRPP/REF gene localization order of the TKS chromosome, they were named TkSRPP1-TkSRPP10 and TkREF1-TkREF2.Subsequently, the physicochemical properties of each member of the TkSRPP/REF genes were analyzed (Table 1), such as the length of the open reading frame (ORF) of the gene sequences, molecular weight, amino acid number, and isoelectric point.The full lengths of the open reading frames of TkSRPP/REF were 381 bp (TkSRPP1) to 2121 bp (TkREF2); the number of amino acids was between 126 (TkSRPP1) and 706 (TkREF2).The results showed that TkREF2 had the longest protein sequence with a molecular weight of 75.809 kDa, while TkSRPP1 had the smallest molecular weight of 14.61 kDa.In addition, the isoelectric points (pl) of the TkSRPP/REF members ranged from 4.47 (TkSRPP1) to 8.79 (TkREF2).TkSRPP1, TkSRPP7, TkSRPP8, and TkREF2 belong to the acidic proteins (pI < 7), and the rest are weak alkalinity proteins (pI > 7).Four TkSRPP/REF proteins had instability index values less than 40.0.The prediction of the aliphatic index of the family genes showed that TkSRPP/REF proteins have high stability, with TkREF2 having the highest aliphatic index of 102.54 and TkSRPP3 having the lowest aliphatic index of 69.30.The grand average of hydropathicity (GRAVY) of the TkSRPP/REF family members was less than 0 except for TkREF1, which encodes hydrophilic proteins.The subcellular localization of the TkSRPP/REF genes family was predicted in the WoLF PSORT website.The results showed that only TkREF1 was localized to the vesicular membrane and all the remaining members were localized in the cytoplasm (Table 1).
We predicted and analyzed the structure of the TkSRPP/REF proteins; the secondary structure of each member contained α-helix, β-turn, and random coil (Table S1).Meanwhile, we predicted the three-dimensional structures of 12 TkSRPP/REF proteins using homology method modeling (Figure S1).All 12 proteins were detected and evaluated using online software.The secondary structure was dominated by α-helices, which accounted for 56.35-76.04%,followed by random coil accounting for 14.67-32.02%,whereas β-turn had the lowest percentage of 2.40-8.73%(Table S1).A variety of secondary structures are grouped together to act as the basic building blocks of a tertiary structure (Figure S1).The large differences in the basic data of the SRPP subfamily and the REF subfamily suggest that members of the two subfamilies have experienced different evolutionary selective pressures, resulting in the exercise of slightly different biological functions.

Phylogenetic Analysis of TkSRPP/REF Proteins
To analyze the evolutionary relationship between SRPP/REF genes in different species, representative species were selected to construct a phylogenetic tree together with TKS.The T. kok-saghyz, T. mongolicum, L. sativa, H. annuus, and G. max SRPP/REF sequences were selected to align using Clustal W software, version 1.81, and a phylogenetic tree based on 1000 bootstrap replicates was constructed using the neighbor-joining method (Figure 1).According to the sequence similarity and phylogenetic analyses, the SRPP/REF genes were mainly divided into five evolutionary branches, with 13, 10, 16, 7, and 6 genes contained in evolutionary branches I to V, respectively.TkSRPP7 and TkSRPP8 were clustered in branch I (Figure 1).TkSRPP4, TkSRPP5, TkSRPP9, and TkSRPP10 were clustered in branch II.TkSRPP1, TkSRPP2, and TkSRPP3 were clustered in branch III.TkREF1 and TkREF2 were clustered in branch V.There were no family members presented in branch IV.In each branch, TkSRPP/REF first clustered with the orthologous species T. mongolicum and then with L. sativa and H. annuus.The SRPP/REF proteins of G. max were separated from the orthologous of other Asteraceae species.In addition, we further screened 111 SRPP/REF protein sequences from 17 species to construct a phylogenetic tree, and these genes were mainly divided into seven evolutionary branches.Branches IV and VI were not TkSRPP/REF genes, and TkSRPP/REF clustered more compactly with members of the Asteraceae family.This phenomenon suggests that TkSRPP/REF is distantly related to maize, rice, Arabidopsis, and other non-Asteraceae species.Furthermore, TkREFs were clustered in branch VII with other REF genes (Figure S2).We analyzed the motifs and conserved the structural domains and gene structures of the TkSRPP/REF genes.It was found that the basic structures of family members with close evolutionary relationships are relatively similar (Figure S3).

Chromosomal Localization of TkSRPP/REF Genes
To better show the evolutionary pattern of the SRPP/REF gene family, the genomic distribution of the SRPP/REF family genes in T. kok-saghyz and its related species T. mongolicum and L. sativa were mapped to chromosomal locations using TBtools software, version v2.096.According to genome data, T. kok-saghyz is diploid, and T. mongolicum and L. sativa are triploid.T. kok-saghyz has 16 chromosomes, and T. mongolicum and L. sativa have 24 chromosomes.The sequence information is all anchored to eight pseudochromosomes.The SRPP and REF genes have similar localization distributions, and the members of the SRPP and REF subfamilies are localized on different chromosomes, respectively (Figure 2).

