Functional Analysis of the HbREF1 Promoter from Hevea brasiliensis and Its Response to Phytohormones

Functional Analysis of the HbREF1 Promoter from Hevea brasiliensis and Its Response to Phytohormones

Promoter sequences are located upstream of the 5′ end of the structural gene, which can be recognized and bound by RNA polymerase and initiate gene transcription. Eukaryotic RNA polymerase requires interaction with transcription factors in order to initiate the process of transcription. These transcription factors possess the ability to locate the specific DNA elements for binding and to attract other proteins to the transcription site [39]. Eukaryotic promoters mainly include two types of regulatory elements: relatively conservative basic elements and cis-elements with great differences between genes [40]. The composition of promoter elements, especially cis-acting elements, is crucial for the temporal and spatial expression of genes and the regulation of transcription levels. In this study, the cloned HbREF1 promoter sequence of 1758 bp has an AT content as high as 75%. The analysis of the promoter sequence indicates that the core element of the TATA box required for transcription is located upstream of the translation start site at −150 bp. There are binding sites for nine types of transcription factors associated with plant abiotic stress and hormone responses in the HbREF1 promoter region. Transcription factor families such as MYB, bHLH, EIL, ERF/DREB, Trihelix, and bZIP play crucial roles in plant growth and development, secondary metabolism regulation, response to hormones, and regulation of abiotic stress [41,42,43,44,45,46]. MYC2, a member of the bHLH family, is a major regulatory factor in the JA signaling pathway. ABA is one of the important hormones involved in plant tolerance of nonbiological stress, and bZIP transcription factors play a significant role in ABA signal transduction. EIL and ERF/DREB are important transcription factors in the ethylene signaling pathway. TGA transcription factors (D subfamily of the bZIP transcription factor) have been reported to participate in SA response. Previous studies have shown that the expression of HbREF1 in latex is induced by ABA, MeJA, and tapping [29,47]. It is speculated that these predicted transcription factors regulate the transcription of HbREF1 in response to hormones and tapping damage.
Deng et al. [48] isolated the sequence of 378 bp upstream of the HbREF1 translation initiation site using the chromosome walking method. Priya et al. [12] fused the promoter sequence of 378 bp upstream of the translation initiation site with GUS and transformed it into tobacco; the results showed that the promoter sequence of 378 bp has the activity to initiate GUS transcription. However, these sequences provide too little information to study the detailed regulatory mechanism of HbREF1, and it was necessary to isolate longer promoter sequences. Transient expression analysis provides a rapid and effective method to identify whether the promoter has the active function of initiating downstream gene expression [49]. The transcriptional activity of the promoter was qualitatively and quantitatively analyzed by detecting the luciferase reporter gene. In this study, five HbREF1 promoter vectors fused to the luciferase reporter gene were constructed, with lengths of 1758 bp, 1300 bp, 718 bp, 583 bp, and 200 bp, respectively. The LUC in vivo bioluminescence imaging result showed that all five HbREF1 promoter vectors exhibited transcriptional activity. The basal transcriptional activity analysis result showed that as the deletion length of the HbREF1 promoter increases, the transcriptional activity becomes stronger. It is speculated that there are multiple elements that inhibited the promoter transcriptional activity in the −1758 bp to −200 bp region, especially in the −1300 bp to −583 bp region, where the inhibitory effect is stronger.
Tungngoen et al. [22] found that treating mature untapped rubber tree bark with ET, auxin, ABA, and SA can induce a transient increase in latex yield. Deng et al. [29] found that there is a JA signal transduction module COI1-JAZ3-MYC2 in the laticifer of rubber trees, which promotes the biosynthesis of NR by upregulating the expression of rubber biosynthesis genes. Previous studies have shown that treatment with ET, JA, and ABA can increase the expression of HbREF1 [11,29,47], while the effect of SA treatment on HbREF1 expression has not been reported. Sequence analysis reveals that the promoter sequence of HbREF1 contains multiple TFBSs for transcription factors that may respond to hormones such as ABA, ET, JA, and SA. In Hevea brasiliensis, the MYC transcription factors, as a core component of the JA signal, bind to the G-box motif of the HbPSK5 promoter and activate the HbPSK5 promoter. Overexpression of HbMYC26 and HbPSK5 in Taraxacum kok-saghyz can promote the formation of latex vessels and increase rubber content [50]. In Taraxacum brevicorniculatum, TbbZIP.1 binds to the ABRE elements of the TbSRPP promoter and regulates the expression of the TbSRPP in an ABA-dependent manner, increasing the latex content [51]. The results of this study suggest that the transcriptional activity of the HbREF1 promoter can be induced by ABA, ET, JA, and SA. An analysis of transcriptional activity after treatment by ABA shows that the ABA response element is located upstream of the −200 bp region (Figure 5). The two TFBSs of bZIP were also predicted to exist upstream of 200 bp (Table 1). It was speculated that HbREF1 regulated by ABA is related to these two motifs. In response to ET treatment, the promoter fragments P−1758, P−1300, and P−718 were strongly activated to induce the LUC gene expression, but the promoter fragments P−583 and P−200 were not effective for the promoter activity (Figure 5). It is shown that the ET response element is located upstream of −583 bp. As shown in Table 1, the TFBSs of EIL and ERF/ERDB are located upstream of −585 bp, and they appear to serve as regulatory factors in the HbREF1 promoter that responds to ET. In response to JA treatment, only P−1758 was strongly activated to induce LUC gene expression, but its deletion derivatives (P−1300, P−718, P−583, and P−200) had no significant effect on promoter activity (Figure 5). It is shown that the JA response element is located in the upstream region of −1300 bp. However, the binding site of bHLH is located in the −1144 bp~−202 bp region. Previous studies have indicated that MYB and ERF/DREB are important regulatory factors in the JA signaling pathway [41,44]. One MYB binding site is located at −1677 bp, and one ERF/DREB binding site is located at −1328 bp (Table 1). The induction of HbREF1 by JA may be subject to collaborative regulation by multiple transcription factors. In SA treatment, it is shown that there is an SA response element located upstream of −718 bp (Figure 5), and one bZIP binding site (TGA) situated at −1107 bp (Table 1) that seems to function as a regulatory element within the HbREF1 promoter, responding to SA.

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