A Basic Helix-Loop-Helix Gene from Poplar is Regulated by a Basic Leucine-Zipper Protein and is Involved in the ABA-Dependent Signaling Pathway
- 457 Downloads
Basic helix-loop-helix (bHLH) transcription factors (TF) comprise a large group of proteins that are involved in many developmental and physiological processes in plants. In this study, a bHLH gene (PkbHLH2), along with its promoter, was cloned from Populus koreana Rehd. A PkbHLH2 promoter::GUS gene fusion construct was generated to investigate the expression of PkbHLH2. The results demonstrated that PkbHLH2 was expressed mainly in leaf stalks, leaf veins and roots. Yeast one-hybrid assays showed that a bZIP gene product (PkbZIP2) can bind specifically to the ABA-responsive elements (ABRE) that exist in the promoter region of PkbHLH2, regulating the expression of PkbHLH2. In addition, the “GC” of the ABRE core motif “ACGTG” was very important for PkbZIP2 recognition, because its mutation to “TT” completely prevented the interaction between PkbZIP2 and ABRE. Furthermore, both PkbHLH2 and PkbZIP2 can be up-regulated by abscisic acid (ABA) and osmotic stress, and share similar expression patterns when exposed to ABA and osmotic stress. These results suggest that PkbZIP2 is an upstream regulator of PkbHLH2, which can control the expression of PkbHLH2 through an ABA-dependent signaling pathway.
KeywordsABA-dependent signaling pathway ABRE motif bHLH bZIP Populus koreana
Basic helix-loop-helix (bHLH) transcription factors (TFs) are the second largest class of plant TFs, and are involved in many developmental and physiological processes in plants (Bai et al. 2011). For instance, there are 133 bHLH genes in Arabidopsis, constituting one of the largest TF families (Heim et al. 2003). The bHLH proteins contain several highly conserved domains that are structurally heterogeneous (Xu et al. 2011). The bHLH domain is characterized by the signature bHLH domain comprising approximately 60 amino acids (aa) with two functionally distinct regions. The basic region at the N-terminus contains 13–17 primarily basic aa and plays a role in binding to the hexanucleotide E-box DNA motif CANNTG. bHLH domains, whose basic region contains at least five basic aa, comprise a highly conserved HER motif (His5–Glu9–Arg13), which is predicted to bind DNA (Feller et al. 2011). The helix-loop-helix (HLH) region at the C-terminal end comprises two amphiphatic a-helices that are connected by a loop of variable length. The HLH motif was found to form homo- or heterodimers with other bHLH proteins (Heim et al. 2003; Toledo-Ortiz et al. 2003), which is necessary for DNA recognition and DNA-binding specificity. Many bHLH proteins are found to contain an acidic region that can facilitate transcriptional activation and/or dimerization (Feller et al. 2011), and this region is usually N-terminal to the bHLH domain (Chinnusamy et al. 2003; Feller et al. 2006). bHLH proteins also play roles in transcriptional activation or repression, and have either a very broad or very restricted expression pattern, which is affected by their dimerization properties (Feller et al. 2011).
Most of the plant bHLH genes determined so far were functionally characterized in Arabidopsis, and only a few bHLH genes have been characterized functionally in other plant species (Zhou et al. 2009). The functions of plant bHLH genes were found to encompass involvement in regulation of epidermal cell development, carpel, anther and fruit dehiscence, flavonoid biosynthesis, phytochrome signaling, hormone signaling and stress responses (Feller et al. 2011). For example, Bai et al. (2011) isolated two bHLH genes (NtAn1a and NtAn1b) from tobacco, and their study showed that NtAn1 and NtAn2 act in concert to mediate the anthocyanin pathway in tobacco flowers, and that NtAn2 can up-regulate the expression of NtAn1. Zhang et al. (2011a) identified a bHLH TF, CrMYC2, from Catharanthus roseus. The results suggested that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is regulated by a TF cascade consisting of the bHLH protein CrMYC2, which regulates the expression of ORCA. Two bHLH gene products from Arabidopsis, MYC3 and MYC4, can interact with JAZ-interacting TF that regulate JA responses, and are activators of JA-regulated programs, which act additively with MYC2 to mediate specifically different subsets of the JA-dependent transcriptional response. These results suggested that they were involved in the regulation of plant defense and development (Fernández-Calvo et al. 2011; Qi et al. 2011). SPT, a bHLH (AtbHLH024) from Arabidopsis, was identified as a positive regulator in the development of carpel and fruit, but it was also shown that it negatively controls seed germination, expansion of leaves, petals and cotyledons (Heisler et al. 2001; Penfield et al. 2005; Groszmann et al. 2010; Ichihashi et al. 2010). A bHLH gene product, ICE1 (AtbHLH116), can bind to several E-box sequences in vitro and has the ability for transcriptional activation, and it also plays a role in cold acclimatization responses and freezing tolerance (Chinnusamy et al. 2003).
