Search

Ethylene plays a key role in the rapid elongation of deepwater rice. We cloned cDNAs corresponding to at least three members of the 1-aminocyclopropane-1-carboxylate (ACC) synthase gene family of deepwater rice ( Oryza sativa ev. Plai Ngam). Genomic DNA gel blot analysis confirmed the presence of a multigene family. Expression patterns were studied by Northern blot on poly(A + ) RNA prepared from plants exposed to abscisic acid, ACC, gibberellic acid (GA), indole-3-acetic acid, jasmonic acid, submergence and wounding. The mRNA levels were suppressed by ACC, GA, submergence and wounding, but remained relatively constant under all other conditions.

Ethylene inhibits hypocotyl elongation in etiolated Arabidopsis seedlings. However, when Arabidopsis was grown in the light in the presence of ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), a marked induction of hypocotyl elongation occurred. This resulted from an increase in cell expansion rather than cell division. The effects of ethylene and ACC were antagonized by the ethylene action inhibitor Ag + . The elongation response was absent or weakened in a set of ethylene-insensitive mutants ( etr1-3 , ein2-1 , ein3-1 , ein4 , ain1-10 , ein7 ). With the exception of ein4 , the degree of inhibition of hypocotyl elongation was correlated with the strength of the ethylene-insensitive phenotype based on the triple response assay. In addition, the constitutive ethylene response mutant ctr1-1 , grown in the light, had a longer hypocotyl than the wild type. Exogenous auxin also induced hypocotyl elongation in light-grown Arabidopsis . Again, the response was abolished by treatment with Ag + , suggesting that ethylene might be a mediator. The results showed that, depending on light conditions, ethylene can induce opposite effects on cell expansion in Arabidopsis hypocotyls.

The early and differential responses of the individual buds along a shoot have remained largely unknown due to the difficulties of analyzing early indicators that allow the monitoring of the effects of subtle changes in the environment on the growth activity of the individual bud. To overcome this problem, we transformed poplar [Populus tremula (L.) x P. alba (L.)] with two chimeric genes, Pcdc2a-gus and PcyclAt-gus, the expression of which is closely linked to cell division in Arabidopsis thaliana (L.) Heynh. We analyzed the expression levels of both chimeric genes in individual buds of the same tree, and under different conditions known to promote or retard growth in the buds. The expression levels of both chimeric genes were found to reflect closely the growth activity of the buds. After decapitation of the shoot, the expression of Pcdc2a-gus and PcyclAt-gus revealed rapid and selective changes in the cell cycle, even when no morphological changes were observed. Furthermore, on the basis of the expression of the chimeric cell cycle genes, different degrees of growth activity and dormancy could be discriminated in the axillary buds. In addition, the expression of PcyclAt-gus was found to be closely associated with the day length, which is critical for dormancy induction in poplar.

We report a modification of a previously published procedure (de Almedia Engler et al., 1994) aimed at the fluorescent detection of RNA-RNA hybrids. Transcripts ofARAth;Cyc1;1 were located in whole mounts of roots ofArabidopsis thaliana afterin-situ hybridization with digoxigenin-and biotin-labeled antisense probes. Digoxigenin- and biotin-labeled hybrids were detected by FISH with an AP-conjugated antibody against digoxigenin or AP-conjugated streptavidin for detection of biotin, with the UV-excitable dye ELF™ as a substrate. Alternatively, biotin-labeled hybrids were detected with HRP-conjugated streptavidin, with fluorescein tyramine as a substrate. Images were recorded via confocal microscopy. We report on the different fixation and reaction conditions needed for obtaining the highest signal-to-noise ratio, as well as on the requirements for mounting media, filter sets, excitation wavelengths, and recording modes used for recording confocal data sets.

An efficient and reproducible Agrobacterium-mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria, which were pretreated in vir gene-inducing conditions, were infiltrated into complete leaf tissue. Histochemical β-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the induced bacteria entered into the intercellular spaces, thus cnabling T-DNA transfer in all cell layers of the leaf. The protocol was optimized for Phascolus vulgaris leaves, but similar results were obtained with other plant species, such as Phaseolus acutifolius, poplar, and tobacco. A β-glucuronidase chimeric gene interrupted by an intron was used as a marker for histological detection of the sectors.

