Functional Characterization of TkSRPP Promoter in Response to Hormones and Wounding Stress in Transgenic Tobacco

Taraxacum kok-saghyz is a model species for studying natural rubber biosynthesis because its root can produce high-quality rubber. Small rubber particle protein (SRPP), a stress-related gene to multiple stress responses, involves in natural rubber biosynthesis. To investigate the transcriptional regulation of the TkSRPP promoter, the full-length promoter PR0 (2188 bp) and its four deletion derivatives, PR1 (1592 bp), PR2 (1274 bp), PR3 (934 bp), and PR4 (450 bp), were fused to β-glucuronidase (GUS) reporter gene and transformed into tobacco. The GUS tissue staining showed that the five promoters distinctly regulated GUS expression utilizing transient transformation of tobacco. The GUS activity driven by a PR0 promoter was detected in transgenic tobacco leaves, stem and roots, suggesting that the TkSRPP promoter was not tissue-specific. Deletion analyses in transgenic tobacco have demonstrated that the PR3 from −934 bp to −450 bp core region responded strongly to the hormones, methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid (SA), and also to injury induction. The TkSRPP gene was highly expressed under hormones and wound-induced conditions. This study reveals the regulation pattern of the SRPP promoter, and provides valuable information for studying natural rubber biosynthesis under hormones and wounding stress.


Introduction
Small rubber particle protein (SRPP) is located on the rubber particle film surface [1][2][3] and forms a subunit of the rubber transferase (RT-ase) complex, which affects rubber chain elongation and rubber quality [4,5]. A previous finding showed that the Parthenium argentatum Gray SRPP protein (GHS) could enhance the rubber biosynthesis activity in vitro [6]. Overexpression of TkSRPP3 in Taraxacum kok-saghyz (T. kok-saghyz) could improve the rubber content in roots. RNA interference in TkSRPP3 gene expression resulted in decreased rubber content and rubber hydrocarbon molecular magnitude in the root [4]. In addition to the above, the reduction of Taraxacum brevicorniculatum (T. brevicorniculatum) TbSRPP expression through RNAi technology could affect the stability of rubber particles and reduce the dry rubber content by 40~50% [7], indicating that SRPP family genes are crucial in rubber biosynthesis.
SRPP also plays a vital role in abiotic stress tolerance. For example, as an SRPP homologue, CaSRP1 gene overexpression in response to water stress in Capsicum plants could enhance drought tolerance and growth of Arabidopsis thaliana [8]. Three SRPP homologues (SRP1, SRP2, and SRP3) were identified in the model plant Arabidopsis thaliana, and their expressions were induced in response to drought as well as high and low temperature conditions to some extent. Compared with the wild-type plants, the overexpressed SRP genes in Arabidopsis showed higher vegetative growth, reproductive growth, and better tolerance to drought stress [9]. However, in producing rubber plants, the study of SRPP genes mainly concentrated on rubber biosynthesis [10,11], and the functional role of its stress tolerance in transgenic research has not been reported.
Previous studies found that SRPP promoters had many cis-acting elements related to abiotic stresses. The latex-specific TbSRPP promoter was regulated in response to external environmental conditions like light, wounds, and cold [12]. The Hevea brasiliensis (H. brasiliensis) SRPP promoter was cloned and induced in response to methyl jasmonate (MeJA), abscisic acid (ABA), gibberellin (GA), cold, heat, and wounding stress [13]. Moreover, the HbSRPP promoter was activated via HbMYC2 protein that regulated jasmonic acid (JA) responsive gene expression [14]. A drought-induced HbNAC1 was found to bind to the cis-element CACG in the HbSRPP promoter to increase the expression of βglucuronidase (GUS) gene [15]. JA and ET-induced HbMADS4 could also activate the HbSRPP promoter [16]. These results indicate that the SRPP promoter can respond to various abiotic stresses.
T. kok-saghyz is an ideal plant for rubber biosynthesis research, containing 10 Tk-SRPP/REF genes in the genome [17]. No functional analysis of the TkSRPP genes for their role in stress tolerance or TkSRPP promoter activation has been determined. In this study, the isolation and functional analysis of the 5 flanking sequence (PR0) of the TkSRPP gene is described. The PR0 promoter activities were conducted with the help of a GUS reporter gene through a histochemical GUS assay in tobacco. Deletion promoters were performed with Agrobacterium-mediated genetic transformation to analyze tissue specificity and hormones and wound response in transgenic tobacco. The gene expression of the TkSRPP promoter was analyzed under hormones-and wound-induced conditions. By analyzing the regulation pattern of the TkSRPP promoter, we can better understand the molecular mechanism by which TkSRPP promotes rubber biosynthesis in response to hormones and wounding stress.

