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.     

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.     

Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns

The induction by cytokinin stress and ethylene of nine different tobacco mosaic virus-inducible mRNA classes (termed A-I) encoding pathogenesis-related (PR) proteins was studied. The induced mRNA levels were compared to basal levels in healthy tobacco plants grown in tissue culture and in a greenhouse. Cytokinin stress and ethylene were found to induce different subsets of the mRNAs, indicating that ethylene is not the primary inducing signal in cytokinin-stressed shoots. mRNAs F, H and G encoding the basic hydrolytic enzymes chitinase, -1,3-glucanase and a basic equivalent of PR-1, respectively, were found to be expressed at high levels in roots of healthy plants. mRNAs D, I and B encoding the acidic equivalents of the proteins proved to be present at low levels in healthy plants. These results indicate that genes encoding basic and acidic isoforms of pathogenesis-related proteins are differentially regulated.     

Changes in the tissue-specific prevalence of translatable mRNAs in transgenic tobacco shoots containing the T-DNA cytokinin gene

Two-dimensional gel electrophoresis of in vitro translation products was used to examine differences between the steady state RNA populations of an untransformed tobacco plant line and a non-rooting tobacco shoot line transformed with a T _l -DNA segment from Agrobacterium tumefaciens carrying the cytokinin gene (T-cyt). The analysis comprised about 240 translation products representing the more abundant mRNAs. Approximately 8% of the translation products were found to have significantly different concentrations, due to both increases and decreases, when the shoot parts of the transformed and untransformed lines were compared. Only a few of these differences were specific for the comparison of transformed and untransformed shoots. Most of the differences were also observed when the shoot and root parts of the untransformed line were compared. This implies that the shoot or root prevalence of several mRNA species in normal plants is altered in transgenic T-cyt shoots. The observed changes in the mRNA population of transgenic T-cyt shoots are discussed in relation to the transformed phenotype and previously cloned mRNAs showing similar changes in tissue-specific prevalence.     

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.     

Fruit-specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense-related gene expression in leaves

This paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit-specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100-fold higher than in control fruit. In the leaves of a fruit-bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR-1 and chitinase mRNAs, which encode defense-related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.     

Expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene with the AGPase S1 Promoter in Tomato Results in Unbranched Roots and Delayed Leaf Senescence

Transgenic tomato plants were produced with the isopentenyl transferase gene (ipt) ligated to a promoter that is active exclusively in sink tissue. Initially, transgenic plants had smaller, round-scale leaves, swollen stems, and exhibited early development of lateral shoots compared to wild type. Expression of the ipt gene resulted in the formation of unbranched roots on cuttings and delayed senescence in excised leaves. Callus and root formation occurred on excised leaves and leaf discs during dark incubation. The retention percentage of chlorophyll, as well as cytokinin in excised leaves or discs was significantly greater than wild type. Transgenic tomato fruit had elevated levels of cytokinins in the first days after fruit set and these levels were maintained longer during fruit development.     

Molecular characterization of messenger RNAs for 'pathogenesis related' proteins la, lb and lc, induced by TMV infection of tobacco

A cDNA library was made to poly(A)-containing RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco plants and clones corresponding to mRNAs for the `pathogenesis-related' (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR-1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3'-non-coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR-1a and PR-1c mRNAs. The data indicate an ~90% homology between the amino acid sequences of PR-1a, -1b and -1c. Using one of the clones as probe it was shown that in the TMV-inoculated lower leaves and the non-inoculated upper leaves of a tobacco plant, the PR-1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.     

Expression of <Emphasis Type="Italic">Lupinus albus</Emphasis> PR-10 proteins during root and leaf development

Based on the NH₂-terminal sequence of three PR-10 isoforms previously identified in Lupinus albus leaves and a conserved amino-acid region in the PR-10 proteins from leguminosae, a pair of oligonucleotides was designed and used to amplify the corresponding cDNA fragment from a L. albus leaves cDNA library. A fragment of DNA of 200 bp was isolated from the polymerase chain reaction (PCR) mixture and subsequently used to screen the cDNA library. A cDNA coding for a PR-10 protein of 158 amino acid residues was cloned and sequenced. Subsequent studies involving Northern and Western blot analysis have shown that the PR-10 protein isoforms are differentially expressed during the development of the healthy lupin plant. High mRNA and protein contents were detected in roots and hypocotyls of both 7- and 20-d-old plants. In young leaves, the mRNA and protein contents were low and increasead in mature leaves. Tissue printing experiments with root sections suggest that the proteins are extracellular and are mainly associated with the vascular tissues in mature roots.     

