Botanical Studies (2011) 52: 249-256.
MOLECULAR BIOLOGY
Molecular characterization of isopentenyltransferase (BoAIPTI) from Bambusa oldhamii expressed in Escherichia coli
Chuan-Shan YEH, Lu-Sheng HSIEH, Chien-Chi YANG*, and Ping-Du LEE*
Department of Biochemical Science and Technology, College of Life Science, National Taiwan Uni-versity, 1 Rooseveit Road Section 4, Taipei 106, Taiiwan
(Received August 20, 2010; Accepted October 19, 2010)
ABSTRACT. Cytokinins (CKs) are a group of phytohormones involved in stages of plant development, such as cell division and various other growth processes. The key enzyme in de novo biosynthesis of naturally oc­curring cytokinins is adenylate isopentenyltransferase (AIPT). This study amplified a PCR product, containing partial sequence encoding putative AIPT, using primers based on conserved protein sequences. Genomic DNA containing a full-length of AIPT was obtained by screening a genomic DNA library. This study used 5'-RACE and 3'-RACE, using gene specific primers, to clone and sequence cDNA encoding BoAIPTI. The open read­ing frame of BoAIPTI was 1035 bp in size, encoding 344-amino acids, with a molecular mass of 37.7 kDa. No introns were found by comparing AIPT from bamboo with AIPT sequences from other species. BoAIPT1 contains a putative ATP/GTP binding motif at the N-terminal as observed in other known AIPT. Recombinant BoAIPT1 expressed in Escherichia coli catalyzed the isopentenyl transfer reaction from dimethylallyldiphos-phate (DMAPP) to the N6 adenylate amino group. Real-time PCR analyses revealed BoAIPT1 expression in­creased simultaneously with green bamboo shoot growth, with the top green shoot section having the highest expression level.
Keywords: Adenylate isopentenyltransferase (AIPT); Bambusa oldhamii; Molecular cloning; Gene expres­sion; Escherichia coli.
INTRODUCTION
meostasis in cells, including rate of de novo synthesis, rate of inter-conversion, rate of transport and rate of cytokinin conjugate breakdown (Sakakibara, 2006).
Two CK biosynthetic pathways have been described in plants, the adenylate pathway, and the tRNA pathway
(Chen, 1997; Mok and Mok, 2001). In the adenylate path­way, isoprenoid CK biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (AIPT; EC 2.5.1.27) using dimethylallyl diphosphate (DMAPP) and adenosine phosphate as substrates. AIPT transfers the isoprenyl group from dimethylallyldiphosphate (DMAPP) to the N6-amino group of AMP, ADP, or ATP to produce iP-nucleotides. In Arabidopsis, prenyl side chains of iP-nucleotides undergo hydroxylation by cytochrome P450 monooxygenases. The CK-activating enzyme, LOG (Lonely Guy, Kuroha et al, 2009), has phosphoribohydrolase activity that releases a ribose 5'-monophosphate moiety from iP-nucleotides to produce active iP (Chen, 1997; Sakakibara, 2006). In the second pathway, tRNA-IPT catalyzes CK formation by isoprenylation of certain tRNA at an adenine adjacent to the 3'-end of the anticodon. Subsequent degradation of prenylated tRNA generates the CK nucleotide (Vreman and Skoog, 1972; Murai et al., 1994). Presently, the first pathway is more commonly accepted.
Cytokinins (CKs) are a class of phytohormones that are involved in a wide variety of plant growth (Tanaka et al., 2006; Sato et al., 2009) and development processes, including cell division, delay of senescence (Gan and Amasino, 1995; Kim et al., 2006), apical dominance shoot meristem function (Higuchi et al., 2004; Kurakawa et ai., 2007), and nutritional signaling (Sakabkibara, 2006; Ar-
gueso et al., 2009).
