Plant Cell Rep DOI 10.1007/s00299-015-1809-6

ORIGINAL PAPER

Molecular characterization of HbCZF1, a Hevea brasiliensis CCCH-type zinc finger protein that regulates hmg1 Dong Guo1 • Hong-Yan Yi1,2 • Hui-Liang Li1 • Chen Liu1,2 • Zi-Ping Yang1,2 Shi-Qing Peng1



Received: 12 March 2015 / Revised: 9 May 2015 / Accepted: 12 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Key message The HbCZF1 protein binds to the hmg1 promoter in yeast and this interaction was confirmed in vitro. The hmg1 promoter was activated in transgenic plants by HbCZF1. Abstract Biosynthesis of natural rubber is known to be based on the mevalonate pathway in Hevea brasiliensis. The final step in the mevalonate production is catalyzed by the branch point enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), which shunts HMG-CoA into the isoprenoid pathway, leading to the synthesis of natural rubber. However, molecular regulation of HMGR expression is not known. To study the transcriptional regulation of HMGR, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the hmg1 (one of the three HMGR in H. brasiliensis) promoter as bait. One cDNA that encodes the CCCH-type zinc finger protein, designated as HbCZF1, was isolated from H. brasiliensis. HbCZF1 interacted with the hmg1 promoter in yeast one-hybrid system and in vitro. HbCZF1 contains a 1110 bp open reading frame that encodes 369 amino acids. The deduced HbCZF1 protein was predicted to possess a

Communicated by S. Schillberg. D. Guo and H.-Y. Yi contributed equally to this work. & Shi-Qing Peng [email protected] 1

2

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China College of Agriculture, Hainan University, Haikou 570228, China

typical C-X7-C-X5-C3-H CCCH motif and RNA recognition motif. HbCZF1 was predominant in the latex, but little expression was detected in the leaves, barks, and roots. Furthermore, in transgenic tobacco plants, over-expression of HbCZF1 highly activated the hmg1 promoter. These results suggested that HbCZF1 may participate in the regulation of natural rubber biosynthesis in H. brasiliensis. Keywords 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase  CCCH-type zinc finger protein  Hevea brasiliensis  Promoter  Natural rubber biosynthesis Abbreviations AbA Aureobasidin A ANOVA Analysis of variance GUS b-Glucuronidase ET Ethrel GFP Green fluorescent protein HMGR 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase EMSA Electrophoretic mobility shift assay GST Glutathione S-transferase MeJA Methyl jasmonate MVA Mevalonate NR Natural rubber ORF Open reading frame qPCR Quantitative polymerase chain reaction RT-PCR Reverse transcription

Introduction Rubber tree (Hevea brasiliensis) is cultivated for the commercial production of natural rubber (NR) due to its good yield of rubber and the excellent physical properties

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of the rubber products (Asawatreratanakul et al. 2003). The economic importance of NR has led to intensive investigations on NR biosynthesis. NR is synthesized from a universal precursor, isopentenyl diphosphate, which can be synthesized through the mevalonate (MVA) pathway in the cytosol (Archer and Audley 1987; Bick and Lange 2003). The 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyzes the conversion of HMG-CoA to MVA, which is a central step in the cytosolic pathway for the biosynthesis of a number of natural products in plants (Bach and Lichtenthaler 1982). HMGR is a rate-limiting enzyme of the MVA pathway in plants (Bach 1986). Plant HMGR is encoded by a multi-gene family, where the different isoforms exhibit spatial and temporal gene expression patterns (Lumbreras et al. 1995; Suzuki et al. 2004, 2009). In rubber tree HMGR is encoded by a small gene family comprising three members (named hmg1, hmg2, and hmg3) (Chye et al. 1991, 1992). hmg1 is expressed predominantly in the laticifers and is likely to encode the enzyme involved in rubber biosynthesis(Chye et al. 1992). The expression of hmg2 and hmg3 is not cell-type specific. hmg2 and hmg3 are possibly involved in isoprenoid biosynthesis of a housekeeping nature (Chye et al. 1992). In Parthenium argentatum, HMGR1 is specifically involved in rubber biosynthesis (Ji et al. 1993). Although a few reports reveal HMGR plays a major role in NR biosynthesis (Chye et al. 1992; Ji et al. 1993; Suwanmanee et al. 2013), little is known about the regulatory mechanism of HMGR at molecular and biochemical levels in the NR producing plant. In this study, we describe a CCCH-type zinc finger protein gene from rubber tree, named HbCZF1, which was identified in a yeast one-hybrid screening using the promoter region of hmg1 as bait and cDNA expression libraries prepared from latex as prey. The HbCZF1 protein binds specifically to the bait sequence in yeast and this interaction was confirmed in vitro. Co-expression experiments demonstrated that HbCZF1 can up-regulate a phmg1-GUS (b-glucuronidase) reporter, indicating that HbCZF1 may be a positive transcription regulator of hmg1 involved in rubber tree NR biosynthesis.