BTH-Induced Accumulation of Extracellular Proteins and Blackspot Disease in Rose

Treatment of rose shoots with 50 µM acibenzolar-S-methyl (BTH) resulted in increased protection against Diplocarpon rosae. This was accompanied by the induction and accumulation of a set of extracellular proteins as shown by SDS-PAGE and 2D-PAGE. Some of these proteins have been identified as PR-1, PR-2, PR-3 and PR-5 proteins by immunoblot analysis probed with tobacco antisera against PR-1c, PR-N, PR-Q and PR-S protein. Most of the extracellular proteins activated by BTH were also induced and found to accumulate in leaves upon infection with Diplocarpon rosae. However, their accumulation was much more pronounced in BTH-pretreated leaves than in water-pretreated leaves upon a challenge inoculation with D. rosae, particularly, the 15 kD PR-1, 36 and 37 kD PR-2 proteins. They may be more important in the expression of disease resistance.     

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the ??cytokinin deficiency syndrome?? were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.     

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots

PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a cDNA library prepared from mRNAs of rice shoots utilizing the product of PCR as probe. Two cDNA clones, prxRPA and prxRPN, were isolated. They encode distinct isozymes of peroxidase. Sequence analysis indicated that the clones encode mature proteins of approximately 32 kDa, both of which possess a putative signal peptide. Comparison of the amino acid sequences of the two rice peroxidases showed that they are about 70% similar to each other but are only 40% to 50% similar to other plant peroxidases. RNA blot hybridization revealed that mRNAs that corresponded to prxRPA and prxRPN cDNAs accumulate at high levels in roots but only at low levels in stems and leaves. In various tissues of rice plants, levels of both mRNAs were stimulated by wounding and by ethephon. These results indicate that at least two isozymes of peroxidase are expressed not only in shoots but also in roots of rice plants, and that the expression of these genes is influenced by ethylene which is the simplest plant hormone.     

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.     

Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid

Cytokinin oxidase plays an important role in the cytokinin regulatory processes. We have cloned a novel putative cytokinin oxidase, DSCKX1 (D endrobium Sonia cytokinin oxidase), by mRNA differential display from shoot apices of Dendrobium Sonia cultured in the presence of BA. The DSCKX1 gene appears to have three alternative splicing forms and its expression of DSCKX1 was induced in a tissue-specific manner by cytokinins. In transgenic orchid plants overexpressing DSCKX1, the elevated level of cytokinin oxidase activity was accompanied by a reduction of cytokinin content. These plants exhibited slow shoot growth with numerous and long roots in vitro. Their calli also showed decreased capability of shoot formation. Conversly, antisense transgenic plants showed rapid proliferation of shoots and inhibition of root growth combined with a higher endogenous cytokinin content than wild-type plants. Thus DSCKX1 appears to play an important role on cytokinin metabolism and the related developmental programmes in orchid.     

Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide

The human CD14, a high affinity receptor for lipopolysaccharides (LPS), is involved in the innate immunity system and the inflammatory response. There is increasing interest in using recombinant approaches to produce purified CD14 protein for therapeutic uses. Plants provide ideal expression systems for the production of recombinant proteins, but the levels of expression of recombinant proteins produced in planta are still not high. To improve expression levels of CD14 the 22-kDa alpha-zein signal peptide (ZSP) from maize was fused to the human CD14 cDNA so that recombinant CD14 could stably accumulate in plant cells. The human CD14 gene and the modified human CD14 cDNA with the 22-kDa ZSP were respectively transformed into tobacco to produce transgenic plants. Western blot analysis confirmed human CD14 accumulation in the transgenic tobacco. The concentration of the recombinant protein in the tobacco leaves was measured by ELISA, and the results suggested that fusion with the 22-kDa alpha-ZSP effectively increased the accumulation of the recombinant protein (rCD14). The concentration of rCD14 in some of the transgenic lines was 19.54???g?g^?1 tobacco leaf (fw), which was about 0.6?% of the total soluble protein. The rCD14 protein showed natural LPS-binding bioactivity by using U937 cells mensuration. Our results suggested that the maize 22-kDa alpha-zein signal peptide could be used to increase the accumulation of recombinant protein in tobacco leaves so that proteins can be produced in abundant biomass.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation

We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar α-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA.