D-enzyme, disproportionating enzyme, or 4-α-glucanotransferase (EC 2.4.1.25) catalyses glucan transfer from one α-1,4-glucan molecule to another, or to glucose. From the wide distribution of D-enzyme in starch accumulating organs of plants, it is believed that D-enzyme is involved in starch metabolism, but the function <I>in vivo </I>is not known. In the present work, D-enzyme was purified from potato tubers, and cDNA and genomic DNA clones for potato D-enzyme were isolated. The biochemical analysis of purified recombinant D-enzyme suggested that high molecular weight starch (amylose and amylopectin) can serve as donor and acceptor, and very long α-1,4-glucans or even highly branched glucans can be transferred by the enzyme. It was also discovered that D-enzyme catalyses an intra-molecular transglycosylation (cyclisation) reaction on amylose and amylopectin, as well as the well studied inter-molecular transglycosylation (disporportionation) reaction. Analysis of D-enzyme gene expression was carried out by northern and western blot analysis, and in transgenic potato plants using the GUS-reporter gene fusion system. These results suggested that D-enzyme mRNA accumulates under circumstances when starch biosynthesis is most active but declines in amount under conditions when starch is broken down. These observations appear to contradict the widely held view that D-enzyme is involved in starch breakdown, and may suggest a function in starch synthesis. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the CaMV35S promoter, and various phenotypic changes were observed. These plants grew slower than wild type, produced less leaves, less mass of tubers and the apical meristems suffered necrosis. Furthermore, tubers from these plants sprouted later and the growth of sprouts was slower than wild type. However no significant difference was found in starch produced in tubers, either in its quantity or quality. From all these results and the available information about starch metabolism, possible roles of D-enzyme in starch metabolism are discussed.

DNA constructs of Agrobacterium-mediated transformation into <I>Nicotiana plumbaginifolia</I> were prepared. The Ca<SUP>2+</SUP>-sensitive luminescent protein, aequorin was placed in frame with the nuclear localisation signal (NLS) peptide of the simian virus SV40 Large T-antigen, <I>Xenopus leavis'</I> nucleoplasmin, and high mobility group protein, HMG1 from <I>Pisum sativum</I>. After analysing individual transformants, nucleoplasmin was shown to successfully target aequorin to nuclei. These plants, together with plants transgenic for cytosolic aequorin, were used to study Ca<SUP>2+</SUP> dynamics when exposed to the environmental stimuli, wind and cold shock. Wind induced immediate Ca<SUP>2+</SUP> transients in the cytoplasm and nucleus, while cold shock induced a Ca<SUP>2+</SUP> transient in the cytoplasm that was followed by a delayed Ca<SUP>2+</SUP> transient in the cytoplasm that was followed by a delayed transient in the nucleus. Wind and cold shock induced calmodulin gene expression that followed distinct kinetics. Rapid Amplification of cDNA Ends (3' RACE) identified the presence of two calmodulin transcripts in tobacco seedlings; <I>NpCaM-1</I> and <I>NpCaM-2</I> of which only <I>NpCaM-1</I> was induced by both stimuli. The transcripts comprise two different nucleotide sequences but encode identical polypeptides. The expression kinetics of <I>NpCaM-1</I> was related to wind and cold shock-induced nuclear and cytosolic Ca<SUP>2+</SUP> changes by using Ca<SUP>2+</SUP> agonists/antagonists. Wind-induced nuclear Ca<SUP>2+</SUP> changes correlated closely to the subsequent expression of and accumulation of <I>NpCaM-1</I> while changes in cytoplasmic Ca<SUP>2+</SUP> levels did not. Cold shock, on the other hand, did not reveal this correlation, and a role for intracellular Ca<SUP>2+</SUP> levels during subsequent expression of <I>NpCaM-1</I> remains therefore speculative. Heat shock induced a prolonged increase in nuclear Ca<SUP>2+</SUP> level. Seedlings pretreated with Ca<SUP>2+</SUP> or EGTA showed respectively enhanced or diminished subsequent thermotolerance, therefore it was suggested that these increases in Ca<SUP>2+</SUP> levels were required for the acquisition of heat-induce thermotolerance.