Materials and methods Plant materials Seven-year-old virgin trees of rubber tree cultivar CATAS7-33-97 and epicormic shoots of the same clone was grown at the Experimental Farm of the Chinese Academy of Tropical Agricultural Sciences on Hainan Island of People’s Republic of China. The shoots were treated by 0.5 % ethrel or 0.1 % methyl jasmonate,

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respectively, according to the Chen’s method (Chen et al. 2012). The latex samples from three of epicormic shoots for each interval were collected by cutting at 2 h, 6 h, 1, 3, and 5 days after treatments. As control, latex samples were collected from three of epicormic shoots treated with water, the solvent of ethephon and from three of epicormic shoots treated with 7 % ethanol, the solvent of methyl jasmonate at the corresponding intervals. Latex samples were collected at 1, 3, 6, 9, 24, and 48 h after treatments from 12 shoots for each interval, and stored at -80 °C for RNA extraction. Rubber tree flowers, leaves, and barks were collected and washed with double-distilled H2O, then immediately frozen into liquid nitrogen. Yeast one-hybrid The hmg1 promoter (approximately 1.0-kb) was amplified by PCR with forward primer 50 -GCAAGCTTGATC ACGTGAGAAAGAATTTG-30 (with the HindIII site) and reverse primer 50 -GACTCGAGGTAAAAATATGCCG GCGCAGGA-30 (with the XhoI site) (Zhang et al. 2006) and then cloned into HindIII and XhoI restriction sites of the yeast one-hybrid bait vector pAbAi (Clontech), generating pAbAi-phmg1. Latex RNA was extracted according to Tang’s method (Tang et al. 2007). The mRNA was purified using the NucleoTrapÒ mRNA Mini Kit (Macherey Nage) according to the instructions of the manufacturer. The first-strand cDNA was reverse-synthesized using the RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas) according to the instructions of the manufacturer. The double-stranded cDNA was amplified by long-distance PCR and size selected ([200 bp) using Chroma SpinTM TE-400 columns (Clontech). The latex cDNA was introduced into the yeast strain Y1HGold (Clontech), along with the pAbAi-phmg1 vector (bait vector) and the SmaI-linearized pGADT7-Rec vector (prey vector). The yeast cells were then cultivated on SD/-Leu medium supplemented with 500 ng/ml Aureobasidin A (AbA) for the selection of transformants at 30 °C for 3 days. To confirm the hmg1 promoter-binding proteins, HbCZF1 was cloned into pGADT7-Rec. The pGADT7HbCZF1 and phmg1-AbAi were co-transformed into the yeast strain Y1HGold, as previously described. pGADT7Rec53?p53-AbAi, pAbAi-hmg1, pGADT7-HbCZF1, and pGADT7-HbCZF1 ?pAbAi were used as controls. Transformed clones were grown on SD/-Leu selective medium containing 500 ng/ml AbA for 3 days at 30 °C. Bioinformatic analyses of HbCZF1 The nucleotide sequence and amino acid sequence of HbCZF1 were analyzed using the BLAST program (http://

Plant Cell Rep

www.ncbi.nlm.nih.gov). Amino acid comparison was performed using DNAMAN software.

stained with SYPRO Ruby EMSA stain for monitoring protein, and photographed.

