Isopentenyl Transferase Research Articles

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T2 generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.

Erratum: Inducible isopentenyl transferase as a high-efficiency marker for plant transformation

Erratum: Inducible isopentenyl transferase as a high-efficiency marker for plant transformation

Inducible isopentenyl transferase as a high-efficiency marker for plant transformation.

Overexpression of the isopentenyltransferase gene (ipt) from the Ti-plasmid of Agrobacterium tumefaciens increases cytokinin levels, leading to generation of shoots from transformed plant cells. When combined with a dexamethasone-inducible system for controlling expression, ipt expression can be used to select for transgenic regenerants without using an antibiotic-resistance marker. The combined system allows efficient cointroduction of multiple genes (in addition to ipt) and produces transgenic plants without morphological or developmental defects.

Leaf Senescence Is Delayed in Tobacco Plants Expressing the Maize Homeobox Gene knotted1 under the Control of a Senescence-Activated Promoter

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.

Simple and effective detection method for gene expression product of transgenic plant

It is a crucial problem in gene engineering whether an exogenous gene could be correctly expressed in cells of transgenic animal or plant, and how the expression products could be detected quantitatively in translational level. It is very difficult to analyze some gene expression products, such as isopentenyl transferase (ipt), in transgenic investigation because they are trace in organisms. A convenient method is described to determine trace content gene expression product which is hard to purify. The method includes predicting and synthesizing the antigenic peptide according to the cDNA sequence, coupling the synthetic antigenic peptide with carrier protein, raising specific antibodies against the synthetic antigen, and detecting the gene expression product by ELISA and Western blot.

Growth and In Vitro Propagation of Peach Plants Transformed with the Shooty Mutant Strain of Agrobacterium tumefaciens

Peach [Prunus persica (L.) Batsch] plants #94-1, #99-1, and #40-1, carrying a cytokinin biosynthesis (ipt) gene following transformation with the shooty mutant strain of Agrobacterium tumefaciens, were evaluated for altered growth habit and axillary shoot formation, both in vitro and in the greenhouse. After 9 weeks of in vitro propagation on four different levels of 6-benzyladenine (BA), only transformant #99-1 exhibited significantly greater axillary shoot formation (on 10 μm BA), and significantly greater fresh mass (on 3,10, and 30 μm BA) than the control #RG-3. Tolerance to a supra-optimal (30 μm) concentration of BA was indicated by fresh mass increases for #99-1 shoot cultures. Delayed senescence on 0 μm BA was exhibited by 87% of the transformants, but by only 12% of the control plants. Greenhouse-grown #99-1 and #40-1 were significantly shorter than #RG-3 plants at 6 weeks and at 1 year, but only #40-1 exhibited significantly greater branching than the controls. Chemical names used: 6-benzyladenine (BA); isopentenyl transferase (ipt).

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt.

Virulence genes of Agrobacterium tumefaciens are under the control of positive and negative transcriptional regulators. We found that the transcriptional regulator Ros controls expression of the plant oncogene ipt, which encodes isopentenyl transferase, in A. tumefaciens. This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant. An ipt promoter::cat reporter gene fusion showed a 10-fold increase in ipt promoter activity in A. tumefaciens ros mutant strains when compared with wild type. Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in ros mutant strains. In vitro studies using purified Ros showed it binds directly to the ipt promoter. Analysis of the deduced Ros amino acid sequence identified a novel type of C2H2 zinc finger. In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C2H2 motif. Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn2+ and DNA binding activities of Ros. The existence of such a regulator in Agrobacterium may be due to horizontal interkingdom retrotransfer of the ros gene from plant to bacteria.

