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HbTGA1, a TGA Transcription Factor From Hevea brasiliensis, Regulates the Expression of Multiple Natural Rubber Biosynthesis Genes

The TGA transcription factors are known to modulate the biosynthesis of secondary metabolites in plants. However, their regulatory function in natural rubber (NR) biosynthesis was not revealed in the rubber tree (Hevea brasiliensis). Here, 14 genes encoding TGA transcription factors (name HbTGA1-HbTGA14) were identified in the rubber tree. HbTGAs were differentially expressed in different tissues. HbTGA1 was expressed at its highest level in latex. We found specific in vitro and in vivo binding of the HbTGA1 protein with promoters of multiple NR biosynthesis genes (HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2). The activation of the promoters of HbHMGS2 and HbCPT6 was significantly suppressed by HbTGA1, while the activities of promoters of HbHMGR2, HbCPT8, and HbSRPP2 were increased by HbTGA1. The promoter activities of HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2 were significantly increased by HbTGA1 under jasmonate stress, while the promoter activities of HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2 were also significantly increased by HbTGA1 under salicylic acid stress. The present study provides insights into the role of TGA transcription factors in regulating the expression of NR biosynthesis genes from H. brasiliensis.


INTRODUCTION
Rubber tree (Hevea brasiliensis Muell. Arg) is an economically essential tropical tree and provides a sole commercial source of natural rubber (NR). NR, cis-1,4-polyisoprene, is an important industrial raw material because of its unique physical properties (Backhaus, 1985;van Beilen and Poirier, 2007). NR is an end-product of a side branch of the plant isoprenoid synthesis pathway (Yamashita and Takahashi, 2020). NR is produced in laticifer cells through a sequential condensation of isopentenyl diphosphates (IPPs) in a cis-1,4 configuration (Cornish, 2001;Chow et al., 2007;Salehi et al., 2021). As a consequence genes involved in IPP synthesis to cis-1,4-polyisoprene are named as NR biosynthesis genes (Tang et al., 2016). The biochemical pathways involving NR biosynthesis are now fully understood (Yamashita and Takahashi, 2020;Salehi et al., 2021), the regulatory mechanism of NR biosynthesis genes has not yet been fully investigated in the rubber tree. Guo et al. Hevea brasiliensis HbTGA1 Transcription Factor The TGA transcription factors (TFs) are members of the subfamily of basic leucine zipper (bZIP) TFs (Li et al., 2019a;Wang et al., 2019). TGA proteins have the conserved bZIP domain-containing N-x 7 -R/K-x 9 -L-x 6 -L-x 6 -L motif, and Yx 2 RL[RQ]ALSS[LS]W motif, which is the unique motif of TGA TFs (Gatz, 2013). TGA TFs regulate gene expression by specific binding of TGACG-motif on gene promoter (Jakoby et al., 2002) and play crucial roles in secondary metabolites biosynthesis (Lv et al., 2019;Han et al., 2020;Huo et al., 2021), plant development , defense against pathogens (Kesarwani et al., 2007;Shearer et al., 2012), and responses to stress (Gatz, 2013;Li et al., 2019b). Given the potential importance of TGA TFs in plant secondary metabolites biosynthesis, we carried out the analysis of the TGA TFs family in the rubber tree and investigated the roles of TGA TFs in NR biosynthesis.

Identification of Rubber Tree TGA TFs
Ten Arabidopsis TGA protein sequences were downloaded from the NCBI database. These protein sequences were used as query sequences against the local rubber tree genome database  through the BLASTp search (E-value 1.0 × e −5 ). Then, candidate rubber tree TGAs were further confirmed for conserved domains in the Pfam database (El-Gebali et al., 2018). The final confirmed genes were named HbTGA1-HbTGA14. The molecular weight and isoelectric point of HbTGAs were predicted by the ExPASy server (Gasteiger et al., 2003).

Chromosomal Distribution of HbTGAs
The start and end points of HbTGAs were calculated from the rubber tree annotation GFF3 files, the chromosomal distribution of HbTGAs was displayed using MapChart v2.3.

Analysis of Gene Structure and Construction of a Phylogenetic Tree
The exon and intron of HbTGAs were investigated using a web server GSDS (Hu et al., 2015). Multiple sequence alignments for HbTGA and other TGA proteins sequences from Arabidopsis, cassava, and Ricinus communis derived from the NCBI database were carried out using Clustal. 1 The phylogenetic tree was constructed based on the HbTGA and other TGA protein sequences from Arabidopsis, cassava, and R. communis using MEGA7.0 software with Neighbor-Joining methods followed by 1,000 bootstrap replicates (Kumar et al., 2016).