Chloroplasts of Nicotiana tabacum have two superoxide dismutases: a Fe- and a CuZn-containing enzyme, encoded by the nuclear genes sodB and sodCp, respectively. As a first step in studying the physiological function of these two enzymes, we compared the expression of sodB and sodCp in different plant organs, in response to hormonal treatments, and upon treatment with paraquat and Norflurazon. The sodCp transcript and active enzyme were detected only in young leaves of mature plants. The sodB transcript was more abundant in young compared to old leaves, but the enzymatic activity was higher in mature and senescent leaves. sodCp and sodB exhibited a different expression pattern upon treatment with abscisic acid, indole-3-acetic acid, kinetin, gibberellin, and 1-aminocyclopropane-1-carboxylate. Paraquat treatment caused a decrease in abundance of both transcripts, although the dose dependency of this decrease differed. Norflurazon-induced photooxidation resulted in a 10-fold increase of sodCp mRNA whereas the sodB transcript level was 25% higher than the control. These differences in expression might explain why both plastid-located superoxide dismutase enzymes are needed, particularly under stress conditions.

Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA. Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs. We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages.

Gravitropic bending of plant organs is mediated by an asymmetric signaling of the plant hormone auxin between the upper and lower side of the respective organ. Here, we show that also another plant hormone, gibberellic acid (GA), shows asymmetric action during gravitropic responses. Immunodetection using an antibody against GA and monitoring GA signaling output by downstream degradation of DELLA proteins revealed an asymmetric GA distribution and response with the maximum at the lower side of gravistimulated roots. Genetic or pharmacological manipulation of GA levels or response affects gravity-mediated auxin redistribution and root bending response. The higher GA levels at the lower side of the root correlate with increased amounts of PIN-FORMED2 (PIN2) auxin transporter at the plasma membrane. The observed increase in PIN2 stability is caused by a specific GA effect on trafficking of PIN proteins to lytic vacuoles that presumably occurs downstream of brefeldin A-sensitive endosomes. Our results suggest that asymmetric auxin distribution instructive for gravity-induced differential growth is consolidated by the asymmetric action of GA that stabilizes the PIN-dependent auxin stream along the lower side of gravistimulated roots.

In Arabidopsis the promoter of the gene encoding S-adenosyl-L-methionine synthetase (SAM-S) Psam-1 confers expression preferentially in the vascular tissue. In search for promoters that drive expression in particular cells of the lignifying tissues in trees, we have analyzed the expression pattern conferred by the Psam-1 promoter in transgenic poplar. Histochemical analyses demonstrated beta-glucuronidase (GUS) activity mainly in phloem and cortex tissue throughout the plant, and in root tips. Fluorimetric assays showed high GUS activity in the tissues outside (phloem, cortex and cork) compared to those inside (xylem and pith) of the cambial layer. In contrast, the endogenous SAM-S activity was high in tissues inside and low in tissues outside of the cambial layer. RNA gel blot analysis demonstrated a high transcript level of the endogenous sam-s gene(s) in tissues both outside and inside the cambial layer. This indicates that the low SAM-S activity in the bark was at least partially due to translational and/or post-translational regulation of the endogenous sam-s gene(s). In dormant transgenics, the tissue specificity was conserved, but the activity levels were up to 10-fold reduced.

Artemisinin is a new very promising antimalarial compound produced in the areal parts of the plant Artemisia annua L. (Asteraceae). There is a great interest in the overproduction of artemisinin by means of transgenic plants. The existing Agrobacterium tumefaciens-mediated transformation procedure uses the expensive decontaminating agent vancomycin. In addition vancomycin exhibits a low activity against the Agrobacteria. This paper describes the use of the penicillin derivatives amoxicillin and ticarcillin in combination with the β-lactamase inhibitor clavulanic acid, on the market as Augmentin® and Timentin® (Smith Kline Beecham Pharma S.A., Genval, Belgium), respectively. Sensitivity studies revealed that those antibiotics still permit plant regeneration. The leaf disc transformation was performed with the C58C1 (pGV2260; pTJK136) Agrobacterium strain. Transgenic tissue was selected on 20 mg l−1 kanamycin. The integration of the foreign genes was proven by polymerase chain reactions and additional DNA sequencing. β-Glucuronidase gene expression analyses were performed. In all cases transgenic callus could be obtained, but the shoot regeneration failed. Decontamination was most efficient when high initial concentrations of Augmentin® and Timentin® (300 and 200 mg l−1, respectively) were used. Those concentrations can gradually be reduced. We may conclude that decontamination by the penicillin derived antibiotics is cheaper and more efficient than vancomycin, but is only suitable when the production of callus is desired, for example for the establishment of cell cultures.

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon, The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch, Nod factors produced by Tn5-insertion mutants in nodZ, noeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry, Fucosylation of Nod factors depended on both nodZ and nolK, Arabinosylation depended on noeC and/or downstream genes, Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia cell, the fucosyltransferase activity was ascribed to the protein encoded by nodZ, In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.

