Molecular Cloning of a Putative Gene Encoding Isopentenyltransferase from Pingyitiancha (Malus Hupehensis) and Characterization of Its Response to Nitrate

Molecular cloning of a putative gene encoding isopentenyltransferase from pingyitiancha (Malus hupehensis) and characterization of its response to nitrate

JING PENG,1 FUTIAN PENG,1,2 CHUNFU ZHU1 and SHAOCHONG WEI1

1 College of Horticulture Science and Engineering/State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong Downloaded from https://academic.oup.com/treephys/article/28/6/899/1634693 by guest on 02 October 2021 271018, P.R. China 2 Corresponding author ([email protected])

Received October 10, 2007; accepted December 4, 2007; published online April 1, 2008

– Summary A putative isopentenyltransferase (IPT) encoding suggest that NO3 substantially affects the de novo biosyn- gene was identified from a pingyitiancha (Malus hupehensis thesis and translocation of cytokinin. Rehd.) expressed sequence tag database, and the full-length Two biosynthetic pathways for cytokinin production are be- gene was cloned by RACE. Based on expression profile and se- lieved to exist in plants: the tRNA pathway and the AMP quence alignment, the nucleotide sequence of the clone, named (ATP/ADP) pathway (Blackwell and Horgan 1994). Recent MhIPT3, was most similar to AtIPT3, an IPT gene in Arabi- experimental evidence demonstrates that isopentyladenine dopsis. The full-length cDNA contained a 963-bp open reading (iP)-type and trans-zeatin (tZ)-type cytokinins are synthe- frame encoding a protein of 321 amino acids with a molecular sized through the action of AMP/ATP/ADP isopentenyltrans- mass of 37.3 kDa. Sequence analysis of genomic DNA re- ferase (IPT), with ATP and ADP being the preferred substrates vealed the absence of introns in the frame. Quantitative of A. thaliana IPTs (AtIPTs) (Kakimoto 2001), whereas real-time PCR analysis demonstrated that the gene was ex- cis-zeatin (cZ)-type cytokinins are synthesized through the ac- pressed in roots, stems and leaves. Application of nitrate to tion of tRNA IPTs (Miyawaki et al. 2006). The first steps in roots of nitrogen-deprived seedlings strongly induced expres- plant cytokinin synthesis result in the production of sion of MhIPT3 and was accompanied by the accumulation of N 6-∆2-isopentenyladenine (iP) riboside 5′-tri-, di- or mono- cytokinins, whereas MhIPT3 expression was little affected by phosphate (iPRTP, iPRDP or iPRMP, respectively) by IPT; ammonium application to roots of nitrogen-deprived seed- these can then be hydroxylated to tZR 5′-tri-, di- or mono- lings. Application of nitrate to leaves also up-regulated the ex- phosphate (tZRTP, tZRDP or tZRMP, respectively) by a cyto- pression of MhIPT3 and corresponded closely with the accu- chrome P450 monooxygenase, CYP735A (Sakakibara 2006). mulation of isopentyladenine and isopentyladenosine in Active cytokinin species are free-base forms, such as iP and leaves. tZ. LOG (LONELY GUY) has phosphoribohydrolase activity and catalyzes the conversion of cytokinin nucleotides, such as Keywords: apple, cytokinin, gene expression, IPT, quantitative iPRMP and tZRMP, to the free base form with the release of a real-time PCR. ribose 5′-monophosphate (Kurakawa 2007). The Arabidopsis genome encodes seven genes belonging to a plant-specific ATP/ADP IPT clade (AtIPT1, AtIPT3, AtIPT4, AtIPT5, Introduction AtIPT6, AtIPT7 and AtIPT8) (Kakimoto 2001). Among these Cytokinin has various physiological effects such as promotion IPT genes, N availability differentially regulates expression of of cell division, chloroplast differentiation and shoot develop- AtIPT3 and AtIPT5, and AtIPT3 is a key determinant of – ment, counteraction of senescence and induction of photosyn- cytokinin biosynthesis in response to rapid changes in NO3 thesis gene expression. Several studies have shown that availability (Takei et al. 2004a). In other plants such as rice cytokinin is a pivotal signaling compound communicating ni- (Oryza sativa L.) and Chinese cabbage (Brassica rapa L.), trogen (N) availability from root to shoot via the xylem vessels several IPT members have been found (Ando et al. 2005, – (Takei