Depending on the structure of the side chain, cytoki-nins are classified into two groups, isoprenoid side chains and aromatic side chains (Mok and Mok, 2001). Natural isoprenoid CKs are classified into one of four basic mol­ecules: isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ). Isoprenoid CKs are commonly found in higher plants, and are much more abundant than aromatic CKs. The metabolic pathway for isoprenoid CKs has been studied in Arabidopsis and in rice (Kakimoto, 2001; Takei et al., 2001; Takei et al., 2004; Sakamoto et al., 2006). Four factors regulate CK ho-
*Corresponding authors: E-mail: pingdu@ntu.edu.tw (Ping-Du LEE); E-mail: ccyang@ntu.edu.tw (Chien-Chi YANG).
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AIPTs in higher plants such as Arabidopsis thaliana, rice, mulberry (Morus alba) and hops (Humulus lupulus L.) have been cloned and characterized (Sakano et al., 2004; Abe et al., 2007). In Arabidopsis thaliana, there are seven AIPT genes (AtlPTl, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, and AtIPT8) (Takei et al., 2001). Us­ing transient expression, four AtIPTs (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) are localized in plastids (Kasahara et al., 2004). Farnesylation of AtIPT3 directs localization of the protein to the nucleus/cytoplasm (Galichet et al., 2001). Researchers have also identified AIPTs in some phytopathogenic microorganisms, such as Agrobacterium tumefaciens (Akiyoshi et al., 1984; Sakakibara et al., 2005). AIPT has been extensively studied in plants, and has recently been successfully applied to agriculture. Pre­vious studies reported on transformation of the AIPT gene into broccoli, which produced a significant delay in senes­cence and yellowing of harvested flower heads and leaves (Chen et al., 2001; Chan et al., 2009).
The monocotyledonous plant, bamboo, Bambusa old-hamii (Family Poaceae) is found mainly in the tropics and the subtropics. Bamboo is often noted for its fast growth. During the rapid growth of bamboo, AIPT may play an important role in CK biosynthesis. We report here on the cloning of cDNA encoding BoAIPT1 and analysis using real-time reverse transcription polymerase chain reaction (RT-PCR) to investigate BoAIPT1 expression levels at dif­ferent shoot growth stages to further understand properties of bamboo AIPT.
DIG-labeled probe (Roche, Germany) for screening the genomic DNA library. Plaques were blotted onto Hy-bond-N+ membranes (Millipore, USA). Hybridization and detection were performed per manufacturer's instructions (Roche, Germany). Positive clone phage DNA was iso­lated (Lambda midi kit, QIAGENE) and sequenced.
cDNA cloning of BoAIPT1
Bamboo shoots were frozen with liquid nitrogen and homogenized, and total RNA was extracted using the TRI-ZOL reagent (Invitrogen, USA). The BoAIPT1 specific primers, RP2F 5'-TGA CTG CTG CTT CCT GTG GG-3' and RP2R 5'-CAG ATG CCT CGG GAG TAG TC-3', were designed based on the coding region. Full length BoAIPT1 cDNA sequences were obtained by the RACE
method (Clontech, USA).
Expression of BoAIPT1 in Escherichia coli
The recombinant plasmid BoAIPT1 cDNA was sub-cloned into the pTrcHis A vector (Invitrogen, USA) using the N-terminal and C-terminal primer pairs: YEH2F 5'-ATC ACA GGA TCC ATG CCA CTC TAC TCT A-3' (the BamHI underlined) and YEH3R 5'-AGA GAA TTC TTA TAC TAT GGC GGC C-3' (the EcoRI underlined). PCR was carried out at 95°C for 0.5 min, 52°C for 0.5 min, 72°C for 1 min, with 30 cycles of reaction and an exten­sion for 5 min at 72°C. A hexahistidine tag was fused to the N-terminus. A single recombinant E. coli (BL21 DE3) colony was used to inoculate 1 L of LB (Luria-Bertani with 1M sorbitol and 2.5 mM betaine) medium, which contained 50 [ig/ml of ampicillin. The cells were cultured at 37°C until OD600 of 0.6 was reached. IPTG (isopropyl-p-D-thiogalactoside) was added to a final concentration of 0.3 mM, and the cells were further incubated at 18°C with vigorous (150 rpm) shaking overnight.