Heterologous expression and purification of HbCZF1

Nuclear localization analysis

The open reading frame (ORF) of HbCZF1 was amplified with forward primer 50 -ACGGATCCATGAATCCATT GAC GCTGGT-30 (with the BamHI site underlined) and reverse primer 50 -GACTCGAGTCATCTCTC TGATTTG CGCC-30 (with the XhoI site underlined), and then fused to the BamHI/XhoI sites in the pET-28a (?) vector (Novagen, USA). After sequence confirmation, the pET-HbCZF1 plasmid was transformed into Escherichia coli strain Rosetta (DE3) competent cells for protein expression. E. coli cells containing pET-HbCZF1 were cultured in LB medium supplied with 100 mg/l Kan at 37 °C. When the OD 600 of the cultures reached 0.5, 1 mM isopropyl-b-Dthiogalactopyranoside (IPTG) was added and the cultures were incubated for another 4 h at 37 °C. The cells were harvested by centrifugation and resuspended in 10 ml of 0.05 M sodium phosphate buffer (pH 7.5) containing 20 mM imidazole, and sonified on ice for 5 min at 50 % pulses using a Ultrasonic Crasher (Ningbo Scientz Biotechnology Co. Ltd., China). The homogenate was centrifuged for 10 min at 10,0009g and 4 °C. Since most of the expressed HbCZF1 proteins existed in the inclusion body, the pellet was resolved in 8 mol/l urea. Then the proteins were isolated as denatured forms through a prepacked 5 ml Hi-trap Ni column (Pharmacia) following the manufacturer’s protocol. For refolding, the purified proteins were dialyzed in turn against 50 mmol/l phosphatebuffered saline buffers containing 6, 4, 3, 2, 1, and 0 mol/l urea (pH 7.4) at 4 °C for at least 4 h for each urea concentration. After removing the precipitates by centrifugation, the dialyzed protein solution was lyophilized, and stored at -80 °C before use. Electrophoretic mobility shift assay (EMSA) The EMSA was performed with the Electrophoretic Mobility Shift Assay kit (Invitrogen, USA) following the manufacturer’s instruction. The DNA–protein binding reaction was performed by incubating double-stranded oligonucleotides with purified protein at room temperature for 30 min in a total volume of 15 ll. The reaction system contained 20 mmol Tris–HCl (pH 7.6), 30 mmol KCl, 0.2 % (w/v) Tween-20, 1 mmol DTT, and 10 mmol (NH4)2SO4. The binding mixture was resolved on a 6 % non-denaturing polyacrylamide gel in 0.5 9 TBE buffer. The gel was first stained with SYBR Green EMSA stain for visualizing DNA, and photographed; the same gel was

The full-length coding sequence of HbCZF1 was amplified with forward primer 50 -GTCCATGGTAATGAATCCATT GACGCTGGT-30 and reverse primer 50 -GCACTAGTT CTCTC TGATTTGCGCCTAT-30 , which contained NcoI and BglII sites, respectively. The PCR products were introduced into the pCAMBIA1302 vector to generate CaMV35S::HbCZF1-GFP afterward, the plasmids of 35S::HbCZF1-GFP and pCAMBIA1302 were introduced into onion epidermal using a PDS-1000/He gene gun (BioRad, USA) at 1100 psi. The transformed onion epidermal was cultured on MS medium in darkness at 25 °C for 24 h and then observed using a laser scanning confocal microscopy (Leica, TCS SP2, Wetzlar, Germany). Isolation of total RNA and quantitative polymerase chain reaction (qPCR) Total RNA was extracted according to Tang’s method (Tang et al. 2007). First-strand cDNA was synthesized using the RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas, Lithuania). qPCR was conducted using the following primers: CZF1 (50 -CACAGCTGTATGTACTTA CCTATC-30 ) and CZF2 (50 -CACAGCTGTATGTACT TACCTATC-30 ) for HbCZF1, and HMG1 (50 -TTTGCCT TTGT TGCCCACTGAG-30 ) and HMG2 (50 -TGCACCA ACAATGGCAATTGGCC-30 ) for hmg1. A housekeeping gene eIF1Aa was used as standard control; it was amplified by primers eIF1Aa1 (50 -GCGTGACTATCAGGACGAC AA-30 ) and eIF1Aa2 (50 -CAAGACCTCCAGCAATA CCCT-30 ). Real-time RT-PCR was performed using the fluorescent dye SYBR Green (Takara, China), and the melt curve analysis of amplification products was conducted using the Stratagene Mx3005P Real-Time Thermal Cycler (Agilent, USA). The Real-time RT-PCR conditions are as follows: 30 s at 95 °C for denaturation, 40 cycles for 5 s at 94 °C, 20 s at 58 °C, and 20 s at 72 °C for amplification. An average of three independent biological replicates of each time was performed. Plant transformation The hmg1 promoter was inserted into the pCAMBIA1381Z vector using the restriction sites BamHI and HindIII to generate phmg1::GUS. The phmg1::GUS was transformed into the Agrobacterium tumefaciens strain GV3103, which was then transformed into tobacco by a leaf disc method. Plants transformed with phmg1::GUS were selected by