et al. 2002). Application of exogenous nitrate (NO3 )in- Sakamoto et al. 2006), whereas only one IPT member has been duced rapid accumulation of cytokinins in roots of barley identified in the genomes of hop (Humulus lupulus L., (Samuelson and Larsson 1993), maize (Sakakibara et al. 1998, Cannabinaceae) and mulberry (Morus alba L., Moracerae) Takei et al. 2001a) and Arabidopsis thaliana (L.) Heynh. (Sakano et al. 2004, Abe et al. 2007). So far, the response of – (Takei et al. 2002). In maize xylem vessels, the translocation IPT to a NO3 signal has been reported only for Arabidopsis. rate of trans-zeatin riboside (tZR) increases markedly in re- We cloned an IPT gene from pingyitiancha (Malus – sponse to NO3 supplements (Takei et al. 2001b). These results hupehensis Rehd.), an important rootstock for apple produc- 900 PENG, PENG, ZHU AND WEI tion in China. Expression of MhIPT3 was up-regulated in re- in public databases using the BLAST tool. Amino acid se- – sponse to exogenous application of NO3 . quence alignments were performed with DNAMAN software. Phylogenetic tree analysis was performed with the ClustalW program. Materials and methods Exogenous application of nitrate and quantitative real-time Plant material and growth conditions PCR Pingyitiancha (Malus hupehensis) seeds were germinated on Nitrogen was applied as either 16 mM KNO3 or 16 mM NHCl4 vermiculite, and the seedlings grown in a greenhouse for for 0.5, 2 or 3.5 h. At harvest, plants were separated into roots, 4 weeks in vermiculite irrigated with distilled water. The stems and leaves. Specific gene primers were designed from greenhouse was maintained at a constant temperature of 24 °C the cDNA sequence of MhIPT3 to be analyzed using the Downloaded from https://academic.oup.com/treephys/article/28/6/899/1634693 by guest on 02 October 2021 and the photoperiod was extended to 16 h with fluorescent Primer 5.0 software (PE Applied Biosystems) following the lights. The 4-week-old seedlings were transferred to a growth manufacturer’s guidelines. For the quantitative real-time PCR chamber providing the same conditions as the greenhouse, but analysis, the r18S gene was used as an internal constitutively the seedlings were grown hydroponically in aerated expressed control (house-keeping gene). The primers (re- Hoagland’s nutrients containing 0.8 mM nitrate for 48 h to verse-R and forward-F) and [5′] 6-FAM and [3′] TAMRA flu- generate N-deprived seedlings. For the root experiments, the orescent-dye-labeled probes (-P) (Table 1) were synthesized N-deprived plants were transferred to a treatment medium by Invitrogen and used at 200 nM final concentration. Each containing 16 mM KNO3,NH4Cl or KCl and allowed to grow real-time PCR was performed in 25 µl final volume on an under the same chamber conditions for 0.5, 2 or 3.5 h. After re- FTC2000 thermocycler according to the manufacturer’s in- moving seedlings from the treatment media, excess liquid was structions. Three replicates were run for each sample. The removed with paper towels, and whole roots were excised, PCR cycles were as follows: 1 cycle of 2 min at 50 °C and frozen in liquid N and stored at –80 °C. For the leaf experi- 10 min at 95 °C, followed by 40 cycles each of 15 s at 95 °C, ments, leaves of the N-deprived plants were brushed with H2O and 1 min at 60 °C. Reactions performed without template or 16 mM KNO3 for 30 min, then excised, frozen in liquid N yielded no product. and stored at –80 °C. Identification of cytokinin species and concentrations by Search for candidate MhIPT ESTs ELISA The amino acid sequence of each of the AtIPTs was used as an Wells of a microtiter plate (MaxiSorp, Nalge Nunc Interna- inquiry for searching the apple EST database with tBLASTn tional) were coated with 50 µl of coating solution (0.1 pg ml–1 (http://www.ncbi.nlm.nih.gov/BLAST/). BSA conjugating ZR or 0.5 pg ml–1 BSA conjugating iPA in

50 mM NaHCO3 pH 9.6) for 2 h at 37 °C. After three washes Total RNA isolation, cDNA synthesis and RACE with phosphate-buffered saline (PBS), the plates were incu- Total RNA was extracted from 0.1 g of roots with TRIZOL bated with 400 pl of blocking buffer (0.5% BSA, 0.05%