Chromosomal Localization of TkSRPP/REF Genes
To better show the evolutionary pattern of the SRPP/REF gene family, the genomic distribution of the SRPP/REF family genes in T. kok-saghyz and its related species T. mongolicum and L. sativa were mapped to chromosomal locations using TBtools software, version v2.096.According to genome data, T. kok-saghyz is diploid, and T. mongolicum and L. sativa are triploid.T. kok-saghyz has 16 chromosomes, and T. mongolicum and L. sativa have 24 chromosomes.The sequence information is all anchored to eight pseudochromosomes.The SRPP and REF genes have similar localization distributions, and the members of the SRPP and REF subfamilies are localized on different chromosomes, respectively (Figure 2).
All the TkSRPP subfamily members are localized on chromosome 4 in the T. kok-saghyz (Figure 2A), and two TkREF genes are localized on chromosome 5.Among them, there were nine TkSRPP members located in the gene cluster at the end of chromosome 4, centered between 1,204,419,836 bp and 123,368,525 bp, except TkSRPP1.These genes were closely aligned in position and functionally synergized with each other to regulate TKS secondary metabolic processes, whereas TkSRPP1, which was located far away from the gene cluster, is located at the other edge of chromosome 4 and positioned between 1,734,758 bp and 1,735,187 bp.TkSRPP1 is far away from other SRPP subfamily members; it is suggested that this gene may possess an independent evolutionary branching in the course of evolution.
Seven TmSRPPs were localized on chromosome 4 (Figure 2B) and one TmREF was localized on chromosome 5.Similarly, TmSRPP1 was localized between 1,018,788 bp and 1,020,349 bp at the end of chromosome 4 away from the gene cluster.TmSRPP2-TmSRPP7 All the TkSRPP subfamily members are localized on chromosome 4 in the T. kok-saghyz (Figure 2A), and two TkREF genes are localized on chromosome 5.Among them, there were nine TkSRPP members located in the gene cluster at the end of chromosome 4, centered between 1,204,419,836 bp and 123,368,525 bp, except TkSRPP1.These genes were closely aligned in position and functionally synergized with each other to regulate TKS secondary metabolic processes, whereas TkSRPP1, which was located far away from the gene cluster, is located at the other edge of chromosome 4 and positioned between 1,734,758 bp and 1,735,187 bp.TkSRPP1 is far away from other SRPP subfamily members; it is suggested that this gene may possess an independent evolutionary branching in the course of evolution.
Seven TmSRPPs were localized on chromosome 4 (Figure 2B) and one TmREF was localized on chromosome 5.Similarly, TmSRPP1 was localized between 1,018,788 bp and 1,020,349 bp at the end of chromosome 4 away from the gene cluster.TmSRPP2-TmSRPP7 genes were localized in the gene cluster at the other end of chromosome 4, clustered between 72,592,019 bp and 72,760,006 bp.In L. sativa, LaREF1 and LaREF2 were localized on chromosome 8.LaSRPPs were localized on chromosome 9.Among them, LaSRPP1 was localized between 5,548,999 bp and 5,550,612 bp at the end of chromosome 9, and the rest of the LaSRPP genes were clustered between 136,106,466 bp and 137,424,082 bp (Figure 2).
genes were localized in the gene cluster at the other end of chromosome 4, clustered between 72,592,019 bp and 72,760,006 bp.In L. sativa, LaREF1 and LaREF2 were localized on chromosome 8.LaSRPPs were localized on chromosome 9.Among them, LaSRPP1 was localized between 5,548,999 bp and 5,550,612 bp at the end of chromosome 9, and the rest of the LaSRPP genes were clustered between 136,106,466 bp and 137,424,082 bp (Figure 2).

Gene Duplication, and Collinearity Analysis of TkSRPP/REF Genes
The SRPP/REF family was analyzed for collinearity and shown on chromosome loops (Figure 3).A total of three collinear blocks, including TkSRPP2/TkSRPP9, TkSRPP3/TkSRPP10, and TkSRPP6/TkSRPP9, were identified.The ratio of Ka/Ks between non-synonymous substitutions (Ka) and synonymous substitutions (Ks) for these three gene pairs was much lower than 1.This means that members of this family underwent a strong purifying selection (Table 2).Furthermore, we predicted replication events for TkSRPP/REF family members.The replication modes are more diverse.Only TkSRPP1 had dispersed distribution, TkSRPP8 had segmental duplication and the rest of the TkSRPP subfamily members underwent tandem duplication( Table S3).However, both members of the TkREF subfamily are singleton genes.The results of replication event analysis indi-

Gene Duplication, and Collinearity Analysis of TkSRPP/REF Genes
The SRPP/REF family was analyzed for collinearity and shown on chromosome loops (Figure 3).A total of three collinear blocks, including TkSRPP2/TkSRPP9, Tk-SRPP3/TkSRPP10, and TkSRPP6/TkSRPP9, were identified.The ratio of Ka/Ks between non-synonymous substitutions (Ka) and synonymous substitutions (Ks) for these three gene pairs was much lower than 1.This means that members of this family underwent a strong purifying selection (Table 2).Furthermore, we predicted replication events for TkSRPP/REF family members.The replication modes are more diverse.Only TkSRPP1 had dispersed distribution, TkSRPP8 had segmental duplication and the rest of the TkSRPP subfamily members underwent tandem duplication(Table S3).However, both members of the TkREF subfamily are singleton genes.The results of replication event analysis indicate that SRPP/REF family members mainly rely on tandem duplication to expand members, and this mode of duplication is significant in the evolutionary process of the family.In addition, we have studied the collinearity analysis of SRPP/REF genes of TKS with other species.The results showed that there were two, three, five, two, and two homologous gene pairs of between T. kok-saghyz and T. mongolicum, L. sativa, H. annuus, A. thaliana, and G. max, respectively (Figure 4).Since H. annuus has a more complex genome, TKS has more homologous gene pairs with it.
cate that SRPP/REF family members mainly rely on tandem duplication to expand members, and this mode of duplication is significant in the evolutionary process of the family.In addition, we have studied the collinearity analysis of SRPP/REF genes of TKS with other species.The results showed that there were two, three, five, two, and two homologous gene pairs of between T. kok-saghyz and T. mongolicum, L. sativa, H. annuus, A. thaliana, and G. max, respectively (Figure 4).Since H. annuus has a more complex genome, TKS has more homologous gene pairs with it.