Although many studies have been performed on bHLH, still little is known regarding the upstream regulator(s) of bHLH, and their spatial expression patterns. In the present study, we cloned a bHLH gene (PkbHLH2) from Populus koreana Rehd, and a PkbHLH2 promoter::GUS gene fusion was generated to investigate the expression of PkbHLH2. Yeast one-hybrid analysis was employed to investigate upstream regulators. Our results show that PkbHLH2 was expressed mainly in leaf stalks, leaf veins and roots. The expression of PkbHLH2 may be regulated by a bZIP gene product, and is up-regulated by ABA and osmotic stress, suggesting that it may be involved in the ABA signal transduction pathway.
Materials and Methods
Plantlets of P. koreana were grown in MS medium. For ABA treatments, these plantlets were treated with 100 μM ABA (supplied in MS medium) for 24 and 48 h, respectively. After treatment, a mixture of leaves and stems, and roots from at least three seedlings were harvested for real-time RT-PCR analyses.
Cloning of the ORF and Promoter of the PkbHLH2 Gene
For cloning of the bHLH gene from P. koreana Rehd, primers were designed according to the sequence of a bHLH (GenBank number: XP_002300555) from poplar as follows: forward primer: 5′-ATGGCTCTGAGCTTCTGTTC-3′; reverse primer: 5′- CTATCCAAGAAATTGCTCC -3′. Total RNA was isolated from P. koreana using a CTAB method (Chang et al. 1993) and was digested with DNaseI (RNase-free) to remove any DNA contamination. Total RNA (2 μg) was reverse transcribed into cDNA using an oligodeoxythymidine primer in a reaction volume of 10 μL, which was then adjusted diluted to 100 μL for use as a PCR template. Primers for amplification of the bHLH promoter (see below) of bHLH were designed according to the promoter sequence of bHLH from poplars: forward primer 5′- ATTCCTGCGTAATGCGTACC-3′, reverse primer 5′- GGAGGATAGAATCTAGAAAA -3′. DNA isolated from P. koreana using a CTAB method (Doyle and Doyle 1987) was used as PCR template for amplifying the bHLH promoter. Multiple sequence alignments of bHLH from poplars and other plant species were performed using CLUSTALX1.81. The promoter sequence was analyzed using the program plantcare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Spatial Expression Analysis of the PkbHLH2 Gene
The primers for amplification of the promoter of the bHLH were as follows: P1: 5′- CCCAAGCTTATTCCTGCGTAATGCGTACCAT -3′ (HindIII site underlined), P2: 5′- CGCGGATCCGGAGGATAGAATCTAGAAAAG -3′ (BamHI site underlined). The PCR products were digested with HindIII and BamHI, and ligated into the pBI121 vector that was also digested with HindIII and BamHI. The construct was transformed into Agrobacterium EHA105. The transformation of Tobacco was performed using the Agrobacterium-mediated method. For histochemical staining, the transformed plants were immersed into GUS staining solution [10 mM Na2EDTA, 50 mM phosphate buffer (pH 7.0), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1 % Triton X-100 and 0.6 mg/mL X-Gluc] and incubated overnight at 37 °C. Stained plants were soaked in 70 % ethanol to remove chlorophyll.