(Invitrogen) reagent and treated with RNase-free DNaseI at Tween 20 in 50 mM NaHCO3 pH 9.6) for 30 min. After wash- room temperature for 15 min in reaction buffer containing ing with PBS-Tween (PBS containing 0.05% Tween 20), 50 µl 20 mM Tris–HCl (pH 8.4), 2 mM MgCl2 and 50 mM KCl. The of cytokinin standard or the semi-purified cytokinin fractions DNaseI was inactivated by adding EDTA (2.5 mM final con- in sample buffer (0.1% BSA, 0.05% Tween 20 in PBS), and centration) and heating to 65 °C for 10 min. Complementary 50 µl of anti-cytokinin antibody (21 ng ml–1 anti-ZR antibody DNAs were synthesized with the SuperScriptIII Kit (Invitro- or 38 ng ml–1 anti-iPA antibody) were added to the wells. After gen) following the manufacturer’s instructions. To generate a 2-h incubation, the plates were washed with PBS-Tween and the full-length cDNA of the putative MhIPT3, gene-specific primers (GSPs) and nested gene specific primers (NGSPs) were designed for 3′- and 5′-RACE according to the instruc- Table 1. Primers and [5′] 6-FAM and [3′] TAMRA fluorescent-dye-la- tions of the SMART RACETM cDNA Amplification Kit beled probe sequences used in quantitative real-time PCR assays of (Clontech, USA) and sequence information from the retrieved genes MhIPT3 and r18S. Accession numbers are given in parenthesis. candidate ESTs. The GSPs and NGSPs used were: 5′-RACE: GSP1, 5′-GGAAGCCGATCACGGCCTAAAAT- Primer Sequence GGA-3′; NGSP1, 5′-ACTTTCCAGTCCCTGTTGCTCCCA- TTAC-3′; and 3′-RACE: GSP2, 5′-AACTTTCCTCTAGCTT- MhIPT3 (DQ792508) ′ ′ CTTCGTCT-3′; NGSP2, 5′-CCGGCAGAAGGAGAAGG-3′. IPT-F 5 -GAAGGAGAAGGTGGTGATCGTA-3 IPT-R 5′-GACTTGCATTTTGTCGGAGTTTA-3′ Both strands of the DNA fragments amplified from the re- IPT-P 5′-AACAGGGACCGGAAAGTCAAGGC-3′ trieved ESTs or 3′- and 5′-RACE reactions were sequenced by the dideoxy chain termination method with an ABI 3730 se- r18S (DQ341382) ′ ′ quencer. The nucleotide and deduced amino acid sequences r18S-F 5 -AAACGGCTACCACATCCA-3 ′ ′ were analyzed with DNAMAN software (Lynnon Corpora- r18S-R 5 -CACCAGACTTGCCCTCCA-3 r18S-P 5′-AGCAGGCGCGCAAATTACC-3′ tion) and DNASTAR programs and compared with sequences

TREE PHYSIOLOGY VOLUME 28, 2008 CLONING OF ISOPENTENYLTRANFERASE GENE AND ITS RESPONSE TO NITRATE 901

the antibodies immunodecorated with goat anti-rabbit IgG To elucidate the phylogenetic relationships of MhIPT3 to conjugating alkaline phosphatase (Bio-Rad). Alkaline phos- IPTs from other plants, we performed a phylogenetic analysis. phatase activity was visualized by adding p-nitrophenyl- In the phylogenetic tree (Figure 3), MhIPT3 forms a cluster

phosphate as substrate in 100 mM CHES, 1 mM MgCl2 with LjIPT3, AtIPT3 and BrIPT3, but a separate cluster with (pH 9.8). Absorbance of the developed color was measured at other IPT enzymes including A. thaliana tRNA (AtIPT2 and 405 nm with a microplate reader (M-SPMax250, Wako Pure AtIPT9). The MhIPT3 appears to be evolutionarily more re- Chemical). The cytokinin concentration was calculated with lated to AtIPT3, AtIPT5 and AtIPT7 than to AtIPT4, AtIPT6 SOFTmax Pro software. and AtIPT8.