This study describes two approaches for the study of Ca<SUP>2+</SUP>-mediated signal transduction in stomatal guard cells. A novel assay for monitoring stimulus-induced changes in guard cell cytosolic Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]<SUB>cyt</SUB>) was developed, using epidermal strips from a transgenic line of <I>Nicotiana plumbaginifolia</I> harbouring apoqequorin, the precursor of the Ca<SUP>2+</SUP>-sensitive photoprotein, aequorin. Results indicated that mechanical, low temperature and abscisic acid (AGA) signals, directly affected stomatal behaviour, promoting rapid closure, and that elevations of guard cell [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> play a key role in the transduction of these signals. Studies with Ca<SUP>2+</SUP> channel blockers and the Ca<SUP>2+</SUP> chelator EGTA, further suggested that mechanical ABA signals primarily mobilise Ca<SUP>2+</SUP> from intracellular store(s), whereas influx of extracellular Ca<SUP>2+</SUP> was a key component in the transduction of low temperature signals. The stomatal response to low temperature was found to be significantly influenced by previous plant growth temperature. The second approach involved the production of transgenic lines of <I>N. plumbaginifolia</I> expressing apoaequorin under the control of two guard cell 'specific' promoters, the ABA-responsivecDeT6-19 promoter from <I>C.plantagineum</I> (Michel <I>et al</I>., 1994; Taylor <I>et al</I>., 1995) and the constitutive lipid transfer protein (LTP1) promoter from <I>A. thaliana</I> (Thoma <I>et al</I>., 1994), enabling stimulus-induced changes in guard cell [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> to be monitored in intact seedlings. ABA-induction of cDET6-19 promoter activity did not affect the ability of stomata to respond to further applications of ABA. Results showed that mechanical and cold shock signals induced similar Ca<SUP>2+</SUP> responses to those observed in epidermal strips, with similar sensitivities to Ca<SUP>2+</SUP> antagonists. Studies with inhibitors of other putative components of the plant Ca<SUP>2+</SUP> signaling pathway also implicated phosphoinositide turnover, possibly stimulated by a coupling G-protein, in the low temperature signaling pathway. The hypothesis that Ca<SUP>2+</SUP> plays a central role in the molecular clock mechanism in plants has also been investigated. Transgenic <I>N. plumbaginifolia</I> seedlings expressing apoaequorin under the control of the CaMV 35S promoter were found to exhibit circadian oscillations in [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> in free-running conditions, confirming previous findings of Johson <I>et al</I> (1995).

No description available.

Radioactive feeding experiments, in which <I>DL</I>-[pyrrolidine-2'-<SUP>14</SUP>C]-nicotine was added to 10 day old cultures, confirmed that the kinetic pattern of this <I>N-</I>demethylation was similar to that of non-radioactive nicotine, and that nornicotine was the major product and was produced intracellularly with a maximum percentage conversion of approximately 70%. The appearance of nornicotine paralleled the disappearance of the added nicotine, although small amounts of four other radioactive-metabolites were observed. One of these metabolites was tentatively identified by GC-MS as <I>N-</I>formyl-3-nornicotine. The properties of (-)-nornicotine produced from (-)-nicotine by 10 day old cell cultures were determined using both, polarimetry and chiral gas chromatography. The results obtained, which were convincingly consistent, showed that the nornicotine produced was exclusively one enantiomer. This provided strong evidence that the bioconversion of nicotine by tobacco cultures does not involve opening of the pyrrolidine ring. It is possible that the mechanism of nicotine bioconversion by cultured cells might differ from that proposed for the plant, in which a partially racemized mixture of (+)- and (-)-nornicotine has been reported. This could be the result of the opening and closing of the pyrrolidine ring during bioconversion. A radioactive enzyme assay procedure developed, using cell-free preparations of tobacco cell cultures, has shown for the first time that the enzyme(s) which catalyses this <I>N-</I>demethylation is present in tobacco. The enzyme(s), which has been fully characterised, has a <I>K</I>m of 7.4<I>μ</I>M and a <I>V</I>max of 7.6 x 10<SUP>-2</SUP><I>p</I>Kat and appears to be NADPH dependent. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity was present in the intermediate pellet whilst the maximum specific activity appeared to be associated with the microsomal fraction. Also, the addition of selected possible methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity, suggesting that the <I>N</I>-demethylation is unlikely to be a transmethylation but satisfies some of the primary criteria for cytochrome P-450 involvement. Studies involving Sephadex G-25 gel fractionation showed that molecules smaller than 5,000MW are not involved in this nicotine <I>N-</I>demethylation. Finally, the possible enzymatic mechanism involved in this <I>N-</I>demethylation is discussed.

Mutants deficient in various enzymes have already contributed much to current understanding of starch metabolism. <i>Arabidopsis thaliana</i> now offers the potential to isolate specific stable null mutants in target genes which should prove invaluable in determining how enzymes work together in transitory starch metabolism. D-enzyme, disproportionating enzyme, or 4-a-glucanotransferase (EC 2.4.1.25) catalyses glucan transfer <i>in vitro</i> from one 1,4-a-D-glucan molecule to another, to itself to form cyclic molecules, or to glucose. <i>In vitro,</i> the preferred substrate of the enzyme is malto-oligosaccharides (MOS). A number of different roles for D-enzyme have been suggested in the breakdown and synthesis of starch. In <i>Arabidopsis</i> D-enzyme was found to be expressed in all plant organs investigated, and at all times over a 24 hour time course. This study uses a reverse-genetics approach to isolate a mutant of <i>Arabidopsis</i> lacking D-enzyme. cDNA and genomic sequences encoding D-enzyme were isolated from <i>Arabidopsis</i> libraries. The sequence was used to design primers for PCR screening of DNA from plants containing random T-DNA insertions. 19 000 plants were screened and one plant line was found in which the D-enzyme gene was disrupted by a T-DNA insert. Western blot analysis and enzyme assays confirmed that the homozygous mutant lacked D-enzyme protein and activity. Mutant plants grew 30% more slowly than wild-type plants and leaves had a higher starch content than the wild-type throughout the diurnal cycle. At the end of the night an appreciable amount of starch remained in the leaves of the mutant that had been degraded in the wild-type leaves. Mutant plants also accumulated high levels of MOS from the start of the dark period, indicating that D-enzyme is responsible for metabolising MOS produced during the nocturnal breakdown of starch. During the day, the mutant was shown to have normal levels of MOS, indicating that, in higher plants, MOS are probably not a substrate for D-enzyme during the synthesis of starch.

Abundance of nitrite in the soil is very variable requiring plants to evolve adaptive growth responses in order the optimise uptake. Nitrate is known to induce its own uptake and assimilation and many nitrate responsive genes have been identified. However, how nitrate is sensed is not known. A genetic screen was designed to identify components of early nitrate signalling and perception. <i>A. thaliana</i> luciferase reporter lines of nitrite reductase NiR, and an ammonium transporter AMT1.1 were characterised and found not suitable for use in a genetic screen. A new transgenic luciferase reporter line driven by the high affinity nitrate transporter NRT2.1 was generated and characterised and found to be suitable for use in a genetic screen. Split root experiments with the NRT2.1 reporter lines showed that this gene responds to local signalling. Pharmacological analysis using <i>A. thaliana</i> cell suspension cultures to identify signalling mechanisms involved in early nitrate signalling revealed a novel sucrose-dependent cyclohexamide effect, where CHX exerts no effect on nitrate gene induction of NiR and NRT2.1 when sucrose levels are high but when sucrose levels are low CHX treatment results in a reduction in the nitrite gene induction response. This may indicate a putative role for new protein synthesis in the nitrate gene induction response when sucrose is limiting. Investigation of the nitrite transport analog, cholate, showed that this molecule is not also a signalling analog as it failed to induce the nitrate responsive genes, NiR and NRT2.1.

The glyoxylate cycle is known to take part in the net conversion of storage lipids to sugar germinating oilseeds. Two enzymes are exclusive for this cycle, malate synthase (MS) and isocitrate lyase (ICL) and their synthesis is coordinately regulated. These enzymes are active during postgerminative growth of seeds but are repressed in mature plants. However, they appear again when plants senesce. Genes for both enzymes are regulated by carbohydrate status. The aim of this study was to examine carbohydrate regulation. Expression of <I>Ms </I>and <I>Icl</I> genes in cucumber roots was low but increased upon excision and dark-incubation during a six day period in the absence of exogenous sugar. However, when sucrose was added to the incubation medium their expression was repressed. Hairy roots obtained using <I>Agrobacterium rhizogenes</I> strain A4 showed the same pattern of expression. Transgenic hairy roots containing <I>Ms </I>and <I>Icl</I> promoters fused to the GUS reporter gene, had a low level of GUS activity. This GUS activity increased dramatically when roots were excised and incubated in the absence of sugar, indicating regulation at the transcriptional level. Histochemical staining showed that GUS activity is concentrated in root tips and lateral root primordia where demand for carbohydrate is presumably greatest. Defoliation and shading experiments were carried out to examine the expression of <I>Ms </I>and <I>Icl</I> in roots of whole plants under natural conditions. In both cases, MS and ICL mRNA increased and roots showed a decline in sugar content. Thus, indication of <I>Ms </I>and <I>Icl</I> expression takes place in roots when supply of carbohydrate from the shoot is impaired. Results are consistent with the hypothesis that gene expression in the roots is controlled by carbohydrate supply from the shoot. Identification of regulatory elements in the <I>Icl</I> promoter required for the sugar response were made possible using transgenic cucumber hairy roots. Deletions of the <I>Icl</I> gene were assayed, which located a 200 bp region necessary for the sugar response more than 1 kbp upstream of the transcriptional start.

In plants the effective partitioning of carbon and nitrogen assimilates between source and sink tissues is essential for growth and development. The regulation of sugar transporters is pivotal not only to the allocation of carbohydrates throughout the plant but in providing sugars, which may act as signals in the modulation of gene expression. Many of the genes associated with the production, distribution, utilisation and storage of assimilates are regulated by sugars. The <i>Arabidopsis thaliana</i> Sugar Transport Protein 1 (AtSTP1) is the principal monosaccharide transporter in <i>Arabidopsis </i>seedlings, accounting for approximately 60% of 3-O-methyl glucose uptake activity. Characterisation of <i>AtSTP1</i> gene expression and transport activity <i>in vitro</i> in plantlets growth in continuous light and in a diurnal growth regime reveal regulation in light, glucose and mechanical stimulation. A rapid accumulation of <i>AtSTP1</i> transcripts upon transfer to the dark was observed in all growth regimes tested. This appears to be a novel feature in sugar transporter regulation. In light grown plantlets, dark-induced <i>AtSTP1</i> transcripts diminished within 1 h of subsequent treatment with exogenous 3 mM D-glucose or white light. Furthermore, a 5 min pulse of white light resulted in a marked repression of dark-induced <i>AtSTP1 </i>transcripts, which is indicative of sugar-independent regulation. The diurnal regulation of the <i>AtSTP1 </i>gene in plantlets entrained a L12 h:D12 h regime is complex, as is the subsequent <i>AtSTP1 </i>response to sugars. The expression pattern of the <i>AtSTP1 </i>gene was different in the shoot and roots; furthermore in the shoots <i>AtSTP1 </i>transcripts displayed circadian rhythm upon transfer to continuous light. It appears that at least three signalling pathways interact in the regulation of STP1 synthesis and activity in <i>Arabidopsis thaliana. </i>These results are discussed in terms of the possible physiological functions of AtSTP1, including retrieval of sugars derived from the cell wall.

No description available.