Yeast One-Hybrid Analysis of the Upstream Regulator of the PkbHLH2 Gene
Two cis-elements of ABRE with the sequence of “ACGTG” were found in the promoter of PkbHLH2, located at −774 and −1,042 bp of the start codon, respectively. For yeast one-hybrid analysis, the promoter sequence contained three tandem copies of the ABRE cis-element cloned into a pHIS2 vector (Clontech , Palo Alto, CA) using the forward and reverse primers 5′-AATTCGCGACGTGGTTGCGACGTGGTTGCGACGTGGTTGAGCT-3 and 5′- CAACCACGTCGCAACCACGTCGCAACCACGTCGCG-3′. A cDNA library was constructed from P. koreana using a Make Your Own “Mate & Plate” Library System (Clontech, Mountain View, CA) according to the user manual. The one-hybrid analysis was performed following the protocol of BD Matchmaker™ Library Construction & Screening Kits User Manual.
A bZIP (PkbZIP2, GenBank number: JN651905) was obtained by yeast one-hybrid analysis. To study the specificity of the interaction between PkbZIP2 and the ABRE cis-element, the core motif of “ACGT” was mutated to “ATTT”, and the interaction between PkbZIP2 and these motifs was performed using yeast one-hybrid analysis.
To further study whether PkbZIP2 is able to regulate expression of PkbHLH2, an 800 bp promoter fragment of bHLH (forward primer: TAATACGTGAGATCGTCCCTAT, reverse primer: CTCGGAGCTCATGTCCCATTTTGAGCAAG, Sac I underlined) was cloned into a pHIS2 vector, and kbZIP2 was cloned into a pGADT7 vector for use in one-hybrid analysis.
Real Time PCR Analysis of PkbZIP2 and PkbHLH2 in Response to ABA Treatment
Primer sequences used for real time RT-PCR
Forward and reverse primer sequences
Cloning of the PkbHLH2 Gene
Cloning the Promoter of PkbHLH2 and Spatial Expression Analysis of PkbHLH2
Identification of the Upstream Regulator of PkbHLH2
To further confirm that PkbZIP2 can regulate the expression of the PkbHLH2 gene, an 800-bp stretch of the promoter region of PkbHLH2 was cloned into a pHIS2 vector. Interaction with PkbZIP2 (inserted into pGADT7) was examined using a one-hybrid assay, and the empty pGADT7 was used as control. The results showed that the PkbZIP2 gene product interacted with the promoter of the PkbHLH2 gene (Fig. 3b), whereas no binding activity was observed using the empty pGADT7, confirming that the interaction between PkbZIP2 and the promoter of PkbHLH2 was specific. These results suggested that PkbZIP2 was an upstream regulator of the PkbHLH2 gene.
Specificity of the Interaction Between PkbZIP2 and the ABRE Motif
To study whether PkbZIP2 binds specifically to the ABRE motif, the core motif of ABRE “ACGTG” was mutated to “ATTTG”, which was inserted into a pHIS2 vector in three tandem copies. Interaction assays between PkbZIP2 and the ABRE motif or its mutated form were performed at different 3-AT concentrations using a yeast one-hybrid assay. PkbZIP2 interacted with the core motif “ACGT”, but not with its mutant form “ATTT” (Fig. 3c), which indicated that the interaction between PkbZIP2 and “ACGT” was specific.
Expression of PkbHLH2 and PkbZIP2 in Response to ABA and Osmotic Treatments
PkbHLH2 has a Conserved HLH Domain and is Expressed in Specific Tissues
In the present study, we cloned a PkbHLH2 gene from P. koreana. Sequence alignments of seven bHLHs from different plant species showed that all of them share a highly conserved HLH domain (Fig. 1a). The HLH domain serves as a TF found in specific DNA-binding proteins. Therefore, the presence of a conserved HLH domain in PkbHLH2 suggested that it may also serve as a TF and that it can interact with other TFs. Other cis-elements, such as ABRE, MRE, MBS, exist in the PkbHLH promoter (Fig. 1b), suggesting that the expression of PkbHLH2 may be involved in stress responses. To examine the spatial expression of PkbHLH2, the GUS reporter gene was fused to the promoter of PkbHLH2 and Arabidopsis plants were transformed. GUS staining revealed that PkbHLH2 was expressed mainly in leaf stalks, leaf veins and main roots (Fig. 2), suggesting that it might perform specialized functions in these tissues.