Spatial expression patterns of MhIPT3 Results and discussion To evaluate the expression of MhIPT3 in different tissues, total RNA was prepared from various tissues of 2-week-old seed- Downloaded from https://academic.oup.com/treephys/article/28/6/899/1634693 by guest on 02 October 2021 Isolation and sequence analysis of full length MhIPT3 lings. An r18S gene was used as an internal standard (Rasori et To isolate the MhIPT3 gene, a 689-bp EST sequence was used al. 2002). Quantitative real-time PCR analysis revealed that to design GSPs for 5′-RACE and 3′-RACE. The entries from MhIPT3 gene expression differed among root, stem and leaf the apple EST database (DT001742) showed good alignment tissues (Figure 4), as also reported for AtIPT3 (Takei et al. with the A. thaliana IPT genes. As a result, a full-length cDNA 2004a). of 1314 bp (DQ792508), named MhIPT3, was obtained by RACE. The nucleotide sequence of MhIPT3 contains a 248-bp 5′-non-coding region, a 963-bp open reading frame encoding a protein of 321 amino acids with a molecular mass of 37.3 kDa, and a 151-bp of 3′-non-coding region (Figure 1). We success- fully amplified MhIPT3 from both the genomic DNA and cDNA of pingyitiancha, and fragment size and sequencing data of both amplification products were similar (data not shown), indicating that introns are absent from the ORF of MhIPT3. Similarly, Takei et al. (2001a) reported that AtIPTs (except AtIPT8) have no introns, and several homologues of AtIPTs identified from genomic DNA and full-length cDNA databases of rice also lack introns, indicating this structural feature of IPTs may be a characteristic of higher plants. In transgenic Arabidopsis expressing a gene for green fluorescent protein (GFP) driven by AtIPT promoters, the AtIPT3::GFP was located in the phloem companion cells (Takei et al. 2004a). According to ProtComp v6.0 (http://ww- w.softberry.com/berry.phtml), the predicted subcellular local- ization of the MhIPT3 protein is primarily in the cytoplasm. The deduced amino acid sequence of MhIPT3 showed 34.22, 53.27, 35.34, 55.29, 35.11, 47.43 and 38.14% identity to the AtIPT isozymes AtIPTl, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8, but it exhibited only 25.10 and 25.52% identity to the tRNA IPT isozymes AtIPT2 and AtIPT9, respectively (Figure 2). On the one hand, MhIPT3 contains a GATGTGKS (amino acids numbers 40–47) sequence known as the ATP/GTP binding motif that is univer- sally observed in ATP-consuming enzymes including ATP- binding cassette transporters (Kakimoto 2001). On the other hand, the putative zinc finger-like motif designated C-X2- C-X12,18-H-X5-H (where X denotes any amino acid residue) which is conserved in A. thaliana AtIPT2 and eukaryotic tRNA IPTs, is not present in MhIPT3, as is also the case for the AtIPT isozymes (Kakimoto 2001, Takei et al. 2001a). Further- more, like other IPTs, MhIPT3 shares a consensus pattern im- mediately downstream of the ATP/GTP binding motif of X - Figure 1. Nucleotide and amino acid sequences of MhIPT3. Primer 5 positions are underlined. Primers: 5′-GSP (NGSP1), gene-specific (V/L/I)-X7-(V/L/I) (V/L/I)-X2-D-X2-Q-X57,60-(V/L/I)(V/L/ primer for 5′-RACE; 3′-GSP (NGSP2), gene specific primer for I)-X-GG-(S,T) (where X denotes any amino acid residue) 3′-RACE; and P1-S and P1-A are the sense and antisense primers, re- (Kakimoto 2001, Takei et al. 2001a). spectively, for the amplification of full-length MhIPT3.

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Figure 2. Multiple alignment of conserved region of amino acid sequences of the predicted translation products of MhIPT3 with those of AtIPTs. Abbreviations and GenBank Accession numbers: Arabidopsis thaliana AtIPT1, AB061400; AtIPT3, AB061401; AtIPT4, NM_118598; AtIPT5, AB062608; AtIPT6, NM_102352; AtIPT7, AB061405; and AtIPT8, AB062614.

