Introduction of genetic material into plant cells.

The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants.

When plant cells decide to divide.

Progression through the cell cycle is central to cell proliferation and fundamental to the growth and development of all multicellular organisms, including higher plants. The periodic activation of complexes containing cyclins and cyclin-dependent kinases mediates the temporal regulation of the cell-cycle transitions. Here, we highlight recent advances in the molecular controls of the cell cycle in plant cells, with special emphasis on how hormonal signals can modulate the regulation of cyclin-dependent kinases.

Crystallization and preliminary X-ray crystallographic analysis of NADPH: azodicarbonyl/quinone oxidoreductase, a plant zeta-crystallin.

Arabidopsis thaliana P1 protein was crystallized using the hanging drop vapor-diffusion method in 0.1 M piperazine-1, 4-bis(2-ethanesulfonic acid) buffer, containing 14% polyethylene glycol 6000 and 0.2 M magnesium acetate at pH 6.5 and 20 degrees C. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a=49.8, b=122.4 and c=149. 9 A. The diffraction data up to 2.9 A were collected by a multiwire area detector.

Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens.

A large number of tobacco SR1 cell clones transformed by the wild-type Agrobacterium C58 have been analysed for the presence of screenable markers such as tumour morphology, opine synthesis and hormone dependence. Distinct phenotypic classes were observed depending upon whether the cell clones were isolated from primary tumours or were obtained via cocultivation of protoplasts. These classes of tobacco SR1-C58 transformants appear to arise from errors in the Ti plasmid (T-DNA) transfer and integration mechanism itself rather than from subsequent T-DNA rearrangements, since 900 subclones, obtained by recloning a wild-type SR1-C58-transformed cell clone, yielded no variation in the phenotypes. A detailed genomic T-DNA analysis showed the presence of characteristic, abnormally short T-DNAs in the teratoma-forming, Acs- class and also in the Nos- class. The abnormal right border in two Nos- clones ends close to a sequence that resembles the normal T-DNA terminus and lies adjacent to the nos promoter, suggesting that this sequence could have functioned as a recognition site directing these particular T-DNA transfers. On the basis of the phenotypic and genomic blotting data it is clear that the short T-DNAs are characteristic of the cocultivation method. Other phenomena causing phenotypic variation, such as the loss of the T-DNA, and the gradual repression of T-DNA gene expression by methylation, are the main causes of aberrations in primary tumours. Moreover, the physical data suggest that early in the transformation cycle of Agrobacterium a replication step of a preselected T-DNA occurs before integration into the plant genome.

Effects of Iron Excess on Nicotiana plumbaginifolia Plants (Implications to Oxidative Stress).

Fe excess is believed to generate oxidative stress. To contribute to the understanding of Fe metabolism, Fe excess was induced in Nicotiana plumbaginifolia grown in hydroponic culture upon root cutting. Toxicity symptoms leading to brown spots covering the leaf surface became visible after 6 h. Photosynthesis was greatly affected within 12 h; the photosynthetic rate was decreased by 40%. Inhibition of photosynthesis was accompanied by photoinhibition, increased reduction of photosystem II, and higher thylakoid energization. Fe excess seemed to stimulate photorespiration because catalase activity doubled. To cope with cellular damage, respiration rate increased and cytosolic glucose-6-phosphate dehydrogenase activity more than doubled. Simultaneously, the content of free hexoses was reduced. Indicative of generation of oxidative stress was doubling of ascorbate peroxidase activity within 12 h. Contents of the antioxidants ascorbate and glutathione were reduced by 30%, resulting in equivalent increases of dehydroascorbate and oxidized glutathione. Taken together, moderate changes in leaf Fe content have a dramatic effect on plant metabolism. This indicates that cellular Fe concentrations must be finely regulated to avoid cellular damage most probably because of oxidative stress induced by Fe.

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts.

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.

Ozone, Sulfur Dioxide, and Ultraviolet B Have Similar Effects on mRNA Accumulation of Antioxidant Genes in Nicotiana plumbaginifolia L.