We describe here a simple and rapid capillary electrophoresis method for the determination of ascorbic acid (L-AA) and isoascorbic acid (D-AA) in vegetative tissues. For optimal yields and stabilization, samples are extracted with cold 3% metaphosphoric acid. Hydrophobic contaminants are then removed by passage through a C18 solid-phase extraction cartridge. The analysis itself is performed on a fused silica capillary with 200 mM borate, pH 9, as the carrier electrolyte, using on-line diode array detection over the range 190-350 nm. Quantitation was performed at 260 nm, the uv-absorption maximum for ascorbate at this pH. This method has a minimum detection limit of 84 fmol/injection and linearity of detector response was observed up to at least 12 pmol/injection. We also describe the influence of electrolyte concentration, pH, and the presence of detergent on separations of L-AA, D-AA, and L-galacturonic acid-1,4-lactone. The protocol has been demonstrated to be suitable for the analysis of L-AA in Arabidopsis, parsley, and mushroom. The method has superior resolution to comparable HPLC separations, a comparable analysis time, but lower sensitivity because of the concentration limitations of the detection system.

Lipo-chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell-associated and secreted radioactively labelled Nod factors in wild-type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild-type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5 alpha. The strain DH5 alpha (pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans. nodl or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nodlJ-encoded transporter showed a specificity for more hydrophilic LCOs.

The molecular interactions between plants and sedentary nematodes are undergoing intense study, not only for reasons of fundamental research but also for the potential benefits to agriculture. The present technology allows the transformation of an increasing number of crop plants, providing new ways to introduce resistance against plant-parasitic nematodes. The ability of sedentary nematodes to induce specialized feeding sites in plant roots is one of the most fascinating aspects of this host-parasite interaction. Molecular approaches have been initiated to identify and characterize plant genes altered in expression after infection by sedentary nematodes. The results obtained indicate that many genes indeed become up-regulated upon nematode infection. Surprisingly, several so-called constitutive promoters that are normally used to achieve high expression in plant cells are completely 'silenced' in the feeding sites within days after nematode infection. Generally, there are two options available for the genetic engineering of nematode resistance: the synthesis of anti-nematode proteins or the localized production of a cytotoxic protein that interferes with the development of feeding cells. Nematode-induced promoters are very useful for the production by plants of sufficiently high levels of anti-nematode proteins at feeding sites. Alternatively, interfering with feeding-cell development is somewhat similar to the hypersensitive response evoked by nematodes in a naturally resistant plant. Here, destruction of specific plant cells can be achieved by the localized expression of a cytotoxin such as barnase, a potent ribonuclease. This approach, however, calls for a highly specific 'non-leaky' promoter, which is active only in the feeding cells. Another possibility is to use a two-component system, where the leakiness of the promoter in other tissues is counterbalanced by the constitutive expression of a neutralizing gene.

The in planta localization of a full-size immunoglobulin G (IgG) antibody and of its derived F ab fragment were determined using immunogold electron microscopy on transgenic Arabidopsis thaliana plants. Although it is widely assumed that antibodies, following assembly in the lumen of the endoplasmic reticulum, migrate via the secretory pathway to the cell surface, until now their subcellular in planta localization has not been demonstrated. Here, we show that both IgG and F ab fragment accumulate within intercellular spaces of leaf mesophyll cells of Arabidopsis . Using immunoblot analysis on leaf intercellular fluid, we demonstrate that these microscopically detected proteins are integral and functional antibodies or F ab fragments. The fact that a full-size IgG is not physically restricted from passage through the cell wall, despite having a molecular mass of 146 kD, questions the generally accepted limitations in cell wall porosity. These results are of particular significance in any plantibody approach that intends to modulate extracellular antigen function.

Summary Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.

By in vitro and in vivo studies with Escherichia coli expressing different combinations of the nodABCS genes of Azorhizobium caulinodans, Nod factor intermediates were identified and their structures determined using mass spectrometry. Substrate-product relationships were studied by time course experiments, and the Nod factor biosynthetic pathway was partially resolved. E. coli strains, harboring nodA and/or nodB, did not produce Nod metabolites, whereas the strain expressing nodC produced chitooligosaccharides. Thus, the first committed step was the production of the carbohydrate backbone. Bacitracin and tunicamycin did not affect this step, suggesting that undecaprenyl pyrophosphate-linked intermediates were not involved. The second step was the deacetylation of chitooligosaccharides by NodB since the E. coli strain expressing nodBC produced chitooligosaccharides, deacetylated at the non-reducing end and since the NodC products were precursors of the NodBC products. A strain expressing nodBCS produced N-methylated oligosaccharides, whereas a strain expressing nodCS produced unmethylated oligosaccharides. Time course experiments showed that methylation occurred after deacetylation. Thus, NodS acted after NodB. The NodBCS metabolites were partially converted to lipo-chitooligosaccharides when the nodABCS genes were expressed, showing that NodA was involved in the acylation and acted after NodS.