Rapid accumulation of MhIPT3 transcript and cytokinins in way has been characterized in Arabidopsis (Sakakibara 2006). – The primary product of cytokinin synthesis is N 6-(D2-isopent- response to NO3 enyl) adenine (iP) nucleotide, which is formed by adenosine Previous studies have highlighted the close correlation be- phosphate-IPT. The isoprene side chain of the iP nucleotide is tween N nutrition and cytokinin concentration in tobacco then hydroxylated by a cytochrome P450 monooxygenase, (Singh et al. 1992), Urtica dioica L. (Wagner and Beck 1993), CYP735A1 or CYP735A2, resulting in the formation of barley (Samuelson and Larsson 1993) and maize (Takei et al. tZ-type cytokinin, a highly active form (Takei et al. 2004b). 2001a). The basic scheme of the cytokinin biosynthetic path- The Arabidopsis genome encodes seven IPT genes (IPT1,

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Figure 4. Patterns of MhIPT3 transcript abundance in different tissues of Malus hupehensis determined by quantitative real-time PCR analy- Downloaded from https://academic.oup.com/treephys/article/28/6/899/1634693 by guest on 02 October 2021 sis of total RNA prepared from roots, stems and mature leaves of 2-week-old hydroponically grown seedlings.

Figure 3. A phylogenetic tree for the conserved region of isopentenyl- Table 2. Effects of foliar application of KNO3 on MhIPT3 gene ex- transferases (IPTs). The tree was made with the ClustalW program. pression and the concentrations of hormones in leaves. Abbreviations: Abbreviations and Genbank Accession numbers: Brassica rapa Z, zeatin; ZR, zeatin riboside; iP, isopentyladenine; and iPA, isopent- subsp. BrIPT1, AB186132; BrIPT3, AB186133; BrIPT7, AB186135; yladenosine. Humulus lupulus HlIPT, AY533024; Lotus japonicus LjIPT1, DQ436462; LjIPT3, DQ436464; Morus alba MaIPT, AY781335; Treatment mRNA (103 ) Z + ZR iP + iPA Oryza sativa OsIPT3, BAE47446; and OsIPT9, AB239806. Control 4.07 Aa 52.01 Aa 7.18 Aa KNO3 treatment 6.97 Bb 84.44 Bb 65.88 Bb IPT3–IPT8) (Kakimoto 2001, Takei et al. 2001a) that have a spatially differentiated expression pattern (Miyawaki et al. 2004, Takei et al. 2004a). Analyses of the individual IPTsre- MhIPT3 transcripts accumulated within 2 h (Figure 5A). The vealed that IPT3, which is expressed in the phloem, is up-regu- time course of MhIPT3 induction corresponded closely with lated by nitrate, but not by ammonium (Miyawaki et al. 2004, the accumulation of iP+iPA and Z+ZR in roots (Figures 5B + Takei et al. 2004a), with a similar expression pattern to the and 5C). Application of exogenous NH4 did not induce gene encoding nitrate reductase (NIA) (Takei et al. 2004a). MhIPT3 expression.

The pattern of IPT3 induction corresponds well to the accumu- Foliar application of KNO3 up-regulated the expression of lation of cytokinins and the nitrate-dependent accumulation of MhIPT3 in leaves and corresponded closely with the accumu- cytokinin is greatly reduced in an IPT3 mutant, indicating that lation of iP+iPA (Table 2). Similarly, Takei et al. (2004a)re- IPT3 is a key determinant of nitrate-dependent cytokinin ported that IPT3 expression in Arabidopsis leaves is induced – biosynthesis (Takei et al. 2004a). In N-limited Arabidopsis, by NO3 , whereas CYP735A expression is negligible in leaf tis- – NO3 feeding caused rapid accumulation of cytokinins in roots sue. Thus, the major form of the leaf-borne cytokinin was the (Takei et al. 2002). To understand the short-term effect of N iP-type, whereas root-borne cytokinin is translocated to leaves sources on the expression of MhIPT3, changes in the accumu- as the tZ-type. The structural difference in the isoprenoid side – lation of MhIPT3 transcripts in roots during induction by NO3 chain between the leaf-borne and root-borne cytokinins might + or NH4 were monitored by quantitative real-time PCR analy- be critical in distinguishing the origin and direction of – sis. When NO3 was resupplied to N-limited seedlings, long-range cytokinin signaling.

Figure 5. Effect of resupplying in- organic nitrogen sources on MhIPT3 expression in roots and on cytokinin accumulation. (A) Accu- mulation of MhIPT3 transcripts in response to exogenous application of nitrate (NO3). (B) Accumula- – tion of iP+iPA during NO3 induction. (C) Accumulation of Z+ZR – during NO3 induction. Abbrevia- tions: Z, zeatin; ZR, zeatin riboside; iP, isopentyladenine; and iPA, isopentyladenosine.

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