The 5 Flanking Sequence of TkSRPP Promoter Cloning and Sequence Analysis
Based on the previous reported transcriptome data of T. kok-saghyz treated with MeJA, the TkSRPP gene was found up-regulated by MeJA [18]. To evaluate the expression pattern of the TkSRPP gene, the 5 flanking sequence of the TkSRPP promoter (Genbank: MZ190894) was cloned by PCR, and its length was 2188 bp at 5 flanking region of the ATG start codon (Figure 1). Based on the above TkSRPP gene sequence, the nucleotide sequence alignment analysis showed a high similarity of 97% between the correct clone sequence and the genome sequence of this promoter (Supplementary File S1). Moreover, the TkSRPP promoter sequence distributed with TATA-box, CAAT-box, and other typical core elements was endowed with the characteristics of typical plant promoters ( Figure 1).

Figure 1.
Physical map of the TkSRPP promoter. The "A" of the translation initiation code "ATG" of TkSRPP was designated as "+1". The TATA-box is highlighted in bold. Putative cis-acting elements are underlined, colored and labeled. The cis-acting element sites on the negative strand and the double strands are shown in blue and pink, respectively. All the stress-responsive motives are represented by different colours.

Cis-Element Analysis of TkSRPP Promoter
The TkSRPP promoter sequence was predicted to identify important cis-regulatory elements using PLACE [19] and PlantCARE [20] databases. From the predicted results, several vital regulatory elements were detected in the TkSRPP promoter (Table 1, Figure   Figure 1. Physical map of the TkSRPP promoter. The "A" of the translation initiation code "ATG" of TkSRPP was designated as "+1". The TATA-box is highlighted in bold. Putative cis-acting elements are underlined, colored and labeled. The cis-acting element sites on the negative strand and the double strands are shown in blue and pink, respectively. All the stress-responsive motives are represented by different colours.

Activity Analysis of Various SRPP Promoters
Based on the correct TkSRPP promoter sequence, deletion fragments of PR0, PR1, PR2, PR3, and PR4 were cloned with sizes of 2188, 1592, 1274, 934, and 450 bp, respectively (Figures 2 and S1a). The recombinant plasmid pCAMBIA1304-PR0/PR1/PR2/PR3/PR4-GUS was further identified by double enzyme (Nco1 and BamH1) digestion, and the results were consistent with the expected fragment size ( Figure S1b,c), suggesting that the five recombinant vectors be successfully constructed for subsequent experiments.  To detect the activity of the PR0 promoter, the GUS histochemical staining was performed on tobacco leaves treated with Agrobacterium harboring PR0 to evaluate the staining degree. The leaves of wild-type tobacco (negative control groups) were not stained while the tobacco leaf with 35S::GUS (positive control groups) was obviously stained to appear as a large blue patch. The experiment showed that the stained degree of tobacco To detect the activity of the PR0 promoter, the GUS histochemical staining was performed on tobacco leaves treated with Agrobacterium harboring PR0 to evaluate the staining degree. The leaves of wild-type tobacco (negative control groups) were not stained while the tobacco leaf with 35S::GUS (positive control groups) was obviously stained to appear as a large blue patch. The experiment showed that the stained degree of tobacco leaf with PR0::GUS was deeper than that of the negative control groups but shallower than that of the 35S promoter (Figure 3), indicating that the transcriptional activity of the PR0 promoter was activated by upstream protein regulators in the tobacco leaf. Furthermore, the GUS activity of other deletion promoters (PR1/PR2/PR3/PR4) was also analyzed with GUS histochemical staining. The PR1, PR2, and PR3 promoter constructs were strongly stained compared with the negative control groups, whereas the PR4 construct was slightly stained in tobacco leaves ( Figure 3). These results indicated that five promoters (PR0/PR1/PR2/PR3/PR4), driven by GUS reporter gene, showed distinct transcriptional activity due to promoter lengths. with GUS histochemical staining. The PR1, PR2, and PR3 promoter constructs were strongly stained compared with the negative control groups, whereas the PR4 construct was slightly stained in tobacco leaves ( Figure 3). These results indicated that five promoters (PR0/PR1/PR2/PR3/PR4), driven by GUS reporter gene, showed distinct transcriptional activity due to promoter lengths.

Genetic Transformation of Different TkSRPP Promoters
To obtain transgenic tobacco plants with the promoter, pCAMBIA1304-PR0/PR1/PR2/PR3/PR4-GUS was transferred into tobacco by the leaf disk method. The callus of the infected tobacco leaves were differentiated into fascicled buds ( Figure S2a), then transferred to the rooting medium to grow into large plants ( Figure S2b), and finally transplanted to the nutrient soil ( Figure S2c). A pair of Kan gene primers and the PR4 promoter primers, respectively, were used to identify transgenic tobacco plants with different SRPP promoters, as partly shown in the picture ( Figure S2d).