Until now most research, and its funding, has been focused on animal and human health care as well as simple microbiological model systems such as Escherichia coli and yeast. Molecular plant studies have generally lagged behind, often simply adapting discoveries from the animal field to plants. Clearly, good health and the efficient tackling of diseases is crucial for the well-being of humans, and good remedies have a high economic value for the pharmaceutical industry. However, one should not forget that plants are an essential component of the large ecosystem that is our planet. They are not only the basic food producers but they are also necessary for a balanced atmosphere (oxygen production) and stable and viable climates. Especially in this period of demographic explosion and growing environmental deterioration, there is a need to rebuild our agricultural systems. Plants also have a wide variety of 'non-food' uses, for instance as energy sources, construction materials, or cosmetics. Last, but not least, they produce a lot of chemicals that can be used as pharmaceuticals. The growing awareness of the importance of plants has coincided with the development of plant molecular biology. Specific features make them ideally suited for gene engineering and genetic studies in general.

A full-length cDNA and the corresponding At-P5S gene encoding the first enzyme of the proline biosynthetic pathway, the Δ 1 -pyrroline-5-carboxylate (P5C) synthetase, were isolated in Arabidopsis thaliana . The At-P5S cDNA encodes a protein of 717 amino acids showing high identity with the P5C synthetase of Vigna aconitifolia . Strong homology is also found at the N-terminus to bacterial and yeast γ-glutamyl kinase and at the C-terminus to bacterial γ-glutamyl phosphate reductase. Putative ATP- and NAD(P)H-binding sites are suggested in the At-P5S protein. The transcribed region of the At-P5S gene is 4.8 kb long and contains 20 exons. Southern analysis suggests the presence of only one At-P5S gene in the A. thaliana genome mapped at the bottom of the chromosome two. Expression analysis of At-P5S in different organs reveals abundant At-P5S transcripts in mature flowering plant. Rapid induction of the At-P5S gene followed by accumulation of proline was observed in NaCl-treated seedlings suggesting that At-P5S is osmoregulated.

A rise in the level of endogenous salicylic acid (SA) during flowering of the thermogenic voodoo lily, Sauromatum guttatum, leads to a pronounced temperature elevation by stimulation of the alternative respiratory pathway. We have studied the thermal response of tobacco (Nicotiana tabacum L.) leaves, a non-thermogenic tissue, to exogenous SA, and its relation to alternative respiration. A reproducible increase in surface temperature of 0.5–1.0°C was registered with high-resolution infrared cameras. The same phenomenon was observed when 2,6-dihydroxybenzoic acid, an active analogue of SA, was used. Non-active SA analogues, such as 3- and 4-hydroxybenzoic acid, did not induce thermogenicity. The thermal effect of SA was abolished with inhibitors of the alternative pathway, such as salicylhydroxamic acid and propyl gallate. Polarographic measurement of the respiratory activity, including that of the alternative pathway in SA-treated plants, showed a significant increase of both total respiration and the alternative pathway compared with non-treated controls. Therefore, we postulate that, as in thermogenic species, SA enhances the activity of total respiration and of the cyanide-resistant pathway in tobacco leaves, subsequently leading to an elevation in surface temperature.

Ivan Couee [1] suggests that our article ‘Fatal attraction: the intuitive appeal of GMO opposition’ [2] defines the societal debates about biotechnology as ‘a battlefield between rationality and irrationality’. Instead, he proposes ‘a framework of mutual respect and interest between citizens and scientists’. However, we believe that this is a false dilemma. While we endorse his plea for mutual understanding, we think that comprehending how concerns and beliefs about GMOs arise from untrustworthy sources facilitates, rather than impedes, the development of a conciliatory framework.

The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana , SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.

A procedure is presented for the detection of mRNA in whole-mount preparations of youngArabidopsis seedlings using digoxigenin (DIG)-labeled RNA probes. It includes tissue preservation with formaldehyde, permeabilization with polyoxyethy lenesorbitan (Tween 20), DMSO, and heptane. Hybridization signal is detected using colloidal gold or alkaline-phosphatase-conjugated anti-DIG antibodies.

