Adenylate Isopentenyltransferase Research Articles

MdIPT1, an adenylate isopentenyltransferase coding gene from Malus domestica, is involved in branching and flowering regulation

Flowering and shoot branching are significant agricultural traits for apple tree breeding. Cytokinin metabolism and signaling pathways play a crucial role in plant development. However, little is known about cytokinin biosynthetic molecular mechanism and function involved in apple flowering and branching. In this study, an adenylate isopentenyl transferase coding gene MdIPT1, homologous to AtIPT3/AtIPT5 in Arabidopsis thaliana, was identified. MdIPT1 was highly expressed in apple floral and axillary buds and was dramatically up-regulated during floral induction and axillary bud outgrowth. The promoter of MdIPT1 showed high activity in multiple tissues and responded to different hormone treatments. The MdIPT1-overexpressing Arabidopsis showed a multi-branching and early-flowering phenotype, with elevated endogenous cytokinin levels and altered expression of genes related to branching and flower formation. Overexpression of MdIPT1 confers the growth vigor of transgenic apple callus on a CKs-deficient medium. Our findings suggest that MdIPT1 is a positive regulator involved in branching and flowering. The data presented herein provide extensive research results on MdIPT1 and will contribute to molecular breeding for new apple varieties.

Comparative analysis of adenylate isopentenyl transferase genes in plant growth-promoting bacteria and plant pathogenic bacteria

Cytokinin is a major phytohormone that has been used in agriculture as a plant-growth stimulating compound since its initial discovery in the 1960s. Isopentenyl transferase (IPT) is a rate-limiting enzyme for cytokinin biosynthesis, which is produced by plants as well as bacteria including both plant pathogenic species and plant growth-promoting bacteria (PGPB). It has been hypothesized that there may be differences in IPT function between plant pathogens and PGPB. However, a comprehensive comparison of IPT genes between plant pathogenic and PGPB species has not been performed. Here, we performed a global comparison of IPT genes across bacteria, analyzing their DNA sequences, codon usage, phyletic distribution, promoter structure and genomic context. We found that adenylate type IPT genes are highly specific to plant-associated bacteria and subdivide into two major clades: clade A, largely composed of proteobacterial plant pathogens; and clade B, largely composed of actinomycete PGPB species. Besides these phylogenetic differences, we identified several genomic features that suggest differences in IPT regulation between pathogens and PGPB. Pathogen-associated IPTs tended to occur in predicted virulence loci, whereas PGPB-associated IPTs tended to co-occur with other genes involved in cytokinin metabolism and degradation. Pathogen-associated IPTs also showed elevated gene copy numbers, significant deviation in codon usage patterns, and extended promoters, suggesting differences in regulation and activity levels. Our results are consistent with the hypothesis that differences in IPT regulation and activity exist between plant pathogens and PGPB, which determine their effect on plant host phenotypes through the control of cytokinin levels.

Determination of cadmium and/or drought stress effects on some plant phytohormone contents and hormone gene expressions in bean (Phaseolus vulgaris L)

Plants can sometimes be under the influence of more than one stress factor. In this case, combined stress factors can cause different responses in plants. In this study, the phytohormonal activity and hormone gene expressions of beans (Phaseolus vulgaris L.) were investigated in different drought levels and soils with different levels of cadmium accumulation. Plants were grown at 4 different cadmium (Cd) levels (Cd: 0, Cd1: 100, Cd2: 150, and Cd3: 200 mg kg$^{-1}$) and 3 irrigation levels (D0: 100%, D1: 75%, and D2: 50% of field capacity). Abscisic acid (ABA), indole acetic acid (IAA), gibberellic acid (GA), salicylic acid (SA), cytokinin, zeatin, and jasmonic acid contents and gene expressions related to them were investigated. As a result of the study, we determined that there was a significant decrease in the content of IAA, GA, SA, cytokinin, zeatin, and jasmonic acid with increasing doses of drought and Cd. Especially with the coexistence of both stress factors, this reducing effect (the reductions in these hormones were 86.6%, 64.1%, 71.9%, 66.7%, 45.5%, and 73.9% as compared to D0, respectively) on the measured parameters was greater. However, ABA content increased significantly with drought and/or Cd stresses, especially with Cd3D2, about a thousand times higher than the control. On the other hand, mRNA transcript levels of some hormone genes (ABA 8'-hydroxylase, adenylate isopentenyltransferase (AIPT3), auxin-related protein (AUX/ IAA), MeJA biosynthesis and gibberellin 20-oxidase and NRAMP2 gene known to be involved in Cd transport, were detected. According to the results, while ABA 8'-hydroxylase, gibberellin 20-oxidase, and NRAMP2 genes were overexpressed in all stress treatments, AIPT3 and AUX/IAA genes were down-regulated and also, MeJA biosynthesis gene varied depending on dose and stress.