MATERIALS AND METHODS
Plant materials
Fresh Bambusa oldhamii was harvested from a bamboo farm in Taipei, Taiwan. Tissue samples were prepared from 15cm etiolated shoot (divided into top, middle, and base sections) and 30 cm green shoot (divided into top, middle, and base sections). The outermost sheath was removed and discarded, except the sheath of the etiolated shoot base, which was stored in liquid nitrogen. The shoots were diced and frozen in liquid nitrogen. All samples were stored at -80°C until used.
Purification of the recombinant BoAIPT1 pro­tein
An E. coli culture BL21 (DE3) harboring the express­ing vector of BoAIPT1 was harvested at 6,500 g for 10 min, and resuspended in a 50 mM potassium phosphate buffer (KPB) pH 7.5, containing 0.15 M NaCl. A cell disrupter was used to disrupt cells on ice. Lysate was cen-trifuged at 40,000 g for 40 min at 4°C. The supernatant was then carefully removed and loaded onto a HisTrapTM FF 5ml column (GE Healthcare, USA). The column was eluted with 10, 50, 100, and 250 mM imidazole buffers. Recombinant protein was concentrated, and its buffer was exchanged with 50 mM KPB by Amicon Ultra (Millipore, Billerica, MA, USA) and stored at 4°C.
Genomic DNA library screening
The genomic DNA library of B. oldhamii was con­structed using the Lambda FIX® II/Xho I Partial Fill-In Vector Kit (Strategene, La Jolla, CA). The degenerate primers, IPT-F 5'-TGC C(G/T)C (A/T)CC A(C/A)C TGA TC(A/G) GC-3' and IPT-R 5'-C(G/A)A GCT C(C/G)G GCA (C/G)GC C(G/T)A T-3', were designed based on the conserved regions of AIPT genes from Arabidopsis, rice and hops. PCR was carried out under the following condi­tions: 95°C for 0.5 min, 48°C for 0.5 min, and 72°C for 0.5 min, with 30 cycles of reaction and a final extension for 5 min at 72°C.
A 254 bp BoAIPT1 fragment was used to generate a
BoAIPT1 activity assay
The protein concentration was assayed by the Brad­ford method (Protein Assay, Bio-Rad), and bovine serum albumin was used as the protein standard. The standard reaction buffer contained 2.0 [ig of the purified recombi-
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nant BoAIPT1, 100 mM Tris-HCl buffer, (pH 7.5), 50 mM
KCl, 10 mM MgCl2, 1 mg/ml bovine serum albumin, and, 1 mM DTT in a final volume of 100 [il.
Isopentenyl reactions were assayed at 30°C for 60 min, and terminated by boiling for 5 min. Half of the superna­tant was removed and treated with calf intestine alkaline phosphatase (CAIP) buffer and incubated for 1 h at 37°C. This was then followed by centrifugation at 13,000 g for 1 min. Reaction products were profiled by reversed phase HPLC (Waters 1525 HPLC system with 2487 absorbance detector) using a Hypersil GOLD aQ column (4 x 250 mm, Thermo) with flow rate of 1.0 ml/min. The separation program was as follows: 100% buffer A (0.1 M acetic acid pH 3.3) for 15 min, followed by a linear gradient of 0-80% of buffer B (acetonitrile) for 35 min. UV absorbance was monitored at 280nm. Product retention times were com-pared to the standards purchased from Sigma.
RESULTS
Isolation and sequence analysis of BoAIPT1
Degenerate primers were designed for BoAIPT1 gene according to conserved regions of AIPTs from other spe­cies (Kakimoto, 2001; Sakano et al., 2004; Sakamoto et al., 2006). A 254 bp DNA fragment was amplified by PCR using genomic DNA as a template. The deduced amino acid sequence was homologous to the corresponding re­gions of plant AIPTs. Thus, DNA was used as a hybrid­ization probe to screen a bamboo shoot genomic library. One potential clone was selected and subjected to further analysis. Nucleotide sequence analysis revealed that the clones contained 11 kb, including an open reading frame that was designated BoAIPT1.