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hygromycin resistance and histochemical staining using X-Gluc as substrate. Plants transformed with phmg1::GUS were named SPs. For the effector construct, the ORF of HbCZF1 was ligated into the pBI121 vector with the restriction sites XbaI and SacI, replacing the b-glucuronidase (GUS) gene. The effector construct 35S:HbCZF1 was directly transformed into the transgenic SPs. Double transformants (DTs, plants with phmg1::GUS and CaMV35S::HbCZF1) were selected by hygromycin and kanamycin resistance, histochemical staining using X-Gluc as substrate, which were tested by reverse transcription (RT-PCR) with transformant cDNA as template. RT-PCR for the analysis of HbCZF1 expression was performed using total RNA from double transformants, and amplified with HbCZF1 specific primers P1 (50 -ATGAATCCATTG ACGCTGGT-30 ) and P2 (50 -TCATCTCTCTGATT TGCGCC-30 ). The NtACT was used as an internal control parallel in the reactions, amplified with NtACT specific primers AF (50 -CAGTGGCCGTACAACAGGTAT-30 ) and AR (50 -ATCCTCCAATCCAGACACTGT-30 ). PCR reaction was carried out in 22 cycles of programmed temperature control for 30 s at 95 °C, 30 s at 55 °C, and 45 s at 72 °C with a 5 min preheat at 95 °C and a 10 min final extension at 72 °C. The PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining.

Positive colonies that could grow again after this initial selection step were further analyzed either by colony PCR or plasmid rescue followed by sequence analysis. Finally, 38 PCR products and 31 plasmids were obtained, sequenced, and BLAST searched against GenBank (http:// www.ncbi.nlm.nih.gov). One cDNA encoding the CCCH zinc finger protein, designated as HbCZF1 (GenBank Accession No. KM230617), was isolated. In order to further check the binding specificity of HbCZF1, the one-to-one interaction analysis showed that only the yeast clone harboring HbCZF1 and pAbAi-p hmg1 or positive control could grow on SD/-Leu selective medium containing 500 ng/ml AbA (Fig. 1). The results show that HbCZF1 is able to interact specifically with the hmg1 promoter. Molecular characterization of HbCZF1 The sequence analysis showed that HbCZF1 possessed 1351 nucleotides and contained an open reading frame encoding a deduced protein of 369 amino acids with a predicted molecular mass of 43.73 kDa and an isoelectric point of 8.69. The deduced HbCZF1 protein contained a typical CCCH motif C-X7-C-X5-C3-H and ist3 RNA combined domain (Fig. 2). The amino acid sequence of HbCZF1 was aligned with those of the other plant CCCH zinc finger proteins that exhibit high sequence identities (i.e., 65, 60, 51, and 51 %) to other CCCH zinc finger proteins from Morus notabilis

GUS activity assay A fluorimetric assay was used to determine GUS activities, with 4-umbelliferyl-D-glucuronide as substrate, using a Glomax multi detection system (Promega, USA). Protein content was determined, with BSA as standard, using the Bradford protein assay. Three duplicates of each transformant line were separately assayed for GUS activity. Quantitative analysis was performed using the average of the duplicate measurements per sample, and the standard deviation was calculated. Data were subjected to analysis of variance (ANOVA).

Results Identification of a CCCH zinc finger protein interacting with the hmg1 promoter In order to further understand the transcriptional regulatory mechanism of hmg1, the yeast one-hybrid experiment was performed to screen novel transcription factors from the rubber tree latex cDNA library using the hmg1 promoter as bait. Screening of 112 transformants of latex cDNA library resulted in 60 positive colonies after re-streaking the primary positive colonies on the same selective medium.

