Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

Agrobacterium‐produced and exogenous cytokinin‐modulated Agrobacterium‐mediated plant transformation

Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called ‘crown gall’, via the transfer and integration of transferred DNA (T‐DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)‐induced tumour‐inducing (Ti) plasmid gene, tzs (trans‐zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline‐type A. tumefaciens strains. The loss of Tzs protein expression and trans‐zeatin secretions by the tzs frameshift (tzs‐fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs‐fs mutant with a wild‐type tzs gene restored wild‐type levels of trans‐zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium‐produced cytokinin. Interestingly, although the tzs‐fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans‐zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.     

ipt基因定位表达对转基因烟草育性的影响

杂种优势是生物界的一种普遍现象。作物杂种优势的利用是培育高产、抗逆、优质新品种的重要手段之一。然而 ,常规育种技术获得配套三系的难度很大 ,由此限制了杂种优势利用的发展。近年来应用转基因技术创造雄性不育植株已有不少报道[1- 5] 。有研究表明 ,自然突变的雄性不育株     

Effects on Fertility in Transgenic Tobacco by Localized Expression of ipt Gene

The isopenteryl transferase (ipt) gene from Agrobacterium tumefaciens (Smith et Townsend) Conn was driven under the tobacco TA29 promoter and introduced into tobacco ( Nicotiana tabacum L.) plants by A. tumefaciens mediated transformation. PCR and Southern blot analysis confirmed that the ipt gene has integrated into the genomes of tobacco plants. The expression pattern of this chimeric TA29-ipt gene in the transgenic plants was studied, and the endogenous cytokinin level in different organs was assayed by ELISA method. The results showed that the cytokinin content in the androecium of transgenic plants increased 3-4 times as compared with the control, and some changes of the fertility of the TA29-ipt transgenic plants have been observed.     

Functional analysis of the ver gene using antisense transgenic wheat

The function of ver203 , a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion‐expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S‐promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen‐tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double‐ and single‐stranded probes, and detection of GUS activity by X‐gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.     

Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1

The plant hormone cytokinin plays a major role in regulating plant growth and development. Here we generated cytokinin-reduction Arabidopsis plants by overexpressing a heterologous cytokinin oxidase gene DSCKX1 from Dendrobium orchid. These transgenic plants exhibited reduced biomass, rapid root growth, decreased ability to form roots in vitro, and reduced response to cytokinin in growing calli and roots. Furthermore, the expression of KNAT1, STM, and CycD3 genes was significantly reduced in the transgenic plants, suggesting that cytokinin may function to control the cell cycles and shoot/root development via regulation of these genes.     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

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

Three 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.     

Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo--1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Expression of isopentenyl transferase gene (<Emphasis Type="Italic">ipt</Emphasis>) in leaf and stem delayed leaf senescence without affecting root growth

A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T₂ progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T₂ progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root growth and morphology of the plant were not affected in the transgenic tobacco.     

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.     

Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37.

The trans-zeatin secretion locus (tzs), from the nopaline Ti plasmid of Agrobacterium tumefaciens strain T37, was cloned and the nucleotide sequence determined. This gene is located in the virulence region of pTiT37. The tzs gene is responsible for the secretion of trans-zeatin into bacterial culture medium and in addition has the cytokinin biosynthetic activity, dimethylallylpyrophosphate:AMP dimethylallyltransferase. Sequence analysis showed an open reading frame of 729 nucleotides, capable of encoding a protein of 27,545 daltons. A single new labelled protein of 27,200 daltons was detected in Escherichia coli maxicells expressing the cloned tzs gene. Significant sequence homology was observed between the tzs and the published tmr sequence from pTiT37.     

Water-stress-induced heat tolerance in geranium leaf tissues: A possible linkage through stress proteins?

Evidence is accumulating in favor of a linkage at the cellular level between various abiotic stresses. We conducted a study to evaluate the effect of water stress on the heat tolerance of zonal geraniums, Pelargonium × hortorum cv. Evening Glow. Water stress was imposed by withholding irrigation until pots reached 30% (by weight) of well‐watered controls, and by maintaining the pot weight by additions of water for another 7 days. Leaf xylem water potential (XWP, MPa), relative water content (RWC. %), and heat‐stress tolerance (HST; LT₅₀, defined as the temperature causing half‐maximal % injury based on electrolyte leakage) were measured in control, stressed, and recovered plants. Proteins were extracted from the leaves following the above treatments, and SDS‐PAGE and immunoblotting were performed by using standard procedures. Immunoblots were probed with antibodies to dehydrin and 70‐kDa heat shock cognate (HSC70) proteins. Data indicate that XWP and RWC, respectively, were −0.378 MPa and 92.3% for control plants and −0.804 MPa and 78.6% for stressed plants. Water‐stressed plants exhibited a significant increase in HST compared to control (LT₅₀ of 55°C vs 51°C). Water‐stress‐induced HST was not due to heat acclimation (leaf warming in stressed plants). Data also indicate that water‐stress treatment did not increase freezing tolerance of geranium leaves. Increased HST was associated with the accumulation of several heat‐stable, dehydrin proteins (25–60 kDa), and both cytosolic and ER luminal (BiP) HSC70 proteins. Leaf XWP, RWC, and HST reversed to control levels concomitant with the disappearance/reduction of dehydrins and HSC70 proteins in water‐stress‐relieved plants. The possibility of a cellular linkage between water stress and heat‐stress tolerance is discussed.     

超量表达IPT和KN1基因对转基因烟草生长发育的影响

为了比较异戊烯转移酶基因IPT和玉米homeobox基因KNI超量表达对植物生长发育的影响,将35S：：IPT和35S：：KNI分别导入烟草。观察发现,过量表达IPT和KNI基因对植株再生频率、形态、生长周期和顶端优势等方面的影响均相似,但在生根和开花结籽方面,35S：：IPT转基因植物所受影响更显著。细胞分裂素含量检测表明,35S：：IPT转基因植物叶片中细胞分裂素的含量高于35S：：KNI转基因植物。     

Heat‐shock proteins and cross‐tolerance in plants

Environmental stresses dramatically affect plant survival and productivity. Because plants are immobile, presumably different strategies are required for protection against transient stresses. Under stress, plants synthesize specific proteins, and their accumulation has a role in protecting the tissue from possible damage. An increasing number of studies show the existence of cross‐tolerance in plants: Exposure of tissue to moderate stress conditions often induces resistance to other stresses. Many varied mechanisms explaining the phenomenon of cross‐tolerance have been proposed, and they often, but not always, suggest that specific proteins are induced by one kind of stress and are involved in the protection against other kinds. Although various cross‐protections have been demonstrated in a number of plants, a common mechanism has not been found. This review discusses heat‐shock proteins and their possible roles in protecting the plant under heat and other stresses.     

The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5'-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP

The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process. To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans-zeatin-synthesizing) protein from A. tumefaciens, was cloned and the protein was overproduced and purified. Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5'-phosphate. This suggests that A. tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants.     

The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates.

The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase. Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins. Furthermore, beta-glucosidase activity present in E. coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein. Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity. Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance. These observations shed new light on the control of growth and differentiation in plants by growth factors.