Nicotiana plumbaginifolia Viv. harbors a single extensin gene, although related hydroxyproline-rich sequences are present in the genome. Northern analysis showed that the gene is highly expressed in roots and to a lesser extent in stems. Expression in leaves is low but mRNA levels are increased upon infection with the incompatible bacterium Pseudomonas syringae. Extensin transcript levels in leaves were slightly enhanced after wounding and salicylic acid treatment. In-situ hybridization experiments showed high accumulation of extensin mRNA in cells which, at certain stages of development, require reinforcement of their cell walls. The cortical cells in stem nodes and roots, which are put under severe mechanical stress by adjacent developing tissues, tend to express the gene to high levels. Immunolocalization of the extensin protein in stems and roots demonstrated a close association of the protein with lignin deposition. Mature tissues contained more extensin than younger tissues. The extensin promoter was fused to the beta-glucuronidase gene.

Arcelins are seed storage proteins present in some wild bean accessions (Phaseolus vulgaris). They are implicated in the resistance phenotype of these wild beans towards the Mexican bean weevil. Arcelin 5, one of six arcelin electrophoretic variants, has been characterised in detail. The purified arcelin-5 protein fraction contained two major polypeptides of 32.2 and 31.5 kDa, designated arcelin 5a and arcelin 5b, respectively, and one minor polypeptide of 30.8 kDa, designated arcelin 5c. The three polypeptides have an identical isoelectric point and are identical for their first nine N-terminal amino acids. Arcelin 5a and arcelin 5b are glycoproteins whereas arcelin 5c is not glycosylated. Native arcelin 5 has a molecular mass corresponding to a dimer form. Using amino acid sequence analysis and PCR techniques, two different arcelin-5 cDNA sequences were obtained, designated arc5-I and arc5-II. Both encode proteins of 261 amino acids with a signal peptide of 21 amino acids. The identity between the two is 99% at the DNA level and 97% at the level of the deduced amino acid sequences. The arc5-I and arc5-II cDNAs encode arcelin 5a and arcelin 5b, respectively. Sequence comparisons and protein characteristics show clearly that arcelin 5 is related to, but distinct from, other arcelin variants and lectins of P. vulgaris.

Summary A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.

An isocitrate lyase (Icl)-encoding gene (icl) from the Gram+ plant pathogen Rhodococcus fascians was identified serendipitously as part of a scrambled fragment after shotgun cloning in the promoter probe vector, pDP1. The Icl protein is 429 amino acids long (47.11 kDa) and has a predicted pI of 4.84; it is 54% similar to the Escherichia coli Icl and 24–27% to eukaryotic homologues. Comparison of the prokaryotic and eukaryotic Ici confirms the earlier proposal of Matsuoka and McFadden [J. Bacteriol. 143 (1988) 4528–4536] that the enzyme has enlarged during evolution.

We have characterized several heat-shock-induced genes in rice (Oryza sativa L.) and compared their expression under a variety of conditions. Three of these genes, which are analogs of the hsp82/90 family, lie within a cloned 18-kilobase (kb) region of the genome. The middle member of this cluster, designated hsp82B, has been fully sequenced. The gene uses a promoter containing six putative heat-shock elements as well as several unusual sequence motifs including a stretch of 11 thymidines alternating with 11 adenosines. The mRNA for this gene reaches its highest relative level of expression within 120 min after plants are shifted to 42 degrees C; no other conditions induce this gene. By contrast, we found that during heat stress the expression of hsp70 correlates well with increases in internal ion concentrations, and can also be induced by excess salt or ethanol at normal growth temperatures. These results appear to indicate that whereas hsp70 is induced by all stresses that lead to protein denaturation-including heat stress-HSP82 mRNA accumulates only upon heat stress.

Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally ...

Popular advice for healthy diets that may promote health and longevity include the daily consumption of at least three servings of fruits or vegetables and the variation of foods to include items derived from different plants and those plants should belong to different botanical families (Thompson H.J. et al., 1999). Ancient civilisations in the Middle East and in America included grain legumes and cereals in well-balanced diets. In the funeral offerings found in the Egyptian pyramids various legume seeds were present, including lentils and grass pea. Apparently, legumes were a food of special consideration to be offered to kings, in contrast to the present day reputation of being the meat of the poor, with 75% of all legumes now being produced in developing countries. Excavations of ancient settlements indicate the use of both cereals and legumes (Mahler-Slasky & Kislev, 2010). A well balanced food basket promoting health and strength may have given an evolutionary advantage. The benefits of legume cultivation for soil fertility were already recognised in the 4th century BC (Flint-Hamilton, 1999). Legumes are important factors in the natural cycle of nitrogen, being able to fix atmospheric nitrogen in symbiosis with Rhizobium bacteria. This enables the leguminous plants to thrive on poor soil, which makes them essential partners in the maintenance of soil fertility, and to produce protein-rich seeds. However, maintenance of optimum rates of nitrogen fixation requires continued attention by plant breeders (Provorov & Tikhonovich, 2003). Legumes are also unusually diverse in their defence against predators by producing a large array of secondary metabolites forming their chemical armoury. Those metabolites include anti-nutrients such as inhibitors of digestion and compounds interfering with predator’s metabolism reaching as far as brain function and hormonal control (Rozan et al., 2000). Interestingly, some of these metabolites are beneficial by their inhibition of human cancer cells or by antioxidant activity that can delay ageing. Although legumes have many beneficial properties, they are not a well balanced food by themselves because of deficiencies in some essential amino acids, and should not be the sole component of the food basket. In combination with cereals that are richer in those essential amino acids which are deficient in legumes such as methionine, cysteine and tryptophan, legumes are beneficial for human health and for the world’s ecology. The optimum protein quality is approximated when 60-70% cereals are mixed with 30-40% cooked legumes. This would produce a combined quality of protein comparable with meat (Bressani & Elias, 1974).