Expression Analysis of SRPP Promoter in Tobacco Different Tissues
The histochemical promoter-GUS assay revealed that AtSRP3 can be highly expressed in the Arabidopsis plants [9]. The rubber tissue-specific HbSRPP promoter in T. brevicorniculatum induced greater GUS gene expression in roots compared with the leaves tissues [12]. Therefore, the tissue specificity of the TkSRPP promoter deserved attention. The whole PR0 transgenic tobacco plant grown for half a month was stained by histochemical staining. It was found that the leaves tissues of PR0 transgenic tobacco were strongly stained compared with the wild-type tobacco, followed by the roots and stems (Figure 4a,b). Moreover, the GUS expression of roots and leaves tissues were approximately 2.73 and 3.73 times that in stems in the PR0 transgenic tobacco, respectively ( Figure  4c). These results showed that the TkSRPP promoter, mainly in the tobacco leaf and root, performed obvious transcriptional activity displayed by the GUS reporter gene.

Genetic Transformation of Different TkSRPP Promoters
To obtain transgenic tobacco plants with the promoter, pCAMBIA1304-PR0/PR1/PR2/ PR3/PR4-GUS was transferred into tobacco by the leaf disk method. The callus of the infected tobacco leaves were differentiated into fascicled buds ( Figure S2a), then transferred to the rooting medium to grow into large plants ( Figure S2b), and finally transplanted to the nutrient soil ( Figure S2c). A pair of Kan gene primers and the PR4 promoter primers, respectively, were used to identify transgenic tobacco plants with different SRPP promoters, as partly shown in the picture ( Figure S2d).

Expression Analysis of SRPP Promoter in Tobacco Different Tissues
The histochemical promoter-GUS assay revealed that AtSRP3 can be highly expressed in the Arabidopsis plants [9]. The rubber tissue-specific HbSRPP promoter in T. brevicorniculatum induced greater GUS gene expression in roots compared with the leaves tissues [12]. Therefore, the tissue specificity of the TkSRPP promoter deserved attention. The whole PR0 transgenic tobacco plant grown for half a month was stained by histochemical staining. It was found that the leaves tissues of PR0 transgenic tobacco were strongly stained compared with the wild-type tobacco, followed by the roots and stems (Figure 4a,b). Moreover, the GUS expression of roots and leaves tissues were approximately 2.73 and 3.73 times that in stems in the PR0 transgenic tobacco, respectively (Figure 4c). These results showed that the TkSRPP promoter, mainly in the tobacco leaf and root, performed obvious transcriptional activity displayed by the GUS reporter gene.

GUS Activity Analysis of TkSRPP Promoters under Hormones and Wounding Stress
Under untreated conditions, the GUS activity of PR0, PR1, PR2, and PR3 (11.01, 9.49, 8.20, and 2.21-fold, respectively; p < 0.01) was significantly higher than that of PR4 in transgenic tobacco (Control in Figure 5). In particular, when the TkSRPP promoter was truncated to the PR3 promoter, the regulated GUS activity decreased significantly ( Figure 5).
To ascertain the distribution of stress-related cis-elements in the TkSRPP promoter, the transgenic tobacco with deletion TkSRPP promoters were treated with ABA, MeJA, SA hormones, and wounding stress. After ABA treatment, the GUS activity significantly increased for the PR0, PR1, PR2, and PR3 (2.08, 2.76, 2.47, and 3.48-fold, respectively; p < 0.01), and PR4 (1.50-fold; p < 0.05) promoters compared with the control (untreated conditions) (Figure 5a), suggesting the key cis-elements that respond to ABA resided in the −1284 to −1 bp region of the promoter. After treatment with MeJA, the GUS activity of the PR0, PR1, PR2, and PR3 promoters significantly increased (1.52, 2.31, 4.84, and 7.53-fold, respectively; p < 0.01) compared with the control, while no significant changes were detected for the PR4 promoter (Figure 5b). It may be that the promoter region from −2188 to −405 bp responds to MeJA positively. After SA treatment, GUS activity increased for PR1 and PR2 (1.69 and 1.88-fold; p < 0.01) compared with the control, while the GUS activity had no significant effect on PR0, PR3, and PR4 (Figure 5c). The results showed that the promoter region from −1592 to −934 bp containing SA-related cis-elements responds to SA. Compared with control, the GUS activity increased more for PR3 (2.33-fold; p < 0.01) under wound stress, whereas the GUS activity of other promoters (PR0, PR1, PR2, and PR4) did not show significant change (Figure 5d). The promoter region from −934 bp to −450 bp harboring cis-elements appears to respond strongly to the wound. Interestingly, the PR3 promoter can be induced by ABA, MeJA, and wounding stress (