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Fig. 1 Activation of hmg1 promoter in yeast by HbCZF1. Yeast cells carrying pGADT7-HbCZF1?phmg1-AbAi, pGADT7-Rec53?p53AbAi, phmg1-AbAi, pGADT7-HbCZF1, and pGADT7-HbCZF1 ?pAbAi were grown in SD/-Leu selective medium containing 500 ng/ml AbA for 3 d at 30 °C

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(EXC01448.1), Cucumis sativus (XP_004147921.1), Fragaria vesca subsp. vesca (XP_004294254.1), and Theobroma cacao (XP_007016121.1).

increase at 3 h, reached the highest level at 6 h, and then remarkably decreased at 12–24 h. Sub-cellular localization of HbCZF1

Expressions analysis of HbCZF1 and hmg1 The HbCZF1 and hmg1 transcript levels in different rubber tree tissues were examined using Real-time RT-PCR. Results showed that HbCZF1 and hmg1 were predominantly expressed in the latex, but little expression was detected in the leaves, barks, and roots (Fig. 3).

To examine the sub-cellular localization of HbCZF1 in plants, a green fluorescent protein (GFP) reporter gene was fused in-frame to the N-terminus of HbCZF1 and

Expression analysis of HbCZF1 and hmg1 in response to MeJA and ET Ethrel (ET) is regularly applied on the trunk of rubber trees to stimulate latex yield. Methyl jasmonate (MeJA) is also a key factor related to the production of rubber trees. To examine the expression patterns of the HbCZF1 and hmg1 genes, the rubber tree shoots were treated by MeJA or ET, respectively. qRT-PCR results indicated that the expression of HbCZF1 was induced by MeJA, but not by ET (Fig. 4a). The expression of hmg1 was induced by MeJA and ET in different patterns (Fig. 4b). After MeJA treatments, the expression of hmg1 began to increase at 1 h, reached the highest level at 12 h, and then remarkably decreased at 24 h. After ET treatments, the expression of hmg1 began to

Fig. 3 Transcription patterns of HbCZF1 and hmg1. Real-time RTPCR was performed on RNA derived from various tissues of the rubber tree. eIF1Aa was amplified for normalization. An average of three independent biological replicates of each time was performed. Data are presented as mean ± SE (n = 3). OR roots, LE leaves, BA bark, LA latex

Fig. 2 Alignment of HbCZF1 and other plant CCCH proteins. The amino acid sequence of HbCZF1 was aligned with Morus notabilis (EXC01448.1), Cucumis sativus (XP_004147921.1), Fragaria vesca

subsp. vesca (XP_004294254.1), and Theobroma cacao (XP_007016121.1). Black green and yellow shadings indicate conserved amino acid residues (color figure online)

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transiently expressed into onion epidermal cells under the control of the 35S promoter. As shown in Fig. 5, the GFP::HbCZF1 green fluorescent signal was detected in the nucleus of onion epidermal cells, whereas GFP alone was present throughout the cell. Taken together, these results indicate that HbCZF1 is a nuclear-localized protein. HbCZF1 can interact with the hmg1 promoter in vitro We first purified the recombinant HbCZF1 protein from E. coli (Fig. 6a). The DNA fragment (approximately 1.0kb) from the hmg1 promoter, and the complementary

Fig. 4 Expression patterns of HbCZF1 and hmg1 respond to JA (a) and ET (b) treatment. Relative transcript abundances of HbCZF1 and hmg1 were examined by qPCR. The y axis is the scale of the relative transcript abundance level. The x axis is the time course of JA and ET treatment. eIF1Aa was amplified as an internal control. An average of three independent biological replicates of each time was performed. Data are presented as mean ± SE (n = 3). The significant difference was assessed by ANOVA (two asterisks corresponding to P \ 0.01)