Transcriptome Analysis Reveals Genetic Factors Related to Callus Induction in Barley

Barley is an important cereal crop worldwide. Its genetic transformation is now limited to very few cultivars because of the high genotype dependence of embryogenic callus. To reveal the key genes or factors controlling the callus induction and plantlet regeneration in barley, we compared the transcriptomic profiles of immature embryos of Golden Promise and ZU9, which differed dramatically in the efficiency of the genetic transformation. The samples were taken at 0, 5, 10 and 20 days of the culture, respectively. In total, 5386 up-regulated and 6257 down-regulated genes were identified in Golden Promise. Several genes, identified exclusively in GP callus, were selected for further investigation. These genes were mainly involved in protein metabolism, energy metabolism, stress response, detoxification and ubiquitin–proteasome. Four YUCCA flavin monooxygenases, one PIN-FORMED, one tryptophan aminotransferase related, three small auxin up RNA, three indole-3-acetic acid and one adenylate isopentenyl transferase, seven cytokinin oxidase/dehydrogenase, three Arabidopsis histidine kinase, three Arabidopsis histidine phosphotransfer protein, and one Arabidopsis response regulator were differentially expressed in the calli of the two barley genotypes, suggesting that biosynthesis, response and transport of auxin and cytokinin might be associated with cell reprogramming during callus induction. The current results provide insights into molecular mechanisms of callus induction at an early developmental stage and are helpful for optimizing the tissue culture system in barley.

A novel adenylate isopentenyltransferase 5 regulates shoot branching via the ATTTA motif in Camellia sinensis

BackgroundShoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated.ResultsIn this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3′ untranslated region (3ˊ UTR) and 5ˊ UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3′ UTR AS variant 2 (3AS2) might act as the predominant AS transcript.ConclusionsTogether, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.

Overexpression of oil palm EgDREB1 in tomato decreased fruit sizeand produced parthenocarpic fruits

Drought-responsive element binding (DREB) is involved in the regulation of stress-responsive gene expressions in plants through abscisic acid (ABA)-independent pathway. In this study, constitutive expression of oil palm (Elaeis guineensis) EgDREB1 driven by double strength cauliflower mosaic virus 35S promoter in tomato (Solanum lycopersicum) reduced seed number, produced parthenocarpic fruits, changed morphology of leaves, and increased root biomass of transgenic plants. Early flowering and fruiting of the transgenic lines were observed in the culture vessels. EgDREB1 was specifically expressed in the fruits and its expression was not detected in vegetative tissues (leaves and roots). Altered expression of several endogenous tomato genes involved in the biosynthesis of phytohormones including jasmonic acid, ethylene, auxin, cytokinin, gibberellin (GA) and ABA were observed compared to wild type plants. The expression of AP2-like-ethylene transcription factor (LeAP2), allene oxide synthase (LeAOS), allene oxide cyclase (LeAOC), aminocyclopropane-1-carboxylic acid synthase (LeACS), 1-aminocyclopropane-1-carboxylate oxidase 1 (LeACO), auxin responsive factor 8 (LeARF8), auxin/indole-3-acetic acid (LeAux/IAA), cytokinin oxidase/dehydrogenase-like (LeSlCKX1), adenylate isopentenyltransferase (LeSlIPT1), gibberellin 2-oxidase 2 (LeGa2ox2), gibberellin 20-oxidase 4 (LeGa20ox4) and ABA-aldehyde oxidase (LeAAO) were different in fruits with reduced seed number compared to parthenocarphic fruits. These results suggest that their expression has significant effects on fruit development in transgenic tomato. EgDREB1 may mediate the expression of some of these genes as dehydration-responsive element binding (DRE) motif were found in their promoter sequences. These data indicate that the EgDREB1 controls fruit development in trsngenic plants by regulating the expression of hormone-associated genes.

Evolutionary variation and expression profiling of Isopentenyl transferase gene family in Arabidopsis thaliana L. and Oryza sativa L.