Gene specific primers were designed for 5'-RACE and 3'-RACE experiments to investigate transcription initia­tion sites and gene structure of BoAIPT1. This study used
RACE to obtain a full-length cDNA of 1797 bp contain­ing BoAIPT1 . The sequence of this cDNA clone contained a 5'-noncoding region of 274 bp, an open reading frame of 1035 bp encoding BoAIPT1 with a molecular mass of 37,599 kDa and 344 amino acids, and a 3' non-coding region of 488 bp. When comparing the BoAIPT1 from cDNA with the genomic DNA, we found that the open reading frames consisted of one exon and no introns. In-trons are absent in AIPT genes from other higher plants, including OsIPT1~OsIPT8 (Sakamoto et al., 2006), HlIPT
(Sakano et al., 2004) and AtIPTs (except AtIPT8) (Takei et al., 2001).
A multiple sequence alignment of BoAIPT1 and AIPT from other species indicated BoAIPT1 possessed an iden­tity of 44-72 % (Figure 1). OsIPT5 and ZmIPT4 were the closest homologs (72%~69% identities) to BoAIPT1. The conserved ATP/GTP-binding sites (P-loop) motif ([A, G]-X4-G-K-[S, T]) in the N-terminal region of the AtIPTs
and OsIPTs were also found in BoAIPT1. The ATP/GTP-
binding motif is universally observed in ATP-consuming enzymes, including ATP binding cassette transporters (Ka-kimoto, 2001). A highly conserved Asp82 is located down­stream of the ATP/GTP-binding motif, which plays an essential role as a general base. There is a cluster of posi­tively charged residues (Lys83, Arg218, Arg261 and Lys 265) in BoAIPT1 at the open end of the ATP-binding channel (Chu et al., 2009).
Preparation of total RNA
Frozen tissues were ground into powder using liquid nitrogen followed by total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manu­facturer's instructions. Genomic DNA was removed with
TURBO DNA-free kits (Ambion, USA).
Real time RT-PCR
Total RNA, isolated from etiolated shoots, green shoots and sheath of etiolated shoot base, etiolated shoots and green shoots, was used as a template. Sequences of the primers for PCR are: RP2F 5'-TGA CTG CTG CTT CCT GTG GG-3' and R257, 5'-GAG CTC CGG CAC GCC GAT-3' for BoAIPT1 and BoActinF1 5'-TGGCATCACACCTTCTACAA-3' and
BoActinR15'-ACCTGGATCTTCATGCTGCT-3' for internal control BoActin. Complementary DNA strands were amplified using iScript One-Step RT-PCR Kit (Bio-Rad). Reverse transcription was carried out as follows: 50°C for 10 min for cDNA synthesis, and 95°C for 5 min for reverse tran-scriptase inactivation. Successive PCR was carried out for 40 cycles at 95°C for 10 sec, and 55°C for 30 sec in a thermal cycler (iQ5 Gradient Real Time PCR, Bio-Rad). All PCR products were subjected to verification using dis-sociation curves and ethidium bromide stained agarose gels to be single fragments. Subsequently, all fragments were cloned into pGEM-T vector for DNA sequencing. Sequence results showed all fragments were identical to the corresponding target gene region. The comparative Ct method was applied for relative quantification of gene expression. This method establishes standard curves (iQ5 Gradient Real Time PCR, Bio-Rad) using arithmetic for-mulae. Relative fold expression changes were calculated as2-aact. To validate the experiments, the amplification efficiencies of the target (BoAIPTI) and reference (BoAc-tin) were approximately equal to those obtained using the AACt calculation.
Cloning and purification of recombinant BoAIPT1 protein
The full-length cDNA of BoAIPT1 was cloned into
pTrcHisA in an attempt to produce recombinant His-tagged protein in E. coli BL21 (DE3). Our study per­formed purification of recombinant hexahistidine tagged BoAIPT1 using affinity chromatography. Protein super­natant was applied to a HisTrap TM FF 5ml column (GE Healthcare, USA), eluted using different concentrations of imidazole buffers, and followed by an SDS-PAGE analy-
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Figure 1. Multiple amino acid sequence alignments of BoIPT1 with AIPTs from other species. The sequence abbreviations and Gen-Bank™ Accession numbers shown here are from Bambusa oldhamii (BoIPT1, HM044217), Arabidopsis thaliana (AtIPT1, AB061400; AtIPT3, AB061401; AtIPT8, AB062614), Humulus lupulus (HlIPT, Q5GHF7), Malus hupehensis (MhIPT, DQ792508), Oryza sativa (OsIPT5, BAC84458) and Zea mays (ZmIPT2, ABY78882; ZmIPT6, ABY78885). The conserved ATP/GTP binding motif, which can be converted in AIPT, is underlined. The degenerate primer pairs, IPT-F and IPT-R, are shown by arrows. The conserved Asp62 is indi­cated by asterisk and dots represent the conserved positively charged residues.