Plastids are DNA-containing organelles unique to plant cells. In Arabidopsis , one-third of the genes required for embryo development encode plastid-localized proteins. To help understand the role of plastids in embryogenesis and postembryonic development, we characterized proteins of the mitochondrial transcription termination factor (mTERF) family, which in animal models, comprises DNA-binding regulators of mitochondrial transcription. Of 35 Arabidopsis mTERF proteins, 11 are plastid-localized. Genetic complementation shows that at least one plastidic mTERF, BELAYA SMERT' (BSM), is required for embryogenesis. The main postembryonic phenotypes of genetic mosaics with the bsm mutation are severe abnormalities in leaf development. Mutant bsm cells are albino, are compromised in growth, and suffer defects in global plastidic gene expression. The bsm phenotype could be phenocopied by inhibition of plastid translation with spectinomycin. Plastid translation is essential for cell viability in dicotyledonous species such as tobacco but not in monocotyledonous maize. Here, genetic interactions between BSM and the gene encoding plastid homomeric acetyl-CoA carboxylase ACC2 suggest that there is a functional redundancy in malonyl-CoA biosynthesis that permits bsm cell survival in Arabidopsis . Overall, our results indicate that biosynthesis of malonyl-CoA and plastid-derived systemic growth-promoting compounds are the processes that link plant development and plastid gene expression.

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H 2 O 2 -induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.

The present millennium has started with unprecedented global menaces with serious implications for mankind. The management of the planet's resources, the consequences of climate change, the problems generated by the food crisis require prompt actions. Actions at political and managerial level that take into account the contributions that science and technology can bring. The main challenges are: food and feed security; a much more sustainable agriculture; improved cash crops as raw material for the chemical and manufacturing industry; and, above all, actions for the preservation of the last surviving wildlife areas. The challenge is to produce better and more. The millennium goals are far from met. The number of undernourished people is reaching 1 billion. We need to produce more, to fulfill the demand of diversified agricultural products, and to guarantee a decent income to the farmers in the developing and emerging countries. To produce better, to satisfy sanitary and environmental requirements, biotechnologists have developed prototype plants that take up fertilisers more efficiently, need less irrigation and are more resistant to biotic and abiotic stresses. It is our mission to ensure that this knowledge is used to a wide range of breeding programmes, to generate the crops of the future. Despite the enormous increase of our knowledge on plant genomes, their dynamics and evolution as well as on gene expression and its link to agronomic traits, we have seen that the best of plant sciences cannot help if society is not confident in the technology. Every effort should be made in creating awareness on how plant biotechnology can play a major role in meeting the main environmental and nutritional challenges we are facing. Society's support of the technology is needed for rationalising and harmonising the regulatory and biosafety policies which presently stop all introductions of transgenic plants by SMEs. It is the duty of the scientists in the public sector to explain to society and to policy makers the important benefits of these novel achievements in plant sciences to the economy, the environment and the global well-being of our societies.

Armin Braun, a plant geneticist in the 1950–60’s at the Rockefeller, was the first to provide experimental evidence for tumor reversion in plants. Through setting up his experiments in plants, Braun made of tumor reversion a philogenetically conserved system. With today’s understanding of the tumor cell plasticity, pluripotentiality and stemness, Braun’s words spelt 50 years ago sound almost magic: “results of this study indicate that the capacity of teratoma tissue of single cell origin to organize is a reflection of the inherent potentialities of pluripotent tumor cells... clones of teratoma tissue of single-cell origin developed organized structures ... a controlled recovery of crown-gall tumor cells could be accomplished”. However, Braun’s findings have never been explained at the molecular level, and even their malignant phenotype remains to be explored. By studying tumor reversion in human cancer cells, we found that Translationally Controlled Tumor Protein (TCTP) is a key gene that needs to be switched off in order to quit the malignant process and revert. Could TCTP be the molecular link to Armin Braun’s findings? Translationally Controlled Tumor Protein (TCTP) knockout in mice causes embryonic lethality, showing that this gene is essential for development. Most interestingly, Brioudes et al. recently reported that TCTP knockout in plants also resulted in a lethal phenotype. TCTP-lacking adult plants could be rescued by “feeding” the embryos, and the same phenotype, i.e., small size plants, were observed using the RNAi approach. Importantly the KO phenotype Could TCTP contribute to Armin Braun’s paradigm of tumor reversion in plants?