oligonucleotides could anneal to form double-stranded structures. EMSA was used to determine the binding affinity of HbCZF1to the hmg1 promoter. As shown in Fig. 6b, the recombinant HbCZF1 bound to probe the hmg1 promoter and resulted in mobility shifts, the numbers of shifted protein-DNA complexes showed a tendency to increase gradually, and free DNA levels decreased accordingly (Fig. 6b, lanes 2–7). Thus, HbCZF1 was able to recognize and interact with the hmg1 promoter in vitro. Activation of the hmg1 promoter in transgenic plants by HbCZF1 To provide evidence that HbCZF1 protein regulates hmg1 transcription, we used double transgenic plants of tobacco to assay their interactions. An approximately 1.0-kb promoter of hmg1 fused with the GUS reporter gene (phmg1::GUS) and the 35S::HbCZF1 recombinant gene were transferred into tobacco plants. Plants transformed with phmg1::GUS (SP1 and SP2), 35S::HbCZF1 plants (SH1 and SH2), and double transgenic plants with phmg1::GUS and CaMV35S::HbCZF1 did not show clear phenotypic changes. The HbCZF1 transcripts were clearly detectable in double transgenic plants (Fig. 7a). In the transgenic plants of phmg1::GUS, the GUS activity in leaves was very low and the stain was weak, whereas GUS activity in leaves was too weak to be detected in 35S::HbCZF1 plants (SH1 and SH2). However, in double transgenic plants (i.e., DT4, 7, 8, 11, 14, 15, 19, and 24), the GUS activity in leaves of double transgenic plants was high (Fig. 7b), and increased by more than 2.5fold compared to the transgenic plants of phmg1::GUS (Fig. 7c). Moreover, there was a good correlation between the transcript levels of HbCZF1 and GUS in leaves of double transgenic plants. Clearly, in transgenic tobacco plants, constitutive expression of HbCZF1 strongly activated the hmg1 promoter.

Discussion CCCH motif-containing proteins comprise a large protein family, and are widely distributed across eukaryotes. The typical CCCH protein usually contains 1–6 CCCH-type

Fig. 5 Nuclear localization of HbCZF1. The upper, the corresponding bright field, fluorescence, merged fluorescence image, and DAPI image of GFP control; the lower panel, the corresponding bright field, fluorescence, merged fluorescence image, and DAPI image of HbCZF1-GFP

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Fig. 6 HbCZF1 binding to the promoter of hmg1 as analyzed by EMSA. a Overexpression of HbCZF1 in E. coli. Expression vector (pET-HbCZF1) was constructed in which the fusion protein was driven by the T7 promoter, made IPTG-inducible, and transformed into E. coli BL21 (DE3). Expression was induced by the addition of 0.1 mM IPTG, and total cell proteins were analyzed after 4 h by SDSPAGE. M molecular markers, 1 E. coli cells harboring pET-HbCZF1 not induced, 2 E. coli cells harboring pET-HbCZF1 after 4 h of

induction, 3 purified HbCZF1 fusion protein. b The hmg1 promoter with HbCZF1 protein stained with SYBR green EMA for visualizing DNA. c The same gel as in B stained with SYPRO Ruby EMSA for visualizing protein. M DNA maker (DL2000). Lane 1 the promoter of hmg1 DNA only. Lanes 2–7 the promoter of hmg1 DNA with increasing amounts of HbCZF1 protein (46, 69, 92, 115,138, and 162 lg). Lane 8 162 lg of HbCZF1, respectively. The arrows indicated the HbCZF1-DNA complex or free DNA

Fig. 7 Activation of d hmg1 promoter in transgenic plants by HbCZF1. a The double transgenic tobacco plants were examined by RT-PCR. b Histochemical staining analysis of T1 double transgenic tobacco (i.e., DT4, 7, 8, 11, 14, 15, 19, and 24), T1 transgenic plants of pHmg1::GUS (SP1 and SP2) and T1 transgenic plants of CaMV35S::HbCZF1 (SH1 and SH2) and wild type (WT) were used

as controls. c Quantitative determination of the GUS activities in the different T1 transgenic tobacco lines (i.e., DT4, 7, 8, 11, 14, 15, 19, and 24). WT, SP (T1), and SH (T1) were used as controls. Three replicates were included for each sample. Data are presented as mean ± SE (n = 3), and the significant difference was assessed by ANOVA (two asterisks corresponding to P \ 0.01)

zinc finger motifs. Most of the characterized CCCH-type zinc finger proteins are associated with RNA metabolism, including RNA cleavage, RNA degradation, RNA

polyadenylation, or RNA export, by binding to RNA (Bai and Tolias 1996; Gao et al. 2002; Lai et al. 2000; Hurt et al. 2009). Most animal CCCH proteins investigated were