Cytokinin is widely known for its critical involvement in various processes of plant growth and development, such as cell division, differentiation, and nutrient metabolism. As a phytohormone, it also plays a vital role in the stress modulation mechanism of the plant. Isopentenyl transferase (IPT) is the key regulatory enzyme of cytokinin biosynthesis. In the present study, all the identified members of Arabidopsis and rice IPT family were analyzed in terms of their chromosomal location, exon-intron distribution, and sub-cellular localization. Based on structural variation and evolutionary conservancy, they could be broadly divided into two families- Adenylate IPT and t-RNA IPT. Further, expression of all AtIPT and OsIPT transcripts were analyzed based on publicly available microarray datasets. All AtIPT genes except AtIPT3 and AtIPT6 showed a high level of expression in all the developmental stages of Arabidopsis. Different members of the AtIPT gene family responded differently depending on the type of stress exposure. A high level of expression was observed in the case of OsIPT2 and OsIPT9 in all the developmental stages and tissues of rice, as compared to the other members. In the case of abiotic stresses, OsIPT5 showed significant up-regulation. Transcript abundance of OsIPT2, OsIPT5, OsIPT7-2 and OsIPT9 fluctuated after biotic stress, hormonal imbalance, chemical toxicity, and nutrient deficiency. These members of the OsIPT gene family might be the crucial stress modulators that were validated through semi-quantitative RT-PCR and their role could be explored further for the generation of abiotic stress tolerant plants.

Light influences cytokinin biosynthesis and sensing in Nostoc (cyanobacteria).

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation.

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Identification and characterization of miRNAs involved in adventitious branches formation of Gracilaria lichenoides in vitro

The main morphological response of Gracilaria lichenoides explants to wounding in vitro was the direct production of adventitious branches (ABs) within a short period rather than callus formation, similar to other seaweeds. However, the underlying mechanism remains unclear because of the lack of genome information. MicroRNAs (miRNAs) play a role in plant growth and development by targeting mRNAs for translational repression or cleavage. Here, we investigated small RNA-mediated regulation of ABs formation by examining the post-transcriptional profiles of different treatment samples of G. lichenoides using the Illumina sequencing platform. The results showed that miR156g-3p, miR2597, miR3447-3p, miR169j-3p and other miRNAs families play significant roles in ABs formation in G. lichenoides through their predicted targets, including phospholipase D (PLD), auxin response factor (ARF), protein phosphatase 2C (PP2C), CTR1 and adenylate isopentenyltransferase (IPT). In addition, we identified 30 putative unique Gracilariales miRNAs in two small RNA libraries that may be involved in ABs induction. Profiling of miRNAs and their target genes of G. lichenoides provide new insight on miRNAs networks involved in ABs formation in seaweed.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Deep resequencing reveals allelic variation in Sesamum indicum.

BackgroundCharacterization of genome-wide patterns of allelic variation and linkage disequilibrium can be used to detect reliable phenotype–genotype associations and signatures of molecular selection. However, the use of Sesamum indicum germplasm for breeding is limited by the lack of polymorphism data.ResultsHere we describe the massively parallel resequencing of 29 sesame strains from 12 countries at a depth of ≥ 13-fold coverage for each of the samples tested. We detected an average of 127,347 SNPs, 17,961 small InDels, and 9,266 structural variants per sample. The population SNP rate, population diversity (π) and Watterson’s estimator of segregating sites (θw) were estimated at 8.6 × 10-3, 2.5 × 10-3 and 3.0 × 10-3 bp-1, respectively. Of these SNPs, 23.2% were located within coding regions. Polymorphism patterns were nonrandom among gene families, with genes mediating interactions with the biotic or abiotic environment exhibiting high levels of polymorphism. The linkage disequilibrium (LD) decay distance was estimated at 150 kb, with no distinct structure observed in the population. Phylogenetic relationships between each of the 29 sesame strains were consistent with the hypothesis of sesame originating on the Indian subcontinent. In addition, we proposed novel roles for adenylate isopentenyltransferase (ITP) genes in determining the number of flowers per leaf axil of sesame by mediating zeatin biosynthesis.ConclusionsThis study represents the first report of genome-wide patterns of genetic variation in sesame. The high LD distance and abundant polymorphisms described here increase our understanding of the forces shaping population-wide sequence variation in sesame and will be a valuable resource for future gene–phenotype and genome-wide association studies (GWAS).Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0225-3) contains supplementary material, which is available to authorized users.