Promoter Analysis of TkSRPP/REF Genes
Promoter cis-acting elements are important transcription initiation binding regions and play an important role in the regulation of gene expression.We used a 2.0 kb sequence upstream of the TkSRPP/REF gene promoter to predict cis-acting regulatory elements via the Plant CARE website (Figure 5).
Multiple classes of cis-acting elements were identified in the promoter regions of 12 TkSRPP/REF genes.These elements were classified into five major categories: promoterrelated and binding sites, light-responsive, hormone-responsive, environment-responsive, and development-related.Among them, promoter-related and binding sites accounted for the largest proportion, ranging from 54.17% (TkSRPP3) to 77.95% (TkSRPP10) of all elements; environment-responsive elements ranged from 2.08% (TkSRPP4) to 11.46% (TkSRPP3); development-related elements ranged from 6.98% (TkSRPP1) to 15.63% (TkSRPP3); light-responsive was also present in all SRPP/REF promoter regions.TkSRPP6 had the highest number of light-responsive elements, which accounted for 13.04% of all elements in this gene.The rest of the genes had light-responsive elements ranging from

Promoter Analysis of TkSRPP/REF Genes
Promoter cis-acting elements are important transcription initiation binding regions and play an important role in the regulation of gene expression.We used a 2.0 kb sequence upstream of the TkSRPP/REF gene promoter to predict cis-acting regulatory elements via the Plant CARE website (Figure 5).
Multiple classes of cis-acting elements were identified in the promoter regions of 12 TkSRPP/REF genes.These elements were classified into five major categories: promoterrelated and binding sites, light-responsive, hormone-responsive, environment-responsive, and development-related.Among them, promoter-related and binding sites accounted for the largest proportion, ranging from 54.17% (TkSRPP3) to 77.95% (TkSRPP10) of all elements; environment-responsive elements ranged from 2.08% (TkSRPP4) to 11.46% (Tk-SRPP3); development-related elements ranged from 6.98% (TkSRPP1) to 15.63% (TkSRPP3); light-responsive was also present in all SRPP/REF promoter regions.TkSRPP6 had the highest number of light-responsive elements, which accounted for 13.04% of all elements in this gene.The rest of the genes had light-responsive elements ranging from 2.11% (TkSRPP9) to 8.80% (TkSRPP2) (Figure 5A,B).In addition, hormone-responsive elements also accounted for a large proportion (Figure 5C), ranging from 3.47% (TkSRPP4) to 14.79% (TkREF1).All family genes contained Abscisic Acid (ABA)-response elements and most genes contained gibberellins (GA3)-, auxin (IAA)-, ethylene (ETH)-, methyl jasmonate (MeJA)-and Salicylic acid (SA)-response elements.The promoter regions of TkSRPP/REF family genes have MYB transcription factor binding sites related to drought inducibility, flavonoid biosynthesis gene regulation, and light response.These results suggest that members of the TkSRPP/REF family are likely to be involved in plant growth and metabolic regulatory processes.

Analysis of TkSRPP/REF Expression Patterns and Physiological Indexes
To further predict the function of TkSRPP/REF genes, we treated TKS with ethylene and detected the expression in different tissues using Real-Time Quantitative PCR.The results showed that the expression of TkSRPP4, TkSRPP6, TkSRPP7, TkSRPP8 and TkSRPP9 was significantly up-regulated after ethylene was induced in the roots.Among them, TkSRPP6 and TkSRPP8 were the most significantly up-regulated compared with the control, which is presumed to play a key role in the ethylene-regulated pathway.The addition of exogenous hormones activated the expression of these genes.TkSRPP3, TkSRPP5, TkREF1, and TkREF2 were gradually down-regulated; TkSRPP1 and TkSRPP2 were significantly down-regulated in the 3 h of treatment, and TkSRPP1 increased with the increase in treatment time.Meanwhile, the expression of TkSRPP2 was significantly up-regulated