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.

There is increasing evidence that the mechanisms controlling the eukaryotic cell cycle are regulated by protein phosphorylation/dephosphorylation cascades. The catalytic subunit of the protein phosphatase 1 is implicated genetically and biochemically in the complex network that regulates mitosis. To investigate further the cell division in plants, we have isolated and characterized two full-length cDNAs from Arabidopsis thaliana, PP1A-At1 and PP1A-At2, encoding polypeptides highly homologous to known protein phosphatase 1 (PP1). DNA gel blot analysis suggests that the protein phosphatases 1 might form a small gene family in Arabidopsis. Northern analysis shows that transcripts are present in all plant organs. In cell cultures, the PP1 mRNA levels are differentially affected by treatment with drugs that block cell division. The expression of PP1A-At1 in a Schizosaccharomyces pombe cdc25ts/wee1- double-mutant strain restores temperature sensitivity, showing that the Arabidopsis phosphatase gene is capable of interacting with genes that regulate the fission yeast mitotic apparatus. However, the dis2-11 S. pombe strain, which has a cold-sensitive allele of the phosphatase 1 gene, is not rescued by expression of the PP1A-At1 gene, suggesting that the plant cDNA is not a functional homolog of the fission yeast gene.

We have established a procedure for automated, kinetic analysis of β-glucuronidase (GUS) activities using a colorimetric or fluorometric microtiter plate reader connected to a computer that directs the measurements and accesses the data. Compared with end-point measurements, the procedure saves time, is more accurate, and needs 20 times less material. It allows a more precise determination of GUS activities over a range of 400,000-fold, with a limit of detection of about 0.01 units of GUS per mL in the colorimetric assay and 0.1 milliunit of GUS in the fluorometric assay. A general protocol for the determination of GUS activities in transgenic plant tissue was worked out and applied to investigate the expression of a chimeric β-glucuronidase gene in stably transformed tobacco calli.

Summary The crucifer Arabidopsis thaliana is an excellent model plant to study the molecular regulation of cell division. Here, we describe the state of the art of cell cycle research in this organism. The expression of the key regulatory gene, cdc2, has been shown to be correlated with cell division and with the cellular competence to divide. Furthermore, plant hormones, such as auxins and cytokinins, have intriguing cell—specific effects on the expression of the cdc2 gene. The results are discussed in view of future developments.

Plant cytokinesis deploys a transport system that centers cell plate-forming vesicles and fuses them to form a cell plate. Here we show that the adaptin-like protein TPLATE and clathrin light chain 2 (CLC2) are targeted to the expanding cell plate and to the equatorial subregion of the plasma membrane referred to as the cortical division zone (CDZ). Bimolecular fluorescence complementation and immunodetection indicates that TPLATE interacts with clathrin. Pharmacological tools as well as analysis of protein targeting in a mutant background affecting cell plate formation allowed to discriminate two recruitment pathways for TPLATE and CLC2. The cell plate recruitment pathway is dependent on phragmoplast microtubule organization and the formation and transport of secretory vesicles. The CDZ recruitment pathway, on the other hand, is activated at the end of cytokinesis and independent of trans-Golgi–derived vesicle trafficking. TPLATE and CLC2 do not accumulate at a narrow zone central of the CDZ. We have dubbed this subdomain the cortical division site and show that it corresponds precisely with the position where the cell plate merges with the parental wall. These data provide evidence that the plasma membrane is subject to localized endocytosis or membrane remodeling processes that are required for the fusion of the cell plate with a predefined region of the plasma membrane.