Prediction of TGA TF Binding Sites on the Promoters of NR Biosynthesis Genes
The 3.0 kb upstream sequences of the start codon of NR biosynthesis genes were obtained as the promoter region from the rubber tree local genome database. TGA TF binding sites on the promoters of NR biosynthesis genes were calculated using PlantCARE (Rombauts et al., 1999).

Extraction of Rubber Tree DNA and RNA
DNA was isolated from the leaves according to method of Allen et al. (2006). RNA was extracted from latex as described previously (Tang et al., 2007).

Subcellular Localization of HbTGA1
The coding sequence of HbTGA1 was amplified with PCR primers (see Supplementary Table S1) from latex cDNA, and cloned into the BglII/NcoI restriction site of pCAMBIA1302, resulting in a HbTGA1 fused with green fluorescent protein (GFP) expression vector, which was further introduced into onion epidermal by Agrobacterium-mediated transformation. The transformed onion epidermal were incubated in darkness for 48 h at 25°C and then visualized under fluorescent microscopy.

Yeast One-Hybrid
The promoter fragments of NR biosynthesis genes harboring TGACG-motif were obtained through PCR amplification with primers (see Supplementary Table S1), respectively. The bait vector was constructed by cloning the promoters of NR biosynthesis genes into the site of SacI/SpeI of pHIS2.1 vector (Clontech). The coding sequence of HbTGA1 was inserted into the pGADT7 vector at the site of EcoRI/BamHI, generating prey vectors. Then, yeast Y187 strain was introduced with bait and prey vectors, respectively. The introduced yeast was cultured on SD/−Trp-Leu medium and SD/−Leu/−Trp/-His medium adding corresponding concentrations of 3-AT obtained by screening experiment for 3 days at 30°C.

Electrophoretic Mobility Shift Assay
To determine the binding of the HbTGA1 protein with promoters of NR biosynthesis genes in vitro, electrophoretic mobility shift assay (EMSA) was performed. Firstly, the coding sequence of HbTGA1 was cloned into the pET28a vector at the site of BamHI/EcoRI, resulting in an HbTGA1-His-tagged protein expression vector. The expression vector was introduced into Escherichia coli BL21 (DE3). HbTGA1-His-tagged protein was purified using a HiTrap affinity column (GE Healthcare) in accordance with the instruction manual. Secondly, total 205 bp-long DNA fragments of the NR biosynthesis genes promoter (except HbHMGS2) were PCR-amplified, which containing the predicted TGACG-motifs with 100 bp upstream and 100 bp downstream. There were two TGACG-motifs between in 217 bp, total 417 bp-long DNA fragments of HbHMGS2 promoter was PCR-amplified, from 100 bp upstream of the first TGACG-motif to 100 bp downstream of the second TGACG-motif. The 205 bp-long DNA fragments which no TGACG-motif was PCR-amplified as negative control. The PCR products were purified using purification kit (Foregene, DE-03011). The primers used for EMSA are listed in Supplementary Table S1. Finally, EMSA was implemented according to the protocol of the EMSA kit (Invitrogen, E33075). In brief, the purified HbTGA1 was incubated with doublestranded promoter nucleotides for 30 min at room temperature. The DNA/protein complex samples were subjected to 12% polyacrylamide gel electrophoresis at 120 V for 30 min. Then, gel was examined using SYBR Green EMSA stain.

Activation of the Promoters of NR Biosynthesis Genes by HbTGA1
To identify if HbTGA1 activates the promoters of NR biosynthesis genes in plant, a dual-luciferase assay system was employed for this purpose. In brief, HbTGA1 was amplified and cloned into a pGreenII62 SK vector, generating an effector vector. The promoter of NR biosynthesis genes was amplified and cloned into a pGreenII0800-luciferase (LUC) vector, as a reporter vector. Then Agrobacterium tumefaciens GV3101 was introduced with effector or reporter vector. The A. tumefaciens containing the reporter and effector were mixed in the ratio of 1:2 (v:v) and then infiltrated into the tobacco following previously described method (Hellens et al., 2005). The infiltrated leaves were collected to measure LUC activities until 72 h after infiltration. For methyl jasmonate (MeJA) and salicylic acid (SA) treatment, tobacco leaves infiltrated with A. tumefaciens were sprayed with 100 μM MeJA, 100 μM SA, or water as control at 48 h after infiltration. The infiltrated leaves were collected and extracted protein for LUC activities assay at 24 h after treatment. The fluorescent values of LUC and the reference Renilla (REN) were measured in accordance with the instruction manual of the Dual-Luciferase Reporter Assay System (Promega, E1910). Experiments were performed in three biological replicates per treatment. Statistical analyses were carried out by one-way ANOVA (SAS6.11). Statistical significance was defined as p < 0.05.
To explore the evolutionary relationships between HbTGAs and TGAs from other plants, a phylogenetic tree was constructed by comparing HbTGAs with TGA proteins from Arabidopsis, cassava, and R. communis. As shown in Figure 2, 42 TGA proteins were separated into five clades. Clades II and IV both contain four HbTGAs, while clade I, III, and V have two members of the HbTGAs family.
The gene structures of 14 HbTGAs were also examined. HbTGAs were interrupted by more than six introns ( Figure 3C). HbTGAs in Clades II, IV, and V contain more than 10 introns, while HbTGAs in Clades I and II have seven introns. The same group of HbTGAs has a similar gene structure and the same conserved domain, which may reflect their similar functions.