Analysis of TkSRPP/REF Expression Patterns and Physiological Indexes
To further predict the function of TkSRPP/REF genes, we treated TKS with ethylene and detected the expression in different tissues using Real-Time Quantitative PCR.The results showed that the expression of TkSRPP4, TkSRPP6, TkSRPP7, TkSRPP8 and TkSRPP9 was significantly up-regulated after ethylene was induced in the roots.Among them, TkSRPP6 and TkSRPP8 were the most significantly up-regulated compared with the control, which is presumed to play a key role in the ethylene-regulated pathway.The addition of exogenous hormones activated the expression of these genes.TkSRPP3, TkSRPP5, TkREF1, and TkREF2 were gradually down-regulated; TkSRPP1 and TkSRPP2 were significantly down-regulated in the 3 h of treatment, and TkSRPP1 increased with the increase in treatment time.Meanwhile, the expression of TkSRPP2 was significantly up-regulated at 6 h and then maintained at a lower level.Interestingly, TkSRPP10 was up-regulated at the 3 h and then decreased, and up-regulated at 24 h again.In the leaves (Figure S2), the expression levels of most genes were increased, such as TkSRPP1 and TkSRPP5, which were highly up-regulated in contrast to those in roots.The results of these experiments suggest that these genes are involved in the regulatory pathway of ethylene metabolism and regulate growth and developmental processes in specific tissues.
To understand the effect of ethylene treatment on the change in the rubber content of T. kok-saghyz, we determined the rubber content of plant roots at different times under treatment.As shown (Figure 6B), the yield of crude rubber extract showed a trend of a slight decrease followed by an increase.The yield peaked at 9.13% at the 24th hour.To evaluate the extent of cellular damage and the activation of the antioxidant defense system in T. kok-saghyz after ethylene treatment, we tested malondialdehyde (MDA) content as well as three key antioxidant enzyme activities: Peroxidase (POD), Catalase (CAT) and Superoxide Dismutase (SOD).TKS treated with ethylene showed an overall increasing trend in CAT activity compared to the control.However, the enzyme activity decreased at 6 h and then gradually increased to a higher level (Figure 6C).POD activity elevated significantly with treatment time (Figure 6D).At 6 h, SOD activities reached the highest level (Figure 6F).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 10 of 20 at 6 h and then maintained at a lower level.Interestingly, TkSRPP10 was up-regulated at the 3 h and then decreased, and up-regulated at 24 h again.In the leaves (Figure S2), the expression levels of most genes were increased, such as TkSRPP1 and TkSRPP5, which were highly up-regulated in contrast to those in roots.The results of these experiments suggest that these genes are involved in the regulatory pathway of ethylene metabolism and regulate growth and developmental processes in specific tissues.
To understand the effect of ethylene treatment on the change in the rubber content of T. kok-saghyz, we determined the rubber content of plant roots at different times under treatment.As shown (Figure 6B), the yield of crude rubber extract showed a trend of a slight decrease followed by an increase.The yield peaked at 9.13% at the 24th hour.To evaluate the extent of cellular damage and the activation of the antioxidant defense system in T. kok-saghyz after ethylene treatment, we tested malondialdehyde (MDA) content as well as three key antioxidant enzyme activities: Peroxidase (POD), Catalase (CAT) and Superoxide Dismutase (SOD).TKS treated with ethylene showed an overall increasing trend in CAT activity compared to the control.However, the enzyme activity decreased at 6 h and then gradually increased to a higher level (Figure 6C).POD activity elevated significantly with treatment time (Figure 6D).At 6 h, SOD activities reached the highest level (Figure 6F).In addition, we treated T. kok-saghy roots with MeJA and examined the expression of TkSRPP/REF genes (Figures 7 and S3).It showed that most of the members were significantly up-regulated through MeJA induction.In roots, the level of up-regulation of these family members was very high, such as TkSRPP4, TkSRPP7, and TkSRPP10 (Figure 7).The expressions of TkSRPP2, TkSRPP6, and TkSRPP7 gradually increased with treatment time.Some genes had the highest expression level at 3 h.Similarly, in the leaves, the expression of some genes reached the highest level at 3 h (Figure S4).The expression of different members varies in different tissues.Only the expression level of TkSRPP3 was reduced in leaves and maintained at a low level (Figure S5).The results indicated that this gene was significantly induced by MeJA and highly expressed in TKS roots.These experimental results suggest that the TkSRPP/REF family is involved in regulating the JA metabolic regulatory pathway.
TkSRPP/REF genes (Figures 7 and S3).It showed that most of the members were significantly up-regulated through MeJA induction.In roots, the level of up-regulation of these family members was very high, such as TkSRPP4, TkSRPP7, and TkSRPP10 (Figure 7).The expressions of TkSRPP2, TkSRPP6, and TkSRPP7 gradually increased with treatment time.Some genes had the highest expression level at 3 h.Similarly, in the leaves, the expression of some genes reached the highest level at 3 h (Figure S4).The expression of different members varies in different tissues.Only the expression level of TkSRPP3 was reduced in leaves and maintained at a low level (Figure S5).The results indicated that this gene was significantly induced by MeJA and highly expressed in TKS roots.These experimental results suggest that the TkSRPP/REF family is involved in regulating the JA metabolic regulatory pathway.
In order to understand the effect of MeJA on the changes in T. kok-saghyz rubber content, we measured the crude rubber extract content of TKS roots under different treatment times.The results showed that the peak value was reached at 3 h after treatment, which was about 1.3 fold of the control (Figure 7B).After MeJA application, the CAT activity of TKS increased significantly in comparison to the control and peaked at 6 h (Figure 7C).POD activity reached three-fold of the control at 24 h (Figure 7D).MDA activity decreased slightly (Figure 7E).SOD activity first rose and then maintained within a range after a gradual decline (Figure 7F).In order to understand the effect of MeJA on the changes in T. kok-saghyz rubber content, we measured the crude rubber extract content of TKS roots under different treatment times.The results showed that the peak value was reached at 3 h after treatment, which was about 1.3 fold of the control (Figure 7B).After MeJA application, the CAT activity of TKS increased significantly in comparison to the control and peaked at 6 h (Figure 7C).POD activity reached three-fold of the control at 24 h (Figure 7D).MDA activity decreased slightly (Figure 7E).SOD activity first rose and then maintained within a range after a gradual decline (Figure 7F).

Subcellular Localization of TkSRPP/REF
To detect the location of TkSRPP/REF proteins in cells, we constructed 35S::eGFP, 35S::TkSRPP1-eGFP, 35S::TkSRPP4-eGFP, 35S::TkSRPP7-eGFP and 35S::TkREF1-eGFP fusion expression vectors and transiently expressed them in Nicotiana benthamiana (Figure 8).The subcellular localization of the TkSRPP/REF genes family was predicted in the WoLF PSORT website.The results showed that only TkREF1 was localized to the vesicular membrane, and all the remaining members were localized in the cytoplasm.N. benthamiana cells were observed using laser confocal microscopy and the fusion proteins all fluoresced green in the cytoplasm.We used mCherry red fluorescence as a cytoplasmic marker.As shown, 35S::TkSRPP1-eGFP, 35S::TkSRPP4-eGFP, 35S::TkSRPP7-eGFP and 35S::TkREF1-eGFP were expressed not only in the cytoplasm, but also in chloroplasts as well as the cytoplasmic membrane.It is hypothesized that this result is related to the function exercised by SRPP/REF proteins in the cytoplasm.

Subcellular Localization of TkSRPP/REF.
To detect the location of TkSRPP/REF proteins in cells, we constructed 35S::eGFP 35S::TkSRPP1-eGFP, 35S::TkSRPP4-eGFP, 35S::TkSRPP7-eGFP and 35S::TkREF1-eGFP fu sion expression vectors and transiently expressed them in Nicotiana benthamiana (Figur 8).The subcellular localization of the TkSRPP/REF genes family was predicted in th WoLF PSORT website.The results showed that only TkREF1 was localized to the vesicula membrane, and all the remaining members were localized in the cytoplasm.N. bentham iana cells were observed using laser confocal microscopy and the fusion proteins all fluo resced green in the cytoplasm.We used mCherry red fluorescence as a cytoplasmi marker.As shown, 35S::TkSRPP1-eGFP, 35S::TkSRPP4-eGFP, 35S::TkSRPP7-eGFP and 35S::TkREF1-eGFP were expressed not only in the cytoplasm, but also in chloroplasts a well as the cytoplasmic membrane.It is hypothesized that this result is related to the func tion exercised by SRPP/REF proteins in the cytoplasm.