We have studied the expression of antioxidant genes in response to near ambient conditions of O3, SO2, and ultraviolet B (UV-B) in Nicotiana plumbaginifolia L. The genes analyzed encode four different superoxide dismutases (SODs), three catalases (Cat1, Cat2, and Cat3), the cytosolic ascorbate peroxidase (cyt APx), and glutathione peroxidase (GPx). The experimental setup for each treatment was essentially the same and caused no visible damage, thus allowing direct comparison of the different stress responses. Our data showed that the effects of O3, SO2, and UV-B on the antioxidant genes are very similar, although the response to SO2 is generally less pronounced and delayed. The effects of the different stresses are characterized by a decline in Cat1, a moderate increase in Cat3, and a strong increase in Cat2 and GPx. Remarkably, SODs and cyt APx were not affected. Analysis of SOD and APx expression in the ozone-sensitive Nicotiana tabacum L. cv PBD6 revealed that induction of the cytosolic copper/zinc SOD and cyt APx occurs only with the onset of visible damage. It is proposed that alterations in mRNA levels of catalases and GPx, but not of SODs and cyt APx, form part of the initial antioxidant response to O3, SO2, and UV-B in Nicotiana.

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

Mutations in the pilz group genes disrupt the microtubule cytoskeleton and uncouple cell cycle progression from cell division in Arabidopsis embryo and endosperm.

Organised cell division and expansion play important roles in plant embryogenesis. To address their cellular basis, we have analysed Arabidopsis abnormal-embryo mutants which were isolated for their characteristic phenotype: mutant embryos are small, mushroom-shaped ("pilz") and consist of only one or few large cells each containing one or more variably enlarged nuclei and often cell wall stubs. These 23 mutants represent four genes, PFIFFERLING, HALLIMASCH, CHAMPIGNON, and PORCINO, which map to different chromosomes. All four genes have very similar mutant phenotypes although porcino embryos often consisted of only one large cell. The endosperm did not cellularise and contained a variably reduced number of highly enlarged nuclei. By contrast, genetic evidence suggests that these genes are not required for gametophyte development. Expression of cell cycle genes, Cdc2a, CyclinA2 and CyclinB1, and the cytokinesis-specific KNOLLE gene was not altered in mutant embryos. However, KNOLLE syntaxin accumulated in patches but no KNOLLE-positive structure resembling a forming cell plate occurred in mitotic cells. A general defect in microtubule assembly was observed in all mutants. Interphase cells lacked cortical microtubules, and spindles were absent from mitotic nuclei although in rare cases, short stubs of microtubules were attached to partially condensed chromosomes. Our results suggest that the cellular components affected by the pilz group mutations are necessary for continuous microtubule organisation, mitotic division and cytokinesis but do not mediate cell cycle progression.

Characterization of a pathogen-induced potato catalase and its systemic expression upon nematode and bacterial infection.

We have isolated a cDNA encoding a catalase (Cat2St) by differential screening of a cDNA library constructed from potato roots infected with the cyst nematode Globodera pallida. Expression analysis confirmed the local induction of Cat2St and showed that it was highest at the adult stage of the parasite. It also revealed that Cat2St was induced in uninfected roots, stems, and leaves of infected plants. Localized and systemic induction of Cat2St was also observed upon root-knot nematode (Meloidogyne incognita) and root bacteria (Erwinia carotovora, Corynebacterium sepedonicum) infections. Based on sequence and expression analysis, Cat2St was found to belong to the recently described class II of dicotyledonous catalases, suggesting that these catalase isoforms could also be pathogen induced. Plant-parasitic nematodes are known to induce, in the roots of their hosts, highly metabolic feeding cells that function as nutritional sinks. Whereas the local induction of Cat2St is probably a consequence of an oxidative stress of metabolic nature, the systemic induction of Cat2St shows striking similarities with the induction of systemic acquired resistance (SAR) genes. The possible role of catalase in compatible plant-pathogen interactions is discussed.

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.

A negatively light-regulated gene from Arabidopsis thaliana encodes a protein showing high similarity to blue copper-binding proteins.

A negatively photo-regulated gene (bcb) has been isolated by means of differential hybridization of a genomic library of Arabidopsis thaliana. In mature plants, a 20-fold increase in the amount of steady-state bcb mRNA can be detected upon 48 h of dark adaption. The expression level of the gene is also dependent upon the developmental stage of the plant. The 21.5-kDa gene product (BCB) shows extensive similarity with blue Cu(2+)-binding proteins such as plastocyanin and stellacyanin. The protein abundance increases only twofold upon dark adaption, which implies the presence of post-transcriptional control. The isolation of a novel negatively photoregulated gene allows us to investigate the complex expression profile of genes responding to the absence of light.

One-step purification and characterization of a lignin-specific O-methyltransferase from poplar.

O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH). Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P. deltoides). The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose. The enzyme uses both CA and 5OH as substrates. We previously have reported the cloning of a corresponding OMT cDNA [Dumas et al., Plant Physiol. 98 (1992) 796-797]. Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone. Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome. RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves.