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sis (Figure 2A) and Western blot analysis (Figure 2B). A major band at about 40 kDa was seen in the Coomassie Brilliant Blue R-250 stained gel (Figure 2A, lane 2). This study also observed the presence of recombinant protein, detected by anti-His antibody, using Western blot (Figure 2B, lane 2). Molecular mass of the purified recombinant protein was estimated to be 40 kDa, which agreed with the molecular mass of BoAIPT1 (Figures 2A and B, Lane 6).
Figure 3. HPLC analysis of the BoIPT1-catalyzed reaction product. (A) HPLC chromatograms of the iPA standard (Sigma-Aldrich). (B) Elution profile of the calf intestine alkaline-phos-phatase-treated isopentenylation reaction product. The arrow indicates iPA retention time.
Optimum protein expression was obtained by incubating
the IPTG-induced culture at 18oC for 18 hr.
Activity assay of recombinant BoIPT1
The enzyme activity of purified recombinant BoIPT1 was analyzed by incubation with DMAPP and ATP. CIAP-treated reaction products were then analyzed by reverse phase HPLC (Figure 3), which clearly demonstrated the formation of an isopentenylated product.
Real-Time RT-PCR
In order to analyze the RNA expression level of
BOAIPT1 at different developmental stages, the total RNA
of etiolated shoots and green shoots and sheath of etiolated shoot base were isolated and analyzed with Real-time RT-PCR (Figure 4). Results revealed a significant difference in the expression levels of both etiolated shoots and green shoots. BOAIPT1 mRNA expression level in the green shoots showed an incremental increase from base to top, with the expression level of the top section 3 times greater than the middle section and 5 times greater than the base section. In etiolated shoots, the RNA expression level of all three sections (top, middle, and base) showed no signif­icant differences. Compared to the top section of the green shoot, the expression level is approximately 5 times less. The results clearly demonstrate that, as a bamboo devel­ops, BOAIPT1 gradually increases, and the green shoots have a higher expression level than the etiolated shoots. The top sections of the green shoots display a higher ex­pression level of BoAIPT1 than the base section.
Figure 2. SDS-polyacrylamide gel electrophoresis of recom­binant BoIPT1 in Escherichia coli by affinity chromatography. Samples from different purification steps were monitored by (A) 15% SDS-PAGE, and the gel was stained with Coomassie Bril­liant Blue R-250, or was preceded to (B) Western blot analysis and detected by using anti-His antibody followed by DAB color development. Lane 1: bacteria lysate without IPTG; lane 2: IPTG induced soluble faction of bacteria lysate; lane 3: eluted sample from buffer containing 10 mM imidazole; lane 4: 50 mM imidazole elution; lane 5: 100 mM imidazole elution; and, lane 6: 250 mM imidazole elution. M, pre-stained protein molecular size marker.
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As the shoots emerge from the ground and are exposed to light, BoAIPT1 expression level increases. This agrees with real-time RT PCR analysis where expression level is lower in etiolated shoots than green shoots, and the base section of the green shoot has a lower expression level than the top section. Cytokinins have been demonstrated to induce shoot growth. When a bamboo emerges from the ground, it grows rapidly. In our study, we found that the green shoot top section has high level of BoAIPT1 expres­sion. This could indicate that the fast growth phenomenon may be related to the high expression level of BoAIPT1.