Discussion
Natural rubber has unique physicochemical properties and is widely used in industrial production, healthcare, civil engineering, construction, and other production research due to its strong abrasion resistance and good ductility [5,33].T. kok-saghyz roots can produce natural rubber (NR), and the small rubber particle protein (SRPP) [34] and rubber elongation factor (REF) [35] are two important regulators in the mechanism of natural rubber production [17].In study, to deeply investigate the function of the SRPP/REF family in TKS, we conducted a genome-wide characterization of the TkSRPP/REF family based on information from the open data, and identified a total of 12 members of the TkSRPP/REF gene family as well as 16 other species (T.mongolicum, H. brasiliensis, L. sativa, H. annuus, G. max, T. brevicorniculatum, C. annuum, S. italica, S. lycopersicum, C. scolymus, O. sativa, Z. mays, A. thaliana, P. vulgaris, M. truncatula and R. communis) of 111 SRPP/REF proteins (Figure S2).The SRPP/REF family was divided into seven branches based on phylogenetic relationships (Figure S2).In different species, its members are unequally distributed in subclades.In particular, most of the family members of TKS are concentrated in subclades I and II with the closest affinity to the homologous species, T. mongolicum.H. brasiliensis are perennial woody plants.So, most of these HbSRPP/REFs are independently comprised in the subclade V.It has been shown that TkSRPP and HbSRPP belong to different evolutionary directions and play different roles in the rubber synthesis mechanism.In this study, we performed phylogenetic analyses for SRPP/REF family members in multiple species.Most of the rubber tree family members are independently located in subclade V and are genetically distant from TKS and other species, which is consistent with previous findings [36,37].
Variation in gene structure is an important feature of family evolution, and the analysis of the basic structure of TkSRPP/REF revealed that almost all family members have a three-exon, two-intron structure, except for TkSRPP1, which has a two-exon, one-intron structure, and TkSRPP5, which has a five-exon, four-intron structure (Figure S3).Due to the significant individual differences in the exon/intron structures of the family members, this difference enriches the gene functions of the TkSRPP/REF family [38].TkSRPP/REF proteins contain a common REF-conserved structural domain at the N-terminus [34].TkREF1 has a special structure containing two incomplete REF-conserved structural domains, and it is hypothesized that TkREF1 plays a key role in the regulation of the NR synthesis [18].The distribution of SRPP/REF family genes in T. kok-saghyz and its related species T. mongolicum and L. sativa were analyzed: the SRPP/REF genes of the three species had the same trend of differentiation.The SRPP subfamily genes and REF subfamily genes were located on different chromosomes, in which most of the genes in the SRPP subfamily existed in gene clusters, and only SRPP1 was located far away from the gene clusters.The density of genes in the genome at the location of SRPP1 is greater than that at the cluster.So, it is assumed that SRPP1 plays an important role in the anabolism process of natural rubber.
We found that among the TkSRPP subfamily, only TkSRPP1 has a deletion of its structural domain.This gene is located on the other end of chromosome 4 of TKS from other members of the SRPP subfamily (Figure 2), which is a dispersed distribution gene (Table 2) with a separate evolutionary branch.The expression of TkSRPP1 is high in several tissues and the expression pattern is more diverse; these results are supported by previous studies [18,39,40] Due to the evolutionary specificity of the TkSRPP1 gene, it is inferred that it has an important regulatory role in NR synthesis or metabolism.The remaining TkSRPPs genes are located in the gene cluster of chromosome IV; segmental duplication as well as tandem duplication modality play an important role in increasing the number of genes [41,42].Most of the TkSRPP/REF family members are tandem duplicated, as this is the main mode of TkSRPP/REF family expansion.Meanwhile, the collinearity analysis of the family members calculated a total of three homologous gene pairs, each with Ka/Ks values less than 1.These genes have undergone a strong purifying selection and evolved in a more conserved manner [42].
Promoter region cis-acting elements are important regulators of plant growth, development, and resistance to biotic and abiotic stresses [43].In order to further clarify the functions of the genes, we have analyzed the promoter regions of the TkSRPP/REF family.The cis-acting elements in the promoter region include various types, among which there are light-responsive elements (G-Box, MRE, etc.), hormone-responsive elements (GAREmotif, ABRE, ERE, etc.), growth and developmental elements (GCN4-motif, O2-site, etc.), defense and stress-responsive elements (STRE, WUN-motif, etc.), and abiotic stress elements (MYB, DRE, MBS, etc.).The TkSRPP/REF promoter region contains different kinds of hormone-response elements, such as ABA, IAA, JA, SA, etc., suggesting that they are likely to play roles in various hormone signaling pathways.In previous reports, SRPP gene expression was up-regulated by drought stress through the transcriptome analysis of Parthenium hysterophorus rubber particles [44].HbSRPP was responsive to abiotic stresses such as hormones, cold, and heat [35].TbSRPP1 was an ABA-sensitive isoform involved in ABA signaling in response to drought, cold, and other abiotic stresses [45].Ethylene is involved in inducing an increase in the phenolic acid content of Salvia miltiorrhiza hairy root [46].Ethylene decreased the lignin content and increased the amount of secondary metabolites such as flavonoids in ramie [47].MeJA activates the expression of Pyrethrin biosynthetic genes by inducing TcMYC2 gene expression.The long-term induction of immature leaves with MeJA increased the accumulation of pyrethrin content [48].Differentially expressed genes and the functional grading of Catharanthus roseus indicated that both MeJA and ethylene may stimulate the expression of genes related to vinblastine and vincristine production [49].Since the gene function of SRPP/REF in TKS is not fully revealed, the prediction of cis-acting elements suggests that this family of genes not only plays a role in the rubber production mechanism, but most of them may respond to various biotic and abiotic stresses.
We used ETH as well as MeJA to treat T. kok-saghyz separately, and the TkSRPP/REF genes were differently expressed in different tissues.For example, the expression of TkSRPP5 in roots was significantly reduced by ETH regulation, whereas the expression of this gene was higher in leaves under the same treatment.In roots (Figure 6), TkSRPP6 and TkSRPP8 were up-regulated by ethylene to a higher extent than in leaves at about 10-fold levels (Figure S4), and it is hypothesized that the reason for their ethylene-induced high expression in roots is to increase latex production [28].Interestingly, the TkSRPP4 and TkSRPP10 genes do not contain ETH regulatory elements.However, both genes tended to be up-regulated in different tissues under ETH treatment.It is hypothesized that exogenous ETH application affected the transcript levels of SRPP upstream genes.In addition, the expression of almost all the genes of this family was significantly up-regulated at different times in both the root and leaf material treated by methyl jasmonate.TkSRPP2 was most highly expressed in leaves at 3 h and in roots at 24 h.Only the expression of TkSRPP3 in leaves was down-regulated by the stimulation of MeJA.It is possible that this gene functions differently in different tissues and is involved in the regulation of a variety of other metabolic regulatory pathways.Whether ETH and MeJA contain some kind of collaborative ability to co-regulate the synthesis of natural rubber remains to be thoroughly investigated.Based on this, we have a more comprehensive understanding of the TkSRPP/REF gene family and found that it may not only be involved in NR synthesis, but also may respond to a variety of biotic and abiotic stresses, and the specific metabolic regulation mechanism remains to be investigated.