IS-like element IS8 in RP4 plasmid and its involvement in cointegration.

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi::RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.

Isolation of genes expressed in specific tissues of Arabidopsis thaliana by differential screening of a genomic library.

The small genome size of Arabidopsis thaliana allows the isolation of genes expressed in specific tissues and under controlled conditions by the differential screening of a genomic library, as has been shown previously for yeast and Drosophila. cDNA probes, based on poly(A)+ mRNA isolated from different Arabidopsis organs, were used in colony hybridizations with 1145 randomly chosen genomic clones, representing 27,000 kb of Arabidopsis DNA. Twenty percent of the clones containing low-copy-number sequences hybridized with one or more of the cDNA probes that were synthesized from mRNA isolated from leaves, stems, seed pods, inflorescences, callus tissue, and light-grown and dark-grown plants. Comparison of the colony hybridizations led to the identification of a large variety of clones which contain differentially expressed genes. The pattern of expression was confirmed by Northern analysis. The advantage of the described method is that it yields directly genomic sequences that contain specifically expressed or induced genes. In particular, it circumvents the construction and differential screening of cDNA libraries for every tissue or environmental parameter to be analyzed.

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana.

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.

The extensin signal peptide allows secretion of a heterologous protein from protoplasts.

Extensins are hydroxyproline-rich glycoproteins which are amongst the most abundant proteins present in the cell wall of higher plants. Here, we describe the structural analysis of an extensin-encoding gene from Nicotiana plumbaginifolia. The encoded protein (46 kDa) has a highly repetitive structure and contains 37% proline, 18.1% tyrosine, 13.4% lysine, 8.1% serine and 7.1% histidine. The extensin-encoding sequence contains a typical signal peptide for translocation of the protein to the endoplasmic reticulum. By using chimeric genes consisting of different 5' parts of the extensin-encoding gene and the neomycin phosphotransferase II-encoding gene (nptII) as reporter gene, we show that the N-terminal part of extensin can mediate the secretion of NPTII from electroporated N. tabacum protoplasts.

Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia.

A cDNA clone for a hormonally regulated beta-glucanase from Nicotiana plumbaginifolia has been isolated by using an oligodeoxynucleotide probe, synthesized to match the previously determined N-terminal amino acid sequence. The cDNA has the complete sequence of the mature protein and contains at least part of a hydrophobic signal peptide. At the amino acid level, the beta-glucanase of N. plumbaginifolia is 73% homologous to a beta(1,3)-glucanase from tobacco and 52% homologous to a beta(1,3;1,4)-glucanase from barley. Southern-blot analysis clearly demonstrated that N. plumbaginifolia contains at least two related genes encoding beta-glucanase. The extent of the complete signal peptide of the cloned beta-glucanase was determined by sequencing part of the corresponding gene. Northern analysis showed that the expression of the beta-glucanase gene is influenced by auxins and cytokinins.

The involvement of poly(ADP-ribose) polymerase in the oxidative stress responses in plants.

In plants many biotic and abiotic stresses can cause secondary oxidative stress. Earlier work showed that, depending on the severity of the oxidative stress, plants can activate either cell protective genes or programmed cell death (PCD). Poly(ADP-ribose) polymerase (PARP) has been implicated as one of the enzymes in the apoptotic pathways induced by DNA damaging agents or oxidative stress. We show that in cultured soybean cells, PARP is involved in responses to mild and severe oxidative stresses, by mediating DNA repair and PCD processes, respectively. Addition of PARP inhibitors reduced the degree of cell death triggered by H2O2. Two windows of NAD consumption after H2O2 treatment were detected. Experiments with transient overexpression of Arabidopsis PARP cDNA promoted DNA repair and inhibited cell death caused by mild oxidative stress. However, following severe stress PARP overexpression increased cell death. Expression of antisense PARP produced the opposite effects: an increase in DNA nicks and inhibition of cell death at high, but not mild doses of H2O2.

The regulation and function of tobacco superoxide dismutases.

Our knowledge of superoxide dismutase (SODs) in tobacco has increased greatly during the past few years. Genes encoding the four identified SOD isoforms of tobacco have been isolated and characterized. Analysis of promoter-beta-glucuronidase fusions has provided information on the cellular expression of SODs in tobacco and has constituted the basis for studying SOD regulation. Constitutive overproduction of SOD has been shown to confer increased tolerance to stress and has started to reveal subtle biochemical differences between SOD isoforms. Thus, thanks to its convenience for molecular and physiological studies, tobacco has come forth in recent years as an excellent model system for studying the regulation and function of SOD in dicotyledonous plants.