Expression Patterns of HbTGAs
The expression patterns of HbTGAs were analyzed using the transcriptome data of different rubber tree tissues (Figure 4). All the 14 HbTGAs were expressed in the evaluated tissues, but there were differences in expression patterns. For example, the expression of HbTGA1 showed the highest level in latex. HbTGA2 and HbTGA3 had higher expression levels in all evaluated tissues, while HbTGA6, HbTGA7, and HbTGA11 had lower expression levels in all evaluated tissues. HbTGA12 and HbTGA13 were expressed most strongly in the root, HbTGA2, HbTGA5, HbTGA9, and HbTGA12 were expressed at their highest levels in the seed. In addition, HbTGA10, 11, 12 were not expressed in latex, HbTGA12 was not expressed in leaf, while HbTGA12 was not expressed in the female flower.

Prediction of the Binding Site of TGA TFs on the Promoters of NR Biosynthesis Genes
The 3 kb upstream fragment of the start codon (ATG) of NR biosynthesis-related gene was isolated as the promoter region. The putative TGA TFs binding site (TGACG-motif) was predicted on the promoters of NR biosynthesis genes. As shown in Figure 5A, the promoters of these natural rubber synthesis genes contain 1-3 TGA TFs recognition sites. The expression of NR biosynthesis genes was analyzed using the transcriptome data of seven rubber tree tissues and organs ( Figure 5B). According to the expression abundance of these genes in latex, HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2 which were expressed at a higher level in latex were selected to further

HbTGA1 Is a Nuclear Protein and Interacts With Promoters of NR Biosynthesis Genes
The subcellular localization of HbTGA1 was analyzed in transiently transformed onion epidermal cells. As shown in Figure 6, HbTGA1 was located in the nucleus. The binding specificity of these promoters of NR biosynthesis genes with HbTGA1 was determined by the yeast one-hybrid experiment (Figure 7). An in vitro EMSA assay further suggested HbTGA1 bound with promoters of HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2 (Figure 8). This finding indicates that HbTGA1 binds to the promoters of these NR biosynthesis genes and activates the transcription in yeast.

HbTGA1 Regulated the Activation of Promoters of Multiple NR Biosynthesis Genes
Since NR is synthesized in laticifers, genes highly expressed in laticifers may participate in NR biosynthesis (Oh et al., 1999). The expression level of HbTGA1 is the most highest among all HbTGAs. To investigate whether HbTGA1 involves in regulating NR biosynthesis-related genes, we transiently expressed HbTGA1 along with a LUC controlled by the promoters FIGURE 2 | Phylogenetic analysis of TGA proteins from Arabidopsis, cassava, and Ricinus communis with Neighbor-Joining method using 1,000 bootstrap replicates. Each clade is demonstrated by different color.
of NR biosynthesis genes in tobacco. The schematic diagrams of the effectors and reporters used in dual-luciferase assay system are shown in Figure 9A. Compared to the control, the expression of HbTGA1 enabled to change the level of the LUC activity controlled by the promoters of NR biosynthesis genes. As shown in Figure 9B, the activation of the promoter of HbHMGS2, and HbCPT6 were significantly suppressed by HbTGA1, decreased by 27 and 55%, respectively; while the activation of promoters of HbHMGR2, HbCPT8, and HbSRPP2, increased to 2.5-, 3.3-, and 1.4-fold, respectively ( Figure 9B). Taken together, HbTGA1 takes part in regulating the expression of multiple genes in the NR synthesis pathway.
Subsequently, we transiently expressed HbTGA1 along with a LUC controlled by the promoters of NR biosynthesis genes in tobacco under MeJA and SA treatment conditions (Figure 10). Compared to the control, the promoter activities of HbHMGS2 and HbCPT6, HbCPT8, and HbSRPP2 were significantly increased by HbTGA1 under MeJA stress, increased to 1.5-, 1.5-, 1.3-, and 1.4-fold, respectively; the promoter activities of HbHMGS2, HbHMGR2, HbCPT6, HbCPT8, and HbSRPP2 were also significantly increased by HbTGA1 under SA stress, increased to 1.3-, 2.0-, 1.6-, 1.4-, and 1.9-fold, respectively. The result revealed that HbTGA1 might participate in the MeJA-and SA-inducible NR biosynthesis in rubber tree.