Plant Materials and Phytohormone
The SHZ variety of Taraxacum koksaghyz was used as test material in this study.Samples were sterilized in 1.5% NaClO solution for 6 min and then placed in sterilized solid medium containing half Murashige and Skoog (1/2 MS).After 4 days of vernalization at 4 • C, they were placed in a growth chamber with a photoperiod of 16 h, a temperature of 25 • C and a relative humidity of 30%.After 14 d of germination period, the plants were transplanted into plastic pots with peat soil/vermiculite/perlite = 3:1:1 in the greenhouse.They were grown under a 16/8 h diurnal photoperiod at 25 • C. Six-month-old T. koksaghyz seedlings were selected as treatment material.Selected materials of the same size and growth were continued in Hoagland's solution for 14 days.The selected seedlings were transferred into Hoagland solution containing 1 mmol/L MeJA and 100 µmol/L ethylene for hormonal treatment of T. koksaghyz roots.Three biological replicates were set up for each treatment with 12 T. koksaghyz seedlings per replicate.After five treatment periods of 0 h, 3 h, 6 h, 12 h and 24 h, the root and leaf materials of each replicate were collected and immediately placed in liquid nitrogen, and then stored at −80 • C for subsequent experimental analyses.
The alkaline boiling method was utilized to determine the rubber content of T. koksaghyz.In total, 0.5 g of dried roots of T. kok-saghyz was taken and placed in a tube.Root tissues were completely immersed in the alkaline solution by adding 1 mol/L NaOH and boiling in a water bath for one hour.After a short time to cool down, a mortar and pestle was used to press the tissue into thin slices.Then, distilled water was added to wash the impurities.Distilled water was washed three times.Following this, 1% HCl solution was added and crude rubber was completely immersed in the acid solution in a boiling water bath for 15 min.The final washing of crude rubber was carried out using 95% ethanol.Finally, the washed crude rubber was dried in an oven at 65 • C until a constant weight.Malondialdehyde content, Peroxidase activity, Catalase activity, and Superoxide Dismutase activity were measured using the Solarbio Physiological Indicator Assay Kit (Solarbio, Beijing, China), Catalase (CAT) Activity Assay Kit (Solarbio, Beijing, China), Peroxidase (POD) Activity Assay Kit (Solarbio, Beijing, China), Malondialdehyde (MDA) Content Assay Kit (Solarbio, Beijing, China), and Superoxide Dismutase (SOD) Activity Assay Kit (Solarbio, Beijing, China).

Identification and Classification of SRPP/REF Gene Family in TKS
To identify the T. koksaghyz SRPP/REF genes, we obtained details of the latest genome of TKS through the website (https://ngdc.cncb.ac.cn/gwh/, accessed on 12 June 2023).The AtSRPP proteins were used as query sequences to retrieve SRPP/REF protein sequences in TKS.The E-value threshold for the BLAST program was set at 1 × 10 −10 to obtain the candidate dataset of TkSRPP/REF proteins.The Hidden Markov Model (HMM) of the conserved structural domain of REF (PF05755) was downloaded from the Pfam website (http://pfam.xfam.org/,accessed on 14 June 2023), with the following threshold: e-values < 10 −5 .All putative SRPP/REF members with conserved REF or REF superfamily structural domains were found in our protein data using the HMM-search module in TBtools software at the following threshold: e-value of 10 −5 .Subsequently, the NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/,accessed on 14 June 2023) [50], InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/, accessed on 14 June 2023) and SMART (https://smart.embl.de/smart/batch.pl,accessed on 14 June 2023) databases were further checked for the integrity of the REF or REF superfamily structural domains of candidate SRPP/REF genes, and redundant sequences that did not contain the complete domains were removed (Table 1).Finally, 12 protein sequences with REF or REF superfamily structural domains were selected if their SRPP/REF protein homology was found to be extreme on the TKS chromosome.Their molecular weights (MW) and isoelectric points (pI) were predicted using ExPaSy (https://www.expasy.org/,accessed on 20 June 2023) and WoLF PSORT (https://www.genscript.com/wolf-psort.html,accessed on 20 June 2023), respectively, as was the subcellular localization.The secondary structures of the TkSRPP/REF proteins were predicted and analyzed using SOPMA website (https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html/,access on on 22 June 2023) (Table S1).The 3D structure of the protein was predicted via homology modeling using SWISS-MODEL (https://swissmodel.expasy.org/,accessed on 22 June 2023) [51][52][53][54][55]. Structure visualization was performed using 3D protein structure visualization PyMOL software, version 2.5.4.