Characterization of Pseudomonas syringae ssp. savastanoi strains isolated from ash

Isolates of Pseudomonas syringae ssp. savastanoi from ash were examined for their ability to produce phytohormones in culture and for pathogenicity, in comparison with isolates from olive and oleander. Nineteen out of 20 ash isolates produced low levels of indole‐3‐acetic acid and its methyl ester but no cytokinins. In contrast, the remaining isolate, NCPPB3474, accumulated high levels of auxins and cytokinins in culture, comparable to those of olive and oleander strains. Hybridization of DNA preparations with tryptophan mono‐oxygenase (iaaM) and isopentenyl transferase (ipt) gene‐containing probes showed sequences of DNA homologous to both probes only in isolate NCPPB3474, and in which the iaaM and ipt genes were located on the chromosome and on a plasmid of about 80 kb, respectively. When assayed for pathogenicity on ash, olive and oleander, 19 of the 20 ash isolates caused disease only on ash but NCPPB3474 caused knots on both ash and olive. Oleander isolates infected all three hosts whereas those from olive caused symptoms only on olive and ash. All the cultures were able to multiply in host plant tissues, but the growth rates and final population densities were correlated to the plant species inoculated and the host origin of the isolates. In particular, the highest population densities were reached by isolates capable of causing symptoms on the inoculated host. The phytohormone production shown by ash, olive and oleander isolates of P. syringae ssp. savastanoi was in accordance with the type of symptoms: cankers accompanied by wart‐like excrescences on ash and knots on olive and oleander. Furthermore, the pathogenic features of these isolates and, in particular, their growth patterns in the different host tissues, support previous evidence on the existence of three distinct pathovars in P. syringae ssp. savastanoi.

Seed-specific expression of the isopentenyl transferase gene (ipt) in transgenic tobacco

The Agrobacterium tumefaciens gene encoding isopentenyl transferase (ipt), a cytokinin biosynthetic gene, was fused to a promoter from a seed-specific gene, vicilin, and introduced into tobacco cells. Intact fertile plants were generated. The expression of the vicilin-ipt gene was shown to be confined to seed and resulted in enhanced levels of cytokinins in the developing seeds and increased seed protein content. Using a simplified quantification method, a significant increase in the levels of endogenous cytokinins was recorded at 16–21 days after flowering. The growth of the transgenic plants and the development of the seeds appeared to be normal.

Tobacco plants carrying a tms locus of Ti-plasmid origin and the HI-1 allele are tumor prone

The autonomous growth of plant tumor cells is believed to result from their persistent loss of the requirement for growth hormones such as auxin and cytokinin. The partially dominant gene Habituated leaf-1 (HI-1) regulates the requirement of cultures tissues of Havana 425 tobacco ( Nicotiana tabacum L.) for cytokinins. The HI-1 allele can partially restore the tumor phenotype in tobacco cells transformed with a Agrobacterium tumefaciens Ti plasmid defective in the isopentenyl transferase locus, which encodes a key enzyme in cytokinin biosynthesis and is required for neoplastic growth. To investigate the oncogenic function of HI-1, we transformed wild-type ( hl-1/hl-1) and Hl-1/Hl-1 tobacco plants with the tms locus derived from the limited-host-range Ti plasmid pTiAg162. This locus encodes enzymes for biosynthesis of the auxin indole-3-acetic acid. Grafting tests and measurements of the hormone requirement of cultured explants show that wound-induced overgrowths arising in tms transformed Hl-1 plants are tumorous. While some wound-induced overgrowths also formed in hl-1/hl-1 transformants, these showed slight hormone-autotrophic growth and weak tumorigenicity in grafting tests. In addition, Hl-1/Hl-1 tms/tms plants, but not hl-1/hl-1 tms/tms plants, spontaneously developed rooty teratomatous overgrowths, showed flowering abnormalities, and formed calli at the base of the stem in young seedlings. Thus, Hl-1 tms plants exhibit a tumor-prone phenotype, and in this regard closely resemble tumor-prone hybrids that arise in certain interspecific crosses of Nicotiana species. Our results show that the interaction of just two genetic elements – the mutant Hl-1 allele of the tobacco host with tms genes of Ti plasmid origin – are sufficient for a tumor-prone phenotype.