GUS Activity Analysis of TkSRPP Promoters under Hormones and Wounding Stress
Under untreated conditions, the GUS activity of PR0, PR1, PR2, and PR3 (11.01, 9.49, 8.20, and 2.21-fold, respectively; p < 0.01) was significantly higher than that of PR4 in transgenic tobacco (Control in Figure 5). In particular, when the TkSRPP promoter was truncated to the PR3 promoter, the regulated GUS activity decreased significantly ( Figure 5).
To ascertain the distribution of stress-related cis-elements in the TkSRPP promoter, the transgenic tobacco with deletion TkSRPP promoters were treated with ABA, MeJA, SA hormones, and wounding stress. After ABA treatment, the GUS activity significantly increased for the PR0, PR1, PR2, and PR3 (2.08, 2.76, 2.47, and 3.48-fold, respectively; p < 0.01), and PR4 (1.50-fold; p < 0.05) promoters compared with the control (untreated conditions) (Figure 5a), suggesting the key cis-elements that respond to ABA resided in the −1284 to −1 bp region of the promoter. After treatment with MeJA, the GUS activity of the PR0, PR1, PR2, and PR3 promoters significantly increased (1.52, 2.31, 4.84, and 7.53-fold, respectively; p < 0.01) compared with the control, while no significant changes were detected for the PR4 promoter (Figure 5b). It may be that the promoter region from −2188 to −405 bp responds to MeJA positively. After SA treatment, GUS activity increased for PR1 and PR2 (1.69 and 1.88-fold; p < 0.01) compared with the control, while the GUS activity had no significant effect on PR0, PR3, and PR4 (Figure 5c). The results showed that the promoter region from −1592 to −934 bp containing SA-related cis-elements responds to SA. Compared with control, the GUS activity increased more for PR3 (2.33-fold; p < 0.01) under wound stress, whereas the GUS activity of other promoters (PR0, PR1, PR2, and PR4) did not show significant change (Figure 5d). The promoter region from −934 bp to −450 bp harboring cis-elements appears to respond strongly to the wound. Interestingly, the PR3 promoter can be induced by ABA, MeJA, and wounding stress (  The GUS activity analysis of five deletion promoters in response to abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA) and wound treatment, respectively. The bars represent standard errors, and the asterisks indicate statistical significance determined by the student's t-test (* p ≤ 0.05, ** p ≤ 0.01).

Expression Analysis of TkSRPP Gene under Hormones and Wounding Stress
Due to the cis-acting element composition effects on the promoter playing an important role in determining the transcription level and function of the gene, qRT-PCR experiments were carried out to analyze the gene expression of this TkSRPP promoter in response to various stresses. The observation showed that the expression levels of the TkSRPP gene in T. kok-saghyz were up-regulated (9.10, 14.59, respectively; p < 0.01) by ABA, MeJA treatment for six hours, consistent with the results of GUS activity of PR0 under above stress conditions. Moreover, compared with the control, the TkSRPP gene expression was induced to rise to 5.33 and 2.03-fold, respectively; p < 0.01 under SA and wound treatment for six hours ( Figure 6); this was not consistent with the results of GUS activity of PR0 under SA and wounding stress conditions attributed to incomplete flanking sequences of the pre-cloned TkSRPP gene. In short, the results indicated that the TkSRPP gene might play a vital role in hormones and wounding stress responses. The GUS activity analysis of five deletion promoters in response to abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA) and wound treatment, respectively. The bars represent standard errors, and the asterisks indicate statistical significance determined by the student's t-test (* p ≤ 0.05, ** p ≤ 0.01).

Expression Analysis of TkSRPP Gene under Hormones and Wounding Stress
Due to the cis-acting element composition effects on the promoter playing an important role in determining the transcription level and function of the gene, qRT-PCR experiments were carried out to analyze the gene expression of this TkSRPP promoter in response to various stresses. The observation showed that the expression levels of the TkSRPP gene in T. kok-saghyz were up-regulated (9.10, 14.59, respectively; p < 0.01) by ABA, MeJA treatment for six hours, consistent with the results of GUS activity of PR0 under above stress conditions. Moreover, compared with the control, the TkSRPP gene expression was induced to rise to 5.33 and 2.03-fold, respectively; p < 0.01 under SA and wound treatment for six hours ( Figure 6); this was not consistent with the results of GUS activity of PR0 under SA and wounding stress conditions attributed to incomplete flanking sequences of the pre-cloned TkSRPP gene. In short, the results indicated that the TkSRPP gene might play a vital role in hormones and wounding stress responses.

Discussion
The involvement of SRPP in the natural rubber biosynthesis has been found [10]. However, the role and function of the SRPP gene in abiotic stress remain inadequately studied. In this study, the 5′ flanking sequence of the TkSRPP promoter was cloned, and it predicted several cis-regulatory elements associated with signal molecules and environmental stresses. To investigate the regulation mechanism of TkSRPP expression, deletion analysis of the TkSRPP promoter in transgenic tobacco was carried out for the critical promoter regions responding to hormones and wounding stress. Moreover, the expression of the TkSRPP gene was enhanced under ABA, MeJA, SA, and wounding stress.