Phylogenetic Analysis TkSRPP/REF Proteins and Gene Structure, Conserved Domains and Motif Composition of TkSRPP/REF Genes
All protein sequences of different taxa of species such as Arabidopsis thaliana, Oryza sativa, Glycine max, T. mongolicum, H. annuus, and L. sativa were downloaded from TAIR (https://www.arabidopsis.org/,accessed on 27 June 2023), Ensembl database (http:// plants.ensembl.org/index.html,accessed on 27 June 2023), and Genome Archive database, respectively (https://ngdc.cncb.ac.cn/gwh/, accessed on 27 June 2023) and the National Centre for Biotechnology Information (https://www.ncbi.nlm.nih.gov/,accessed on 27 June 2023).Then, the sequences of SRPP/REF proteins in Arabidopsis thalian, Oryza sativa, and other species were obtained according to the methods described above (Table S1).To explore evolutionary relationships, multiple sequence comparisons with default parameters were performed between 12 T. kok-saghyz, 10 T. mongolicum, 7 Glycine max, 11 H. annuu, and 12 L. sativa SRPP/REF proteins using Clustal W [56], followed by construction of phylogenetic trees with the neighbor-joining (NJ) method using MEGA11 software, version 11.0.11[57].To assess the reliability of the phylogenetic tree, the bootstrap value was set to 1000 repetitions.The evolutionary tree was visualized and annotated using the online software iTOL (https://itol.embl.de/,accessed on 1 July 2023).The motif composition and distribution of TkSRPP/REFs were identified using MEME [58] to resolve their conserved motifs.The parameters were as follows: optimum motif width set to ≥6 and ≤50; number of motifs: 10.

Chromosomal Distribution and Duplication Analysis of TkSRPP/REF Genes
Chromosome distributions in the identified SRPP/REF genes of T. kok-saghyz, T. mongolicum and L. sativa were obtained and visualized using Gene location visualize of TBtools software.Gene duplication events with default parameters were analyzed using the Mcscanx [59] to analyze collinearity between SRPP/REF by intraspecies versus interspecies (T.kok-saghyz, T. mongolicum, and L. sativa).Non-synonymous (Ka) and synonymous (Ks) substitutions were calculated for each duplicate TkSRPP/REF gene using the Simple Ka/Ks Calculator(NG) of TBtools software.

Promoter Analysis of TkSRPP/REF Genes
A 2000-bp sequence upstream of the start codon (ATG) was intercepted from the TKS reference genome using TBtools.Then, cis-acting elements in the promoter region were predicted using the online PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 July 2023) [60].Visualization and analysis of cis-acting elements of TKS SRPP/REF genes was performed using HeatMap of TBtools.

Quantitative Real-Time PCR (qRT-PCR) Analysis
Quantitative Real-Time PCR (qRT-PCR) was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to extract total RNA from the samples.cDNA was synthesized using a FastQuant First Strand cDNA Synthesis Kit (Tiangen, Beijing, China) according to the manufacturer's protocol.qRT-PCR was performed using a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland), SYBR ® Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology, Hunan, China) and Roche LightCycler instrument.Each treatment had three biological replicates.RT-PCR primers were designed using prime5 software, version v5.00 (PREMIER Biosoft, San Francisco, CA, USA) (Table S2).TkACTIN was used as an internal reference gene; the primers used for qRT-PCR are shown in Supplementary (Table S3).Data were analyzed using the 2 −∆∆Ct calculation method.Statistical analysis was performed using SPSS 22.0 software (SPPS Inc., Chicago, IL, USA).Statistical differences between measurements at different times or with different treatments were analyzed using Duncan's multiple range test.Differences were considered significant at a probability level of p < 0.05.

Subcellular Localization of TkSRPP/REFs
Nicotiana benthamiana seeds were obtained from the Key Laboratory of Xinjiang Phytomedicine Resource and Utilization of Ministry of Education in Shihezi University (Shihezi, China).To study the transient expression of TkSRPP/REFs in N. benthamiana leaves, the full-length CDS of TkSRPP/REFs has been PCR-amplified using primers containing Sma I and Spe I restriction endonucleases (Table S2) and ligated into the vector pCAMBIA1300-eGFP cleaved by Sma I and Spe I to generate pCAMBIA1300-35s-eGFP.The constructed vector was transformed into Agrobacterium rhizogenes GV3101 and infiltrated into 4-week-old Nicotiana benthamiana leaves, and the fluorescence signals in the epidermis of N. benthamiana leaves were observed using a confocal microscope (Nikon, Tokyo, Japan) after 48 h of dark incubation.The experiments used pCAMBIA1300-35S-mCherry-NOS (Puint, Shaanxi, China) as a marker for the cytoplasm.

Conclusions
In this study, a comprehensive analysis of the T. kok-saghyz SRPP/REF gene family was carried out, and a total of 10 TkSRPPs and 2 TkREFs were identified and classified.The basic physicochemical features, three-dimensional structural models, phylogeny, conserved structural domains, gene structure, chromosomal location, and cis-acting elements of the TkSRPP/REFs were analyzed by using genomic and bioinformatics techniques.Phylogenetic and homology analyses of SRPP/REF genes in different plants were used to provide new perspectives on the evolution of the SRPP/REF gene family in TKS.Finally, the tissue expression patterns of TkSRPP/REFs genes were analyzed under ethylene and MeJA treatments.This study provides useful information for the study of the SRPP/REF gene family in T. kok-saghyz, and these findings will contribute to a better understanding of the biological functions and molecular mechanisms of the SRPP/REFs in rubber substitute plants, and provide a basis for functional studies and molecular breeding for genetic improvement in T. kok-saghyz.

Figure 1 .
Figure 1.Phylogenetic analysis of TkSRPP/REFs proteins in T. kok-saghyz, T. mongolicum, L. sativa, H. annuus and G. max.Phylogenetic trees were plotted using the neighbor-joining (NJ) method with 1000 bootstrap replicates.Fifty-two genes were divided into five clades (I-Ⅴ) and identified with different colors.The red pentagram represents T. kok-saghyz, the purple circle represents T. mongolicum, the yellow triangle represents L. sativa, the green square represents H. annuus and the blue circle represents G. max.

Figure 1 .
Figure 1.Phylogenetic analysis of TkSRPP/REFs proteins in T. kok-saghyz, T. mongolicum, L. sativa, H. annuus and G. max.Phylogenetic trees were plotted using the neighbor-joining (NJ) method with 1000 bootstrap replicates.Fifty-two genes were divided into five clades (I-V) and identified with different colors.The red pentagram represents T. kok-saghyz, the purple circle represents T. mongolicum, the yellow triangle represents L. sativa, the green square represents H. annuus and the blue circle represents G. max.