Binding and catalysis of Humulus lupulus adenylate isopentenyltransferase for the synthesis of isopentenylated diadenosine polyphosphates

Various plant developmental processes involve phytohormones such as cytokinins. Isopentenyltransferase (IPT) reaction is the key rate-limiting step in cytokinin biosynthesis that transfers the isopentenyl (iP) group from dimethylallyl diphosphate to the N6-amino group of adenine. Here, a series of diadenosine polyphosphates (Ap n A) were screened as possible substrates of IPT, among which diadenosine tetraphosphate, diadenosine pentaphosphate and diadenosine hexaphosphate showed higher affinity than did the authentic substrates ADP and ATP. In addition, formation of mono-isopentenyl Ap n A and di-isopentenyl Ap n A was observed. Judging by the existing biosynthetic and hydrolytic systems for Ap n A in plants, Ap n A and isopentenyl-Ap n A may occur in the plant cells, with functional importance.

Crystal structure and substrate specificity of plant adenylate isopentenyltransferase from Humulus lupulus: distinctive binding affinity for purine and pyrimidine nucleotides

Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA–IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA–IPTs act on specific sites of tRNA. Here, we present the crystal structure of an AIPT–ATP complex from Humulus lupulus (HlAIPT), which is similar to the previous structures of Agrobacterium AIPT and yeast tRNA–IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ∼ ADP > GTP > CTP > UTP. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA–IPT, HlAIPT has evolved with a different binding strategy for adenylate.

Enzymatic formation of unnatural cytokinin analogs by adenylate isopentenyltransferase from mulberry

A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned from young leaves of mulberry ( Morus alba) by a homology-based RT-PCR. A recombinant enzyme expressed in Escherichia coli catalyzed prenyl transfer from DMAPP to the N 6 amino group of ADP and ATP, respectively, while AMP was a poor substrate of the enzyme. Interestingly, M. alba AIPT also accepted dADP, dATP, CDP, and GDP as the prenyl acceptors, and IPP, HMBPP, and GPP as the prenyl donors, to produce a series of cytokinin analogs. In particular, it was remarkable that the enzyme accepted HMBPP to produce trans-zeatin riboside phosphates, which suggested that trans-zeatin may be also produced from adenosine phosphates and HMBPP. Finally, alanine-scanning mutagenesis of conserved D49, Y54, F93, F120, Y153, F157, W159, Y170, Y217, and Q255, resulted in significant loss of enzyme activity except Y170A, confirming the functional and structural importance of the residues.

Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop ( Humulus lupulus L.)

A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop ( Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38–51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N 6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.

A new method for enzymatic preparation of isopentenyladenine-type and trans-zeatin-type cytokinins with radioisotope-labeling.

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-( E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor-ligand assays and cell metabolism studies.

Identification of Cytokinin Biosynthesis Genes in Arabidopsis: A Breakthrough for Understanding the Metabolic Pathway and the Regulation in Higher Plants.

The primary biosynthetic reaction of cytokinin is thought to be the isopentenylation of an adenine nucleotide such as AMP with dimethylallylpyrophosphate. For many years, the nature of the enzyme catalyzing this reaction in higher plants had not been identified despite the physiological importance of these compounds. However, the completion of the genomic sequence of Arabidopsis thaliana, a model plant for genetic research, has provided us with new opportunities to solve these problems. Recent studies have revealed the cytokinin biosynthesis enzyme is encoded by a small multigene family that is structurally related to both bacterial adenylate isopentenyltransferase and tRNA isopentenyltransferase. Interestingly, biochemical studies of some of the gene products indicate that ADP and ATP, rather than AMP, are preferentially used as substrates for this biosynthetic reaction. These findings require reconsideration of the currently accepted cytokinin biosynthetic pathway. In addition, there is an increasing body of evidence suggesting that the expression of these cytokinin synthesis genes is affected by the availability of nutrients.

Identification of Genes Encoding Adenylate Isopentenyltransferase, a Cytokinin Biosynthesis Enzyme, inArabidopsis thaliana

The initial step in the de novo biosynthesis of cytokinin in higher plants is the formation of isopentenyladenosine 5'-monophosphate (iPMP) from AMP and dimethylallylpyrophosphate (DMAPP), which is catalyzed by adenylate isopentenyltransferase (IPT). Although cytokinin is an essential hormone for growth and development, the nature of the enzyme for its biosynthesis in higher plants has not been identified. Herein, we describe the molecular cloning and biochemical identification of IPTs from Arabidopsis thaliana. Eight cDNAs encoding putative IPT, designated as AtIPT1 to AtIPT8, were picked up from A. thaliana. The Escherichia coli transformants expressing the recombinant proteins excreted cytokinin species into the culture medium except for that expressing AtIPT2 that is a putative tRNA IPT. A purified recombinant AtIPT1 catalyzed the formation of iPMP from DMAPP and AMP. These results indicate that the small multigene family contains both types of isopentenyltransferase, which could synthesize cytokinin and mature tRNA.