The Key Region Function of SRPP Promoter
The interaction of transcription factors with cis-acting elements is essential in the regulation of plant gene expression responding to hormones and environmental stimulus [21]. Data showed that the GUS activity of deletion TkSRPP promoters was significantly increased after ABA treatment (Figure 5a). Moreover, the ABA response elements (EBOX-BNNAPA, MYCCONSENSUSAT, DRE core, RYREPEATBNNAPA) were also found in the TkSRPP promoter (Figure 1), among which EBOXBNNAPA elements were widely distributed and contained an E-box motif (CANNTG) that acts as a binding site of bHLH transcription factors in response to ABA, such as the CmLOX08, GmRD26 and TPS21 promoter [22][23][24][25]. Therefore, the TkSRPP promoter region may have a large number of ABArelated cis-elements that respond positively to ABA treatment. The TkSRPP promoters (PR0, PR1, PR2, and PR3) were upregulated by the MeJA treatment (Figure 5b), suggesting that the MeJA-responsive elements were widely distributed within this region (from −2188 bp to −450 bp). However, the predictive analysis of the TkSRPP promoter only found one TGACG-motif (TGACG) (Figure 2b), a cis-acting regulatory element in the MeJA responsiveness [26,27]. There might be other potential MeJA-inducible elements that were not predicted through the TkSRPP promoter. The SA treatment led to a

Discussion
The involvement of SRPP in the natural rubber biosynthesis has been found [10]. However, the role and function of the SRPP gene in abiotic stress remain inadequately studied. In this study, the 5 flanking sequence of the TkSRPP promoter was cloned, and it predicted several cis-regulatory elements associated with signal molecules and environmental stresses. To investigate the regulation mechanism of TkSRPP expression, deletion analysis of the TkSRPP promoter in transgenic tobacco was carried out for the critical promoter regions responding to hormones and wounding stress. Moreover, the expression of the TkSRPP gene was enhanced under ABA, MeJA, SA, and wounding stress.

The Key Region Function of SRPP Promoter
The interaction of transcription factors with cis-acting elements is essential in the regulation of plant gene expression responding to hormones and environmental stimulus [21]. Data showed that the GUS activity of deletion TkSRPP promoters was significantly increased after ABA treatment (Figure 5a). Moreover, the ABA response elements (EBOXBNNAPA, MYCCONSENSUSAT, DRE core, RYREPEATBNNAPA) were also found in the TkSRPP promoter (Figure 1), among which EBOXBNNAPA elements were widely distributed and contained an E-box motif (CANNTG) that acts as a binding site of bHLH transcription factors in response to ABA, such as the CmLOX08, GmRD26 and TPS21 promoter [22][23][24][25]. Therefore, the TkSRPP promoter region may have a large number of ABA-related cis-elements that respond positively to ABA treatment. The TkSRPP promoters (PR0, PR1, PR2, and PR3) were upregulated by the MeJA treatment (Figure 5b), suggesting that the MeJA-responsive elements were widely distributed within this region (from −2188 bp to −450 bp). However, the predictive analysis of the TkSRPP promoter only found one TGACG-motif (TGACG) (Figure 2b), a cis-acting regulatory element in the MeJA responsiveness [26,27]. There might be other potential MeJA-inducible elements that were not predicted through the TkSRPP promoter. The SA treatment led to a significant increase for GUS expression of the PR1 and PR2 promoters (Figure 5c). Moreover, the presence of a TCA-box motif in this promoter region ranged from PR2 to PR3 (Figure 1) is a cis-acting element in SA responsiveness [28]. Thus, we speculated that the upstream sequence of the PR1 promoter may have a repressive element of SA response. The role played by TCA motif in regulating the expression of TkSRPP is similar to the role of SA as a signal molecule in VpTNL1 from Vitis pseudoreticulata [29]. The HbSRPP promoter with a wound response element can regulate GUS activity through wound treatment [13]. The GUS activity of the PR3 promoter was regulated by the wound (Figure 5d), indicating the existence of wound-inducible elements in the region from −934 to −450 bp, consistent with the results of promoter prediction (Figures 1 and 2). Differences in induction intensity of various TkSRPP promoters under various hormones and wounding stress may be related to the distribution of specific cis-elements in their sequences. Although the sequence of the cis-acting element cannot be precisely identified, the TkSRPP promoter does harbor fruitful cis-acting elements associated with ABA, MeJA, SA, and wounding stress.