Figure 2 .
Figure 2. Chromosomal location of SRPP/REF genes in the T. kok-saghyz genome (A), and T. mongolicum genome (B) and L. sativa genome (C).The scale on the left represents the chromosome size, the length of chromosomes is measured in Mb.The chromosome number was indicated to the left of each chromosome.TkSRPP/REF gene numbers are shown on the right of each chromosome.The scale bar on the left indicates the genes of TkSRPP/REF are marked in red.Gene density of chromosomes from lower to higher is indicated from blue to red within the bar, respectively.

Figure 2 .
Figure 2. Chromosomal location of SRPP/REF genes in the T. kok-saghyz genome (A), and T. mongolicum genome (B) and L. sativa genome (C).The scale on the left represents the chromosome size, the length of chromosomes is measured in Mb.The chromosome number was indicated to the left of each chromosome.TkSRPP/REF gene numbers are shown on the right of each chromosome.The scale bar on the left indicates the genes of TkSRPP/REF are marked in red.Gene density of chromosomes from lower to higher is indicated from blue to red within the bar, respectively.

Figure 3 .
Figure 3. Collinearity analysis of TkSRPP/REF gene.The gray lines in the background represent colinear modules in the T. kok-saghyz genome.The red line indicates a TkSRPP/REF gene pair with collinearity.The outer bar chart represents gene density in chromosomes, the line chart represents GC content.

Figure 3 .
Figure 3. Collinearity analysis of TkSRPP/REF gene.The gray lines in the background represent colinear modules in the T. kok-saghyz genome.The red line indicates a TkSRPP/REF gene pair with collinearity.The outer bar chart represents gene density in chromosomes, the line chart represents GC content.

Figure 4 .
Figure 4.The colinear relationship between the TkSRPP/REF gene of T. kok-saghyz and the SRPP/REF genes of other species.The gray lines in the background represent colinear modules in the genomes of T. kok-saghyz and other plants.The highlighted red line indicates a colinear SRPP/REF gene pair.

Figure 4 .
Figure 4.The colinear relationship between the TkSRPP/REF gene of T. kok-saghyz and the SRPP/REF genes of other species.The gray lines in the background represent colinear modules in the genomes of T. kok-saghyz and other plants.The highlighted red line indicates a colinear SRPP/REF gene pair.

Figure 5 .
Figure 5. Analysis of cis-acting elements of TkSRPP/REF gene.(A) The different colors on the heatmap represent the number of different cis-acting elements in each TkSRPP/REF gene.(B) The different colors on the stacked graph represent the percentage of each type of cis-acting element in different TkSRPP/REF genes.(C) Different colors on the stacked graph represent the percentage of different hormone elements in different TkSRPP/REF genes.

Figure 5 .
Figure 5. Analysis of cis-acting elements of TkSRPP/REF gene.(A) The different colors on the heatmap represent the number of different cis-acting elements in each TkSRPP/REF gene.(B) The different colors on the stacked graph represent the percentage of each type of cis-acting element in different TkSRPP/REF genes.(C) Different colors on the stacked graph represent the percentage of different hormone elements in different TkSRPP/REF genes.

Figure 6 .
Figure 6.TkSRPP/REF expression pattern and physiological indexes under ethylene treatment.(A) Relative expression of TkSRPP/REF genes; (B) rubber content; (C) Catalase (CAT) activity; (D) Peroxidase (POD) activity; (E) Malondialdehyde (MDA) content; (F) Superoxide Dismutase (SOD) activity.The data are the average ± SD of three biological replicates.The error bar displays the mean ± SE of three independent replicates.The average values represented by the same letter showed no significant difference when p < 0.05, as determined using Duncan's multiple range test.

Figure 6 .
Figure 6.TkSRPP/REF expression pattern and physiological indexes under ethylene treatment.(A) Relative expression of TkSRPP/REF genes; (B) rubber content; (C) Catalase (CAT) activity; (D) Peroxidase (POD) activity; (E) Malondialdehyde (MDA) content; (F) Superoxide Dismutase (SOD) activity.The data are the average ± SD of three biological replicates.The error bar displays the mean ± SE of three independent replicates.The average values represented by the same letter showed no significant difference when p < 0.05, as determined using Duncan's multiple range test.

Figure 7 .
Figure 7. TkSRPP/REF expression pattern and physiological indexes under MeJA treatment.(A) Relative expression of TkSRPP/REF genes; (B) rubber content; (C) Catalase (CAT) activity; (D) Peroxidase (POD) activity; (E) Malondialdehyde (MDA) content; (F) Superoxide Dismutase (SOD) activity.The data are the average ± SD of three biological replicates.The error bar displays the mean ± SE of three independent replicates.The average values represented by the same letter showed no significant difference when p < 0.05, as determined using Duncan's multiple range test.

Figure 7 .
Figure 7. TkSRPP/REF expression pattern and physiological indexes under MeJA treatment.(A) Relative expression of TkSRPP/REF genes; (B) rubber content; (C) Catalase (CAT) activity; (D) Peroxidase (POD) activity; (E) Malondialdehyde (MDA) content; (F) Superoxide Dismutase (SOD) activity.The data are the average ± SD of three biological replicates.The error bar displays the mean ± SE of three independent replicates.The average values represented by the same letter showed no significant difference when p < 0.05, as determined using Duncan's multiple range test.

Figure 8 .
Figure 8. Subcellular localization.EGFP was the empty vector used as the control.All proteins were transiently expressed in N. benthamiana epidermal cells, and GFP signals were observed after 48 h of immersion.The cytoplasm was visualized with mCherry-labeled cytoplasm marker.Bars, 20 µm.

Table 2 .
Homologous gene pair Gene 1 and Gene 2, Ka, Ks and Ka/Ks of the TkSRPP/REF gene family.

Table 2 .
Homologous gene pair Gene 1 and Gene 2, Ka, Ks and Ka/Ks of the TkSRPP/REF gene family.