Endocytosis is crucial for various cellular functions and development of multicellular organisms. In mammals and yeast, ADP-ribosylation factor (ARF) GTPases, key components of vesicle formation, and their regulators ARF-guanine nucleotide exchange factors (GEFs) and ARF-GTPase-activating protein (GAPs) mediate endocytosis. A similar role has not been established in plants, mainly because of the lack of the canonical ARF and ARF-GEF components that are involved in endocytosis in other eukaryotes. In this study, we revealed a regulatory mechanism of endocytosis in plants based on ARF GTPase activity. We identified that ARF-GEF GNOM and ARF-GAP VASCULAR NETWORK DEFECTIVE 3 (VAN3), both of which are involved in polar auxin transport-dependent morphogenesis, localize at the plasma membranes as well as in intracellular structures. Variable angle epifluorescence microscopy revealed that GNOM and VAN3 localize to partially overlapping discrete foci at the plasma membranes that are regularly associated with the endocytic vesicle coat clathrin. Genetic studies revealed that GNOM and VAN3 activities are required for endocytosis and internalization of plasma membrane proteins, including PIN-FORMED auxin transporters. These findings identified ARF GTPase-based regulatory mechanisms for endocytosis in plants. GNOM and VAN3 previously were proposed to function solely at the recycling endosomes and trans -Golgi networks, respectively. Therefore our findings uncovered an additional cellular function of these prominent developmental regulators.

T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.

OXIDATIVE STRESS . . . . . . . . . . . . . .. . . . . .... . . . . .. . . . . .. . . . ... .. . . . . .... . . .. .. . . . . . ... . 84 RESPONSE OF SOD TO ENVIRONMENTAL STRESS . . . . . . . . . . . . . . . .. . . . .... . . .... . . . . . . 87 Photoinhibition . . . . . . . . . . .. . ... . . .. . . . .. . . . . ..... . . . , ... ", , ... ,' , ... . ,., . . . "" . .. . ,'.' . . ,' . . . . , ., 87 Paraquat and Other Herbicides . . .. , . . .... , ..... , . . . . ... . . . .. . . . . . . . . . . . . ,.. 91 Atmospheric Pollutants . . " .... , .... , " .... " , , . . , .... , ' 94 Waterlogging and Drought .. ", . . ... , . . . . . . , .. "", . . ", . . . . . ", . . . .. ", . . .. , . . . ... """", . . " 97 The Defense Response to Pathogens . ... . . . . "" ... "" .... " ...... " ,."" .... . , 98 The Phenomenon of Cross-Tolerance . . . , , .. . . . . ...... . . , . . . . . . , . . .... , .. . . . . . . . .. .. 101 The Mechanism of SOD Regulation ........ , , ...... , " 102 GENETIC ENGINEERING OF SOD IN PLANTS .. ' ...... ' . . . . . . . . . . . . . . . . . . . . . . . .. . . . , .... ' 104 Cu/ZnSOD and MnSOD Overexpression . . . . . .. . . . . . , . . . . . . .. . . . . . .. . . . . . , .... . . . . .. . . ,.... . . 105 Prospects for Stress Tolerance through Genetic Engineering of SOD . . . . ,,, .. , . . . . . . . 106

Summary We have isolated the phosphoglycerate kinase gene (pgk) of Trichoderma viride and characterized its expression. Comparison of genomic and cDNA clones allowed the correct deduction of the intron boundaries and the 3′-end cleavage site of this gene. Primer extension analysis showed that transcription initiated at three start points between -296 and -298bp upstream of the translational start codon. The promoter sequence contained a number of cis-acting sequences commonly found in eukaryotic promoters. The pgk transcript analysis of T. viride grown on defined carbon sources showed that neither rate nor growth phase greatly affects pgk expression. By contrast, when Trichoderma spp. were grown in the presence of cell walls of a phytopathogenic fungus as carbon source, pgk messenger levels dropped markedly. This suggests that pgk mRNA accumulation is specifically repressed in the simulated mycoparasitic state.

Nodulation of leguminous plants by Rhizobium, Bradyrhizobium, and Azorhizobium spp. is dependent on the induction by the plant host of different bacterial nodulation (nod) loci. The transcription of these nod loci is activated in the presence of plant-produced flavonoids upon binding of the NodD protein--a LysR-type activator--to specific sequences present in the nod promoters. Originally, a 47-base-pair (bp) region called the nod box was shown to be the target sequence for binding of NodD. From the comparison of the nod box sequences of (brady)rhizobia with a more divergent nod box from Azorhizobium caulinodans, we now propose a modular build-up of the nod box with the sequence A-T-C-N9-G-A-T as the binding target of the NodD protein (the NodD box). More generally, we show that LysR-type-regulated promoters contain the characteristic sequence T-N11-A as the core of an inverted repeat and propose this to be the "LysR motif" involved in specific binding to LysR-type proteins. Data obtained upon site-specific mutagenesis of this motif in the NodD box sustains this proposal. Further, we provide strong arguments that the inducer flavonoid, involved in transcriptional activation of Azorhizobium nod genes, interacts directly with the NodD protein, thereby increasing its binding affinities for the NodD box.

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans -zeatin, and cis -zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188–5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis - and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.