SRPP Is a Typical Stress-Induced Gene in Plants
The functions of homologous family genes seem to be similar in the growth and development of plants and the process of stress responses [30]. The SRPP family genes can promote the biosynthesis of natural rubber, such as T. kok-saghyz, T. brevicorniculatum, and H. brasiliensis [2,4,7,31]. Moreover, the SRPP family genes could respond diversely to ethylene stimulation among different rubber tree clones [32]. Young Arabidopsis thaliana plants overexpressing TbSRPP2 and TbSRPP3 were endowed with higher tolerance to drought stress than wild-type plants [31]. Deletion analysis revealed that HbSRPP and TbSRPP promoters could respond to hormones and abiotic stresses [12,13]. In this study, TkSRPP promoters were predicted to have a large number of cis-elements associated with the stress response ( Figure 1) and can be regulated by ABA, MeJA, SA, and injury in transgenic tobacco ( Figure 5). The TkSRPP gene has high expression when induced by ABA, MeJA, SA, and wound stresses ( Figure 6).
Although only one abiotic stress (wound) was performed in this study, the predicted TkSRPP promoter found several drought-related cis-elements (MBS, CBFHV, DRECRT-COREAT) and other stress responsive elements (Figure 1). We thus speculated that the TkSRPP promoter has the same function as the HbSRPP and the TbSRPP promoter involved in the biological process of a variety of hormones and abiotic stress. In addition, Capsicum annuum and Arabidopsis, the non-rubber-producing plants, also have SRPP homologous genes, but these genes (AtSRPs, GaSRP) might participate in the stress response, such as salt and drought resistance [8,9]. In short, the homologous SRPP genes in rubber-producing plants and non-rubber-producing plants are a class of stress-inducing genes, but whether they have a similar function in rubber synthesis needs further study.

The TkSRPP Gene Is Expected to Promote Rubber Biosynthesis under Hormones and Wounding Stress
JA is a broad-spectrum plant hormone that is essential for the regulation in plant secondary metabolism [33]. Studies have shown that external JA application could promote rubber biosynthesis and laticifer cell differentiation [14,34]. For instance, the expression of the SRPP gene involved in rubber biosynthesis was up-regulated by MeJA induction in the transcriptome of H. brasiliensis and T. kok-saghyz [18,35]. Meanwhile, some transcription factors that respond to JA or MeJA treatments might regulate SRPP gene expression, such as HbMYC2, HbMADS4, HbWRKY83, etc. [14,16]. In this study, the TkSRPP promoter region (from 1256 to 456 bp) could enhance GUS activity induced by exogenous MeJA ( Figure 5). Moreover, the results of qRT-PCR showed that the TkSRPP gene expression could also be up-regulated by MeJA treatments (Figure 6) and the TkSRPP gene showed high expression in RNA-seq data of latex tissue [17]. Thus, the TkSRPP gene can be identified as a key candidate gene to study the regulation of rubber biosynthesis through JA signaling pathways.
JA performs a critical role in regulating plant responses to wounding stress [36]. Previous findings revealed that JA-mediated wound signaling promotes plant regeneration [37,38]. Compared with control, the expression of TkSRPP gene was up-regulated (2.03-fold) under wound treatment for 6 h, but this was obviously lower than MeJA treatment ( Figure 6). Moreover, the GUS activity could reflect that the TkSRPP promoter is more easily regulated by MeJA than the wound ( Figure 5). This may be because wounding stress promotes endogenous JA synthesis and then regulates the expression of the TkSRPP gene by activating JA signaling pathway. The predicted TkSRPP promoter found a woundresponsive element in the region from −934 bp to −450 bp (Figure 2b), and the GUS activity of PR3 promoter had significantly higher than that of control under wounding stress, while other deletion promoters showed no significant change (Figure 5d). The above results indicated that the induction of wounding stress on the regulation of TkSRPP promoter is a complex biological process, which needs to be further studied.
More strong evidence suggests that plant responses to various stresses depend on the crosstalk among hormonal signaling pathways, rather than on the single role of each one [39]. It is exemplified by case that the synergistic effect of both JA and ABA signaling suppressed seed germination of Arabidopsis [40]. The transcription factor ANAC032 from Arabidopsis was characterized by dual roles that directly repress JA signaling and activate SA signaling under the Pseudomonnas syringane infection [41]. Here, we noticed that the GUS activity in the deletion analysis showed similar expression patterns treated by ABA and MeJA ( Figure 5), suggesting that both may play a synergistic role in the regulation of the TkSRPP promoter. On the contrary, under SA treatment conditions ( Figure 5), the GUS activity of only two deletion promoters (PR1 and PR2) had significantly higher than that of the control. However, the GUS activity of the PR0 promoter did not change, which may be due to the existence of JA response elements in the PR0-PR1 region inhibiting the regulation of SA. The crosstalk between the three hormones (ABA, SA, and MeJA) co-regulates the expression of the TkSRPP gene involved in rubber biosynthesis and the mechanism and reason for this need to be further studied. Collectively, the regulation pattern of the SRPP promoter in response to SA, ABA, MeJA and wounding stress were revealed, which provides a theoretical basis for us to better understand how these conditions are involved in rubber biosynthesis process.