Selection of marker-free transgenic plants using the isopentenyl transferase gene.

We have developed a new plant vector system for repeated transformation (called MAT for multi-auto-transformation) in which a chimeric ipt gene, inserted into the transposable element Ac, is used as a selectable marker for transformation. Selectable marker genes conferring antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants, have three major problems: (i) the selective agents have negative effects on proliferation and differentiation of plant cells; (ii) there is uncertainty regarding the environmental impact of many selectable marker genes; (iii) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The MAT vector system containing the ipt gene and the Ac element is designed to overcome these difficulties. When tobacco leaf segments were transformed and selected, subsequent excision of the modified Ac produced marker-free transgenic tobacco plants without sexual crosses or seed production. In addition, the chimeric ipt gene could be visually used as a selectable marker for transformation of hybrid aspen (Populus sieboldii x Populus grandidentata). The chimeric ipt gene, therefore, is an attractive alternative to the most widely used selectable marker genes. The MAT vector system provides a promising way to shorten breeding time for genetically engineered crops. This method could be particularly valuable for fruit and forest trees, for which long generation times are a more significant barrier to breeding and genetic analysis.

Effects of seed-specific expression of a cytokinin biosynthetic gene on canola and tobacco phenotypes.

The Agrobacterium tumefaciens isopentenyl transferase gene (ipt), a cytokinin biosynthetic gene, was placed under the control of 1.9 kb of promoter sequence from the 2S albumin AT2S1 gene isolated from an Arabidopsis thaliana library. The construct was introduced into canola (Brassica napus) and tobacco (Nicotiana tabacum). ipt transcripts were followed during embryo development of transgenic plants by northern hybridizations. The phenotype of transformed plants from the T1 generation was analysed and we observed an increased branching of inflorescences in tobacco and canola plants expressing the ipt gene. Comparing with controls, the average number of capsules and siliques in AT2S1-ipt plants was 82.6 and 24.8% higher, respectively. This result was correlated with an increase in cytokinin levels in transgenic plants, as revealed by RIA. Indeed, cytokinin contents of T1 AT2S1-ipt B. napus seeds were found 2.2-fold higher than cytokinin contents of control seeds, and T1 AT2S1-ipt N. tabacum capsules contained 2.6-fold more cytokinins than control capsules. In tobacco, the average seed weight per capsule was lower in AT2S1-ipt plants while the seed number per silique and the average seed weight were not modified in canola carrying this construct. The average seed yield per plant was not significantly increased in AT2S1-ipt tobacco or canola plants.

Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase ( ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

ORGANELLE-SPECIFIC LOCALIZATION OF IPTASE IN TRANSGENIC NICOTIANA

Nicotiana transformed with the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens was fixed for 1 h in 1% glutaraldehyde and 4% formaldehyde. Ultrathin sections were collected on nickel grids. Grids were treated with polyclonal anti-IPTase antibody raised in rabbits and visualized with 10 nm, protein-A–labeled colloidal gold. Gold label was found throughout the cell, including the cell wall, vacuole, rough ER, and organelles. Cell wall and vacuole labeling appears to be due to non-specific binding and is greatly reduced by a BSA block. Mitochondria and chloroplasts also showed gold label, but not greater than established background levels. Labeling above background levels on the rough ER, free polysomes, and further label in the free cytoplasm indicate a cytoplasmic role for IPTase.

Cytokinin metabolites and gradients in wild type and transgenic tobacco with moderate cytokinin over‐production