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shown to play important roles in post-transcriptional regulation of mRNAs by binding to the AU-rich element in the 30 -UTR (Blackshear 2002; Guo et al. 2004; Kelly et al. 2007; Stumpo et al. 2009). Compared to the largely wellcharacterized CCCHs in animals, only a small number of CCCH proteins have been functionally characterized in plants. These CCCH proteins have been implicated to participate in a wide range of plant developmental and abiotic stresses and defense responses (Lin et al. 2011; Peng et al. 2012; Sun et al. 2007), including seed germination (Kim et al. 2008), embryo development (Li and Thomas 1998; Grabowska et al. 2009), plant architecture determination (Wang et al. 2008), and leaf senescence (Kong et al. 2006; Jan et al. 2013). Several plant CCCH proteins were also identified as RNA-binding proteins (Li et al. 2001; Cheng et al. 2003; Addepalli and Hunt 2007, 2008; Pomeranz et al. 2009, 2011). Compared to the well described protein-RNA interactions of CCCH proteins, knowledge on protein-DNA interactions of the CCCH zinc finger proteins is still very poorly understood. PEI1 is a CCCH gene from Arabidopsis and is able to bind to specific DNA sequences (Li and Thomas 1998). AtTZF1 can bind to both DNA and RNA in vitro (Pomeranz et al. 2009) and seems to have effects on plant growth, development, and abiotic stress responses which may be related to GA and ABA metabolism (Lin et al. 2011; Pomeranz et al. 2011). In rice, OsLIC (Wang et al. 2008) and C3H12 (Deng et al. 2011) displayed binding activity to both double-stranded and single-stranded DNA. It was the first report that OsGZF1 (a CCCH zinc finger from rice) has a function in regulating the GluB1 promoter and controls accumulation of glutelins during grain development (Chen et al. 2014). In this study, HbCZF1 interacts with the hmg1 promoter in the yeast and in vitro, indicating HbCZF1 can bind to DNA. Moreover, HbCZF1 increased the hmg1 promoter in transgenic tobacco. The results strongly indicate that hmg1 is a target gene of HbCZF1, and that HbCZF1 is a transcriptional activator of the hmg1. In rubber tree, the general metabolic pathway leading to NR biosynthesis is now clear (Sando et al. 2008). Some of the NR biosynthesis genes have been cloned and further characterized, notably the rubber elongation factors (Dennis and Light 1989; Priya et al. 2007) HMGR (Chye et al. 1991, 1992), small rubber particle protein (SRPP) (Oh et al. 1999), and cis-prenyltransferase (Asawatreratanakul et al. 2003). HbWRKY1 may be a negative transcription regulator of SRPP involved in NR biosynthesis in rubber tree (Wang et al. 2013). Li et al. also reported that WRKY proteins may be involved in the transcriptional regulation of nature rubber biosynthesis (Li et al. 2014). Moreover, rubber biosynthesis in laticifers may be mainly regulated by JA signaling (Tian et al. 2010; Zhao et al. 2011). The

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expression of HbCZF1 induced by MeJA indicated HbCZF1 might play a role in response to JA. To our best knowledge, this study is the first to demonstrate that CCCH zinc finger protein participates in the regulation of NR biosynthesis, which will provide greater insight into the regulation of NR biosynthesis in rubber tree. Additionally, HMGR is considered as a key enzyme for biotechnological purposes and can be utilized to increase terpenes content in plants. In plants, overexpression of HMGR in the isoprenoid pathway has been attempted to increase triterpenoid productivity (Chappell et al. 1995; Munoz-Bertomeu et al. 2007). As a result, up-regulation of HMGR could improve terpenes productivities in the transgenic plants (Schaller et al. 1995; Harker et al. 2003; Hey et al. 2006; Post et al. 2012). Isolation of more transcription factors regulating the NR biosynthesis pathway and further elucidation of regulatory machinery of HMGR will be a great help in manipulating NR metabolism. Author contribution statement SQP, DG, and HYY conceived and designed the experiments. DG and HYY performed the experiments. HYY, DG, and ZPY analyzed the data. HLL and CL contributed reagents/materials/analysis tools. SQP and DG wrote the paper. SQP revised the manuscript. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 31170285) and the Major Technology Project of Hainan (ZDZX2013023-1). Conflict of interest of interest.

The authors declare that they have no conflict

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Molecular characterization of HbCZF1, a Hevea brasiliensis CCCH-type zinc finger protein that regulates hmg1.

The HbCZF1 protein binds to the hmg1 promoter in yeast and this interaction was confirmed in vitro. The hmg1 promoter was activated in transgenic plan...
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