Plant Materials, Growth Conditions
The leaves tissue samples were obtained from T. kok-saghyz growing in the culture room at 21 ± 2 • C, under a 16/8 h day/night photoperiod for two months. The Nicotiana benthamiana plants for agro-infiltration were cultivated in a culture room at 25 • C under a 16/8 h day/night cycle for six weeks. The tobacco (Nicotiana tabacum) was grown in a sterile bottle at 25 • C under a 16/8 h day/night photoperiod.

Cloning of the HbSRPP Genomic Sequence
The 5 flanking sequence of the SRPP promoter was obtained from the T. kok-saghyz genome of the Genome Warehouse (GWH; http://bigd.big.ac.cn/gwh/, accessed on 10 March 2021) under the accession number PRJCA000437 [17], based on sequence alignment of the TkSRPP gene using Blast 2.11.0 software (ftp://ftp.ncbi.nlm.nih.gov/blast/ executables/blast+/LATEST/, accessed on 3 January 2021). Genomic DNA was isolated from T. kok-saghyz leaves as per the method proposed by [18]. The primers (Table S1) for cloning of the promoter region were designed using Primer 5.0 software. PCR experiment was performed using the total DNA of leaf as a template, and the DNA polymerase is PrimeSTAR Max DNA Polymerase (TAKARA, Beijing, China). The PCR program was as follows: 30 cycles of 10 s at 95 • C, 20 s at 55 • C, and 2.5 min at 72 • C, with a final extension at 72 • C for 8 min. PCR products were detected by 1% agarose gel electrophoresis and recovered with the agarose gel DNA recovery kit (TIANGEN, Beijing, China). The recovered product was connected with the pMD-19T vector (TAKARA, Beijing, China) at 16 • C for 8 h according to the manufacturer's instructions. The recombinant plasmid was then transformed into E. coli DH5α strain, and several positive single colonies were selected and sequenced for confirmation.

Construction of the TkSRPP Promoter::GUS Plasmids
First, for construction of the TkSRPP promoter driving GUS fusion genes, the 5 flanking sequence (−2188 bp) of SRPP gene and four deletion fragments encompassing different lengths of the TkSRPP promoter (−1592 bp, −1274 bp, −934 bp, and −450 bp to −1 bp, respectively) were obtained by PCR amplification using five sets of specific PCR primers with BamHI and NcoI sites (Table S1). These fragments' amplification was performed using pMD-19T-PTkSRPP plasmid as a template to obtain their sequence fragments. Then, five fragments were respectively cloned into the pMD-19T vector. The recombinant plasmids of 19T vectors were digested by BamHI and NcoI, and five fragments were connected with the pCAMBIA1304-35S::GUS vector ( Figure 2) using T4 DNA Ligase (TAKARA, Beijing, China). Finally, all of the structures were transformed into the E. coli DH5α strain, and positive clones were verified by sequencing, colony PCR, and enzyme digestion reaction.

Histochemical Straining Assays
Histochemical GUS assays were performed as described elsewhere [18]. The suspension (1 mL) of Agrobacterium tumefaciens strain (GV3101) harboring a TkSRPP promoter plasmid was inoculated into 50 mL LB liquid medium supplemented with 70 mg/L rifampicin (rif) and 50 mg/L kanamycin (Kan) at 28 • C with shaking until OD600 value was 0.8. Agrobacterium cells were harvested after centrifugation at 5000× g for 5 min, suspended in infiltration solution (10 mM MES, pH 5.7, 10 mM MgCl2, and 20 µM AS), incubated at 28 • C for 3 h at dark. The final bacterial suspension was injected into tobacco lower epidermal leaf via a needleless syringe and the whole tobacco leaf was infused with Agrobacterium. The tobacco plants were then cultivated in a constant temperature incubator under a 16/8 h day/night cycle at 25 • C for 48 h. The treated tobacco leaves were prepared for subsequent GUS staining experiments.
Histochemical staining was performed following the procedures described by Jefferson [22]. The tobacco leaves with Agrobacterium-mediated transient transformation were incubated in GUS staining solutions, including 80 mM sodium phosphate buffer (pH 7.0), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 1 mg/mL 5-Bromo-4chloro-indolyl-b-D-glucuronide (X-Gluc), and 0.1% Triton X-100 for 24 h at 37 • C. After staining, the tissues were transferred to 70% ethanol to decolorize at 37 • C for about two days. Stained tissues were examined and taken photos with a scanner.

Transgenic Plant Generation
Various truncation promoters were transformed into tobacco by stable genetic transformation to verify the functions of deletion promoters. The sterile tobacco leaves were cut into 1 × 1 cm area segments with scissors and then placed in the Murashige and Skoog (MS) medium in the dark for two days. Of the recombinant plasmids carried by the Agrobacterium tumefaciens (GV3101), five were transformed into tobacco by the leaf disc method [42]. Transgenic tobacco plants were screened using Kan (100 mg/L) and identified by a pair of primers of the kan gene and the TkSRPP promoter primers (Table S2).