DISCUSSION
A number of investigates have proved the involvement of TGA TFs in several biological processes (Shearer et al., 2012;Gatz, 2013;Canales et al., 2017;Sun et al., 2017;Lv et al., 2019;Wang et al., 2019;Han et al., 2020;Huo et al., 2021). However, the TGA TFs family and their role in the rubber tree were scarcely understood. Here, we identified 14 TGA TFs members in the rubber tree. The HbTGAs could be clustered together with TGAs from Arabidopsis, cassava, and R. communis in the same clade, suggesting that the evolution of TGA genes is conserved.
Transcription factors regulate gene expression and play key roles in biological processes. In rubber tree, a few TFs take part in regulation of the expression of NR biosynthesis genes. For example, three TFs, including HbWRKY1, HbWRKY14, and HbMADS4, downregulated the expression of HbSRPP Li et al., 2016Li et al., , 2020. HbCZF1 upregulates the expression of hmg1 . HblMYB19, HblMYB44, and HbWRKY27 Frontiers in Plant Science | www.frontiersin.org upregulated the expression of HbFPS1 Qu et al., 2020). HbRZFP1 downregulated HRT2 expression . HbMYC2b activates HbSRPP expression . These data showed TFs play critical roles in NR biosynthesis. It was found that TGA TFs regulate plant secondary metabolites biosynthesis. In Artemisia annua, AaTGA6 regulates artemisinin content by directly binding to the promoter of AaERF1. In Tripterygium wilfordii, TwTGA1 binds with promoters of PMT and MPO1 and activates their expressions, and modulates secondary metabolites biosynthesis (Han et al., 2020). In our study, Y1H and EMSA assays showed that HbTGA1 bound to the promoter of NR biosynthesis genes. The activation of promoters of multiple NR biosynthesis genes was regulated by HbTGA1. These results suggested HbTGA1 might modulate the expression of NR biosynthesis genes and participate in NR synthesis in rubber tree. The functions of HbTGA1 participating in NR synthesis in rubber  tree needs to be explored in rubber tree in future. Additionally, TGA factors have been shown to interact with other TFs to modulate their activity (Gatz, 2013), the potential regulators of HbTGA1 also needs to further be investigated. In plant TGA, TFs have key roles in secondary metabolites biosynthesis through SA and JA signaling pathways (Lv et al., 2019;Huo et al., 2021). For example, TwTGA1 was reported to increase the MeJA-inducible triptolide synthesis by upregulating the expression of TwTPS27a/b in T. wilfordii (Han et al., 2020;Huo et al., 2021). AaTGA6 was reported to modulate SA-inducible artemisinin synthesis in A. annua (Lv et al., 2019). In this study, the activation of promoters of multiple NR biosynthesis genes was significantly increased by HbTGA1 under SA and JA. In addition, SA could also induce a transient increase in NR yield in rubber tree (Tungngoen et al., 2011), suggesting SA signaling pathway might play role in regulating NR biosynthesis. However, there is still a lack of direct evidence to show SA signaling regulatory involvement in NR biosynthesis and the core module of SA signaling needs to further clarify in rubber tree. JA signaling has been reported to modulate NR biosynthesis in rubber tree (Deng et al., 2018). Being the target of SA signaling, HbTGA1 can connect the SA pathway with the JA pathway (Gatz, 2013). Thus, it will be of great interest to further study the regulatory mechanisms of HbTGA1 modulating MeJA-and SA-inducible NR biosynthesis. HbTGA1 might become a biotechnological tool in rubber tree breeding.
FIGURE 7 | HbTGA1 binding with the promoters of NR biosynthesis genes in yeast. Yeast harboring bait and prey vectors were grown in SD/−Trp-Leu medium and SD/−Leu/−Trp/-His selective medium adding corresponding concentrations of 3-AT obtained by screening experiment for 3 days at 30°C.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.909098/ full#supplementary-material A B FIGURE 9 | Effect of HbTGA1 on the activation of the promoter of NR biosynthesis genes. Schematic maps of e-effector and reporter (A). The promoter activity was determined by a transient dual-luciferase (LUC) assay (B). The relative LUC activities (LUC/REN) were normalized to the reference Renilla (REN) LUC. Error bars represent SD of three technical replicates. *p < 0.05, **p < 0.01.