A binary T‐DNA plant expression vector carrying a promoterless isopentenyl transferase (ipt) gene was constructed and used to transform Nicotiana tabacum L. Several primary transformants were obtained that displayed a range of phenotypes characteristic of cytokinin over‐production. Two of the transformants with moderately altered phenotypes, both of which produced viable offspring and expressed the ipt gene at a low level, were selected for use in studies of the regulation of cytokinin metabolism. Both lines were found to contain high concentrations of zeatin‐7‐glucoside (Z7G), indicating that Z7G can accumulate in plants even when the rate of endogenous overproduction of cytokinins is low. This supports the hypothesis that 7‐glucosidation is an important step in the regulation of zeatin (Z) levels. Very sharp gradients in concentration of cytokinin riboside and ribotides, related to age of tissue and distance from the apex, were found in both wild type and transfomed plants, which could be important in developmental regulation and could also account for some of the discrepancies between reported cytokinin levels in various plants. Intriguingly, however, although the combined level of zeatin riboside and ribotide was much higher in the transformed plants than in wild type, the combined level of isopentenyl riboside and ribotide was lower.

THE EFFECTS OF LIGHT QUALITY ON ENDOGENOUS CYTOKININ LEVELS IN IPT-TRANSGENIC TOBACCO

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis, seed germination). It is unclear, however, if and how these two systems interact. The coaction between phytochrome and cytokinins was investigated by using Nicotiana plumbaginifolia plants transformed with the isopentenyl transferase (ipt) cytokinin gene and treated with end-of-day (EOD) red (R) and far-red (FR) light. The ipt gene was under control of either a constitutive cauliflower mosaic virus promoter (35S-plants) or an inducible, heat shock promoter (HS-plants). When treated with EOD FR light, whole plants were characterized by decreased chlorophyll concentrations and increased fresh weights. When treated with EOD R light, 35S-plants contained high concentrations of zeatin riboside (ZR) compared to plants treated with EOD FR light. When treated with EOD FR light, HS-plants contained high concentrations of ZR compared to plants treated with EOD R light. Both cytokinin responses were photoreversible. The reasons for the differences between the 35S- and HS-plant responses are not known. Results appear to implicate interactions between phytochrome and cytokinins in plant growth and development.

Transformation of Nicotiana alata Link and Otto. with an Autoregulatory Senescence-inhibitor Gene Construct

Leaf explants of Nicotiana alata Link and Otto. were surface disinfested and cultured on Murashige and Skoog (MS) medium containing 2.66 μm N6-benzyladenine (BA) to promote shoot proliferation. After 5 weeks, proliferated shoots were removed and remaining callus saved. Callus was inoculated with Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase which catalyzes cytokinin synthesis. Following inoculation, the callus was cocultivated for 6 days on BA medium. Selection for transgenics was done on BA medium plus 100 mg Kanamycin and 400 mg Ticarcillin (antibiotics) per liter. Proliferating shoots were rooted on MS medium containing antibiotics. Rooted cuttings were transplanted to soil, acclimated and flowered in the greenhouse. Transgenics were outcrossed to a commercial N. alata hybrid. Seed was germinated in vitro on half-strength MS medium plus antibiotics. Segregation of transgenics to nontransgenics was 1:1. Evaluation of leaf senescence on 5-month-old plants showed 2 to 14 times fewer senesced leaves on the transgenic than the nontransgenic plants.

Cytokinins in plant senescence: From spray and pray to clone and play

AbstractThree approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence‐inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.

PCR amplification of the fas‐1 gene for the detection of virulent strains of Rhodococcus fascians

Thirty‐six isolates of the fasciation‐inducing bacterium Rhodococcus fascians were evaluated for the presence and location of the fas‐1 gene, which codes for an isopentenyl transferase, the committed step in cytokinin biosynthesis. The presence of fas‐1 was determined by PCR using a set of primers to the most conserved regions of the gene and by Southern hybridization to genomic digests using the PCR fragment as a probe. Both methods revealed the presence of the gene in 18 virulent isolates and the absence of the gene in 18 avirulent isolates. Thus, there is a strong relationship between the presence of the gene and virulence of the organism. The location of fas‐1 was determined by probing blots of linear and circular DNA. For most of the virulent isolates, the gene was localized to a 200 ± 10 kb linear plasmid. Three virulent isolates lacked a plasmid of this size, but contained fas‐1 either on a linear plasmid of 130 kb or on a large circular plasmid.