Tissue Specificity of TkSRPP Promoter
The T2 transgenic tobacco with the TkSRPP promoter and the wild-type tobacco plants were grown on MS in sterile culture bottles. The GUS staining experiments were conducted to analyze the tissue-specific functions of the TkSRPP promoters in transgenic tobacco. The GUS expression of different tissues (leaf, stem, and root) of the TkSRPP transgenic tobacco was also performed by qRT-PCR to assess the tissue specificity of the TkSRPP promoter.

Abiotic Stress Treatments
The T2 transgenic tobacco plants were transplanted into flowerpots and grown for two months, under a 16/8 h day/night photoperiod. The leaves of transgenic tobacco were sprayed with 100 µM abscisic acid (ABA), 1 mM salicylic acid (SA), and 1 mM methyl jasmonate (MeJA), respectively, with deionized water treatment as control groups. The injury stress used scissors to cut the transgenic tobacco leaves into strip wounds with a distance between them of 2 cm. The transgenic tobacco plants were used as control groups under normal conditions. Three biological replicates samples were collected after six hours and were used to measure the GUS activity. In addition, the three months wild-type T. kok-saghyz plants were treated with ABA, SA, MeJA and wound for six hours in the same way as above. The leaf tissues of three biological duplicates were collected for liquid nitrogen quick-freezing and stored in the −80 • C refrigerator.

GUS Fluorimetric Assays
The Stable expression of GUS activity in the treated tobacco leaves was measured as described elsewhere. The tobacco leaf tissue (0.5 g) was homogenized in 1 mL extraction buffer [50 mM sodium phosphate buffer (pH 7.0), 10 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.1% (w/v) sodium dodecyl sulfate, 10 mM b-mercaptoethanol]. The supernatant (50 µL) centrifuged at 13,000 rpm for 15 min at 4 • C was collected and mixed with 390 mL pre-warmed (37 • C) GUS assay solution (1 mM methyl-4-umbelliferyl-D-glucuronide in extraction buffer) and incubated at 37 • C to collect the liquid sample (100 µL) every 30 min. The liquid samples were added to the (900 µL) stop buffer (0.2 M Na 2 CO 3 ). The fluorescence signal was measured with an ultraviolet and visible spectrophotometer (f97xp; lengguang tech., Shanghai, China) at excitation and emission wavelengths of 365 nm and 455 nm. Stop buffer and 0 nM to 100 nM were used for 4-methylumbelliferone (4-MU) calibration and standardization. The total protein concentration was measured with bovine serum albumin as a standard, following the methods of the previous report [43]. The GUS activity was determined by nM of 4-MU generated per min per mg soluble protein and performed with three duplicates.

qRT-PCR
The total RNA was extracted with an EasyPure ® Plant RNA Kit (TRAN, Beijing, China). The first strand of cDNA was synthesized with the EasyScript ® One-Step gDNA Removal Kit and cDNA Synthesis Supermix (TRAN, Beijing, China) following the instructions of manufacturers. Meanwhile, Primer 5.0 software was used to design the primers (Tables S3  and S4) for qRT-PCR. The qRT-PCR reaction was performed on the Roche 480 platform with LightCycler 480 SYBR Green Master Mix (Roche, Shanghai, China). The qRT-PCR procedure was performed using the following profile: preincubated at 95 • C for 5 min, followed by 40 cycles of denaturation at 95 • C for 10 s, annealed at 58 • C for 10 s, and extended at 72 • C for 30 s. The TkGAPDH [18] and NbEF1 [44], respectively, were used as reference genes. The full experiments were performed with three biological and experimental duplicates to analyze quantitative data with the 2 −∆∆ct methods [45].

Statistical Analysis
The data of three independent experiments were analyzed with a one-way analysis of variance. The value p ≤ 0.05 was considered significant and charts were generated with GraphPad Prism 8 software.

Conclusions
In this study, we isolated and characterized the 5 flanking sequence of the TkSRPP promoter associated with many abiotic response elements. Based on the performance of the transgenic tobacco plants, we concluded that the TkSRPP promoter plays a vital role in responding to SA, MeJA, ABA, and wounding stress. Moreover, the results of its gene expression also confirmed the above conclusion. These results enhance our understanding of the role of the TkSRPP promoter in the regulation of SA, MeJA, ABA, and wounding responses, and provide useful information for further study of the rubber biosynthesis mechanisms by the TkSRPP promoter in response to hormones and wounding stress.  Table S1: Primer sequences of different TkSRPP promoter cloning; Table S2: Identification of different SRPP promoter transgenic tobacco; Table S3: Quantitative Primer sequences of TkSRPP gene; Table S4: Quantitative Primer sequences of GUS gene.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.