We will continue to analyze BoAIPT1 to gain a further understanding of its biochemical properties and applica­tion for agricultural uses. In other monocot plants, such as Zea mays and Oryza sativa, more than one AIPT gene has been identified (Sakamoto et al., 2006; Brugiere et al., 2008). However, based on the bamboo genomic library screening, only one AIPT has been successfully screened. Presently, we are continuing the screening process.
Figure 4. Expression profile of BoAIPTI analyzed by real­time RT-PCR. Relative expression level of BoAIPT1 at different growth stages, ES (base, middle and top) and GS (base, middle and top). Sheath of ES base (control). The data shown are means 士SE (n=3).
DISCUSSION
Acknowledgements. This work was supported by grants from the National Science Council of the Republic of Chi­na, Taiwan (NSC No. 97-2313-B-002-011-MY3). We are
grateful to Prof. Ai-Yu Wang for providing the Bambusa oldhamii genomic library.
BoAIPT1 is an intronless gene that shares a similar gene structure with other plant AIPTs (Takei et al., 2001;
Sakano et al., 2004; Abe et al., 2007). In rice and Arabi-
dopsis, approximately 20% of genes are intronless. One proposition is that intronless gene families develop from gene duplication or reverse transcription. The conservation of the lack of introns shows that evolution of AIPT is alike regardless of gene structure or biochemical characteristics.
From the alignment results, we observed that BoAIPT possesses the conserved ATP/GTP-binding sites (P-loop) motif and positively charged amino acid cluster (Lys83,
Arg218, Arg261, and Lys265) (Chu et al., 2009). We also no­ticed the second amino acid of the cluster is always either Arg or Lys in all the different plant AIPTs. The zinc finger­like motif, designated as C-X2-C-X{12,18}-H-X5-H, (where X denotes any amino acid residue, and X{m, n} m to n amino acid residues in number) (Kakimoto, 2001; Sakamoto et al., 2006), which is conserved in eukaryotic
tRNA-IPT is absent in the BoAIPT1 C-terminal. From ac­tivity analysis, we determined that BoAIPT1 uses DMAPP and ATP as its substrate to synthesize iPTP, an iP precur­sor.
Results from real-time RT-PCR analysis showed that, when bamboo emerges from underground, the expression of BoAIPT1 increases as well, predominantly in the green shoot top section. Expression level in the green shoot tip can be 5-times greater than the expression level in etio­lated shoot.
The promoter sequence of BoAIPT1derived from chro­mosome sequencing underwent Plant Cis-acting Regula­tory DNA Elements (PLACE) promoter bioinformatics analysis. Results revealed that the BoAIPT1 promoter has three light-induced regions, suggesting that the BoAIPT1 expression level may be induced by the presence of light.
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以大腸桿菌系統分子鑑定綠竹異戊烯基轉移酶
葉傳山  謝陸盛  楊健志  李平篤
國立台灣大學微生物與生化學研究所
細胞分裂素Cytokinins (CKs)爲植物賀爾蒙，具有調控植物發育的功能，例如細胞分裂與植物生長
過程的調節，CKs生合成路徑關鍵酵素爲腺嘌呤核苷酸異戊烯基轉移酶Adenylate isopentenyltransferase
(AIPT)。本硏究中，根據蛋白質保守性序列設計出DNA引子，進行PCR得到部分綠竹AIPT DNA
序列。由染色體基因庫篩選出基因全長核酸序列。利用專一性引子進行5'-RACE與3'-RACE得到全
長cDNA序列名爲BoAIPTI ， BoAIPTI讀碼框內序列含1,035 bp ，可表現出具有344個胺基酸，分
子量37.7 kDa的蛋白質。BoAIPTI基因不含插入序列（intron)。分析BoAIPT1與其他物種AIPT相似
在N端具有ATP/GTP結合區。大腸桿菌表現BoAIPT1重組蛋白具有活性可將DMAPP中異戊烯側鏈
(isopentenyl side chain)轉移至腺嘌呤核苷酸中N6位置。Real-time PCR分析結果顯示BoAIPTI表現情況
因綠竹生長而增加，且莖頂具有最高表現量。
關鍵詞：綠竹；腺嘌呤核苷酸異戊烯基轉移酶；分子選殖；大腸桿菌表現系統。