Both PkbZIP and PkbHLH Belong to the ABA-Dependent Signaling Pathway
ABA is an important plant hormone that affects many aspects of plant growth and developmental processes, and plays a crucial role in plant responses to stress (Zhang et al. 2006). There are two stress signaling pathways in plants, the ABA-dependent and ABA-independent signaling pathways. Correspondingly, there are two major cis-acting elements included in stress inducible genes, ABRE and DRE/CRT. ABRE is recognized by ABA-dependent regulatory mechanisms, and the DRE/CRT element is recognized by ABA-independent regulatory mechanisms (Liao et al. 2008).
The results of the present study revealed two ABRE motifs in the promoter region of PkbHLH2. This suggested that PkbHLH2 may belong to an ABA-dependent signaling pathway. bZIP transcription factors, which are present in all eukaryotes, contain a basic region that binds DNA, and a leucine zipper dimerization motif (Tang et al. 2012). Some bZIP proteins have been found to specifically interact with the ABRE motif (Busk and Pages 1998; Rock 2000; Kim et al. 2004). The promoters of all ABA-responsive genes contain an ABA-responsive element, ABRE, that binds specifically to the bZIP family of TFs. This binding can result in the up- or down-regulation of expression of ABA-induced genes (Zhang et al. 2006). Nieva et al. (2005) cloned two maize bZIP genes that were found to mediate the expression of the ABA-inducible gene, rab28. The activity of rab28 was modulated by ABA, suggesting that bZIP genes mediate gene expression through the ABA-dependent signaling pathway.
However, there are no reports that bZIP may mediate stress responses via regulation of expression of other TFs. In the present study, the results obtained using a yeast one-hybrid system indicated that PkbZIP2 did interact with the ABRE motifs that were present in the promoter region of PkbHLH2 (Fig. 3a). In addition, PkbZIP2 interacted with the promoter of PkbHLH2 (Fig. 3b), confirming that it was able to regulate the expression of PkbHLH2.
To study the specificity of the interaction between ABRE and PkbZIP2, we mutated ABRE core motif “ACGTG” to “ATTTG”. The results revealed that when “CG” was changed to “TT”, the ability of PkbZIP2 to interact with the mutant was totally abolished (Fig. 3c). This result highlights the importance of the “CG” in the core motif of “ACGTG” for bZIP recognition.
In conclusion, our studies showed that both PkbZIP2 and PkbHLH2 are involved in the ABA-dependent signal transduction pathway as both can be up-regulated by ABA and osmotic stress. In the ABA dependent signal transduction system, PkbZIP2 was the upstream regulator of PkbHLH2, which perceives altered ABA level, is up-regulated by enhanced ABA levels, and binds to ABREs to induce expression of PkbHLH2 and other target genes.
This work was supported by National Natural Science Foundation of China (31000312).
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
- Fernández-Calvo P, Chini A, Fernández-Barbero G, Chico JM, Gimenez-Ibanez S, Geerinck J, Eeckhout D, Schweizer F et al (2011) The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses. Plant Cell 23:701–715PubMedCrossRefGoogle Scholar
- Qi T, Song S, Ren Q, Wu D, Huang H, Chen Y, Fan M, Peng W et al (2011) The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell 23:1795–1814PubMedCrossRefGoogle Scholar
- Zhang H, Hedhili S, Montiel G, Zhang Y, Chatel G, Pré M, Gantet P, Memelink J (2011a) The basic helix–loop–helix transcription factor CrMYC2 controls the jasmonate–responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus. Plant J 67:61–71PubMedCrossRefGoogle Scholar