No description available.

It is proposed that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of fatty acids in Arabidopsis seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings. Double mutant seeds in which both genes are knocked out are dormant, and unable to utilise their stored lipid. Germination can be achieved by removing the seed coat and supplying sucrose. The seedlings are resistant to 2,4-dichlorophenoxybutyric acid (2,4DB), indicating a defect in peroxisomal β-oxidation. Beyond the seedling stage, double mutants also show arrested growth phenotype, and crucially are unable to produce seeds. The double mutant phenotypes can be restored by complementation with a cDNA encoding CSY with either its native PTS2 targeting sequence or a heterologous PTS1 sequence. These results suggest that peroxisomal CSY is not just a glyoxylate cycle enzyme but is also required for fatty acid respiration and to break seed dormancy. It is hypothesised that peroxisomal malate dehydrogenase (PMDH) serves to oxidise NADH produced by β-oxidation, and does not oxidise malate to provide oxaloacetate for the glyoxylate cycle. The Arabidopsis genome encodes eight putative NAD⁺-dependent malate dehydrogenase enzymes, two of which are predicted to be PMDHs. Double mutant seeds in which these two <i>PMDH </i>genes are knocked out are unable to establish as seedlings unless exogenous sucrose is supplied. In this respect the double mutant is similar to a range of β-oxidation mutants. Seedlings are impaired in breakdown of stored lipid and insensitive to 2,4­-DB, showing that β-oxidation is defective. The metabolism of [2-¹⁴C]-acetate into sugars and organic acids is normal in double mutant seedlings, indicating that the glyoxylate cycle is still active.

No description available.

The aim of this project was to study the effects of vanillin and some of its putative precursors on fruits and cultured cells of <I>Capsicum frutescens</I> Mill. and in particular to determine the role of vanillin in capsaicin biosynthesis. When ferulic acid, the putative precursor to vanillin, was supplied to suspension culturers of <I>C. frutescens,</I> no vanillin or capsaicin were detected. Instead, caffeic acid (the precursor to ferulic acid) and an unknown compound, A, were detected in the cells and medium of suspension cultures respectively. When vanillin was supplied to suspension cultures of <I>C. frutescens,</I> vanillyl alcohol and a compound tentatively identified as a vanillic acid glycoside were detected in the medium and cells of suspension cultures respectively. When vanillin was supplied to fruits of <I>C. frutescens</I> prior to the onset of capsaicin synthesis, vanillyl alcohol and a vanillic acid glycoside were produced. In fruits actively accumulating capsaicin, vanillyl alcohol and a vanillin glycoside (B-d-glucosidovanillin) appeared after the addition of vanillin. However, both were transient metabolites and the fate of most of the added vanillin is still unknown. Certain amounts of added vanillin appeared to increase the amount of [<SUP>14</SUP>C]phenylalanine incorporated into capsaicinoids, glycosylated and saponified cell wall phenolics in fruits of <I>C. frutescens. </I>This led to the tentative conclusion that vanillin stimulated phenylpropanoid metabolism thus increasing the flux of [<SUP>14</SUP>C]phenylalanine down the pathway. A similar but less well-defined stimulation occurred in suspension cultures where there was an increase in the incorporation of [<SUP>14</SUP>C]phenylalanine into glycosylated phenolics on the addition of vanillin. There was also some indication that vanillin may act as a precursor to lignin as the addition of vanillin caused a decrease in the incorporation of [<SUP>14</SUP>C]phenylalanine into lignin in both fruits and suspended cells of <I>C. frutescens.</I>

The expression and role of MS and ICL during senescence and following detachment is investigated. After 7 weeks plant growth immunoblot analysis indicate the coordinate accumulation of MS and ICL. However, this increase in MS and ICL does not correlate with the decline in the thylakoid lipids MGDG and DGDG. A detectable decline in these lipid classes is observed after 2 weeks plant growth. Further analysis of changes in chlorophylls, carotenoids, protein and total RNA content confirm that senescence of cucumber cotyledons is initiated very soon after full greening. These data indicate that the glyoxylate cycle does not appear to play any significant role in the disassembly of chloroplast membranes during the earlier stages of senescence. In detached cucumber cotyledons the synthesis of MS and ICL is detectable in dark incubated cotyledons within 48 hours. This appears to be primarily due to an increase in transcripts that encodes these proteins. However, the demonstrable increase in MS and ICL levels occurs prior to any detectable decline in chlorophylls, carotenoids and galactolipids. This indicates that the glyoxylate cycle may play an additional role to the metabolism of products of chloroplast membrane degradation. The role sucrose may in controlling the synthesis of MS and ICL was also investigated. The presence of 25 mM was sufficient to greatly reduce the synthesis of MS and ICL under conditions where the synthesis of these proteins would normally occur in detached cucumber cotyledons and protoplasts.

Endogenous intraprotoplasmic ascorbate in rose cell suspension cultures as a model system ranged from 0.05 mmol kg^-1 in 0-d-old cultures to 1.1 mmol kg^-1 in 5-d-old cultures. Apoplastic ascorbate was estimated as 0.5 and 8 μM in 0- and 5-d-old cultures respectively, indicating that ascorbate is endogenous to, and may be metabolised within, the apoplast. Exogenous (apoplastic) 1 mM L-[1-¹⁴C]ascorbate was almost completely consumed (metabolised and/or taken up) by rose cultures within 8 hours of administration. Total ¹⁴C was removed from medium but slower than ascorbate. The calculated concentration of metabolites of ascorbate showed that metabolites were formed in the medium and then removed from the medium in 5-d-old cultures. Removal of metabolites could be due to either uptake by or binding to cells. The nature of the metabolites of 0.5 mM [1-¹⁴C]ascorbate was examined in 5-d-old rose culture and spent medium by electrophoresis at pH 6.5. Ascorbate was metabolised both enzymically in spent medium and non-enzymically in boiled spent medium. Three ¹⁴C-metabolites were identified as dehydroascorbate, diketogulonate and oxalate. Other acidic ¹⁴C-metabolites (C, D, E and F) have not as yet been identified. F is highly mobile during electrophoresis at pH 2.0, showing that it has a low pK. C, D and E are also mobile at pH 2.0 but less so than F. E and C are interconvertible non-enzymically during storage and can E be regenerated by treatment with NaOH, suggesting that C is a lactone of E. ¹⁴C-F was converted to [¹⁴C]oxalate by whole culture and by spent medium but not by boiled spent medium, indicating an enzyme-catalysed reaction. The enzyme was partially inhibited by 100 mM azide but not by antioxidants. [¹⁴C]Oxalate was produced from¹⁴C-F by alkali hydrolysis indicating the presence of an oxalyl ester group. The metabolism of apoplastic ascorbate, described in this thesis, is very different from its intraprotoplasmic metabolism. I have identified novel metabolites and propose a novel pathway for the metabolism of apoplastic ascorbate.

The project aim was to determine which of the competing pathways predominate(s) <i>in vivo</i> in the formation of the UDP-GlcA, GDP-GalA and UDP-Man utilised for plant cell wall synthesis. The studies conducted on the ³H:¹⁴C ratios of plant cell wall residues at 8 h indicate that UDP-Gal is not a significant direct precursor of UDP-GalA. The ³H:¹⁴C ratios of the intermediary metabolites were studied over time. The results show that the ³H:¹⁴C ratio kinetics of UDP-GalA and UDP-Gal are vastly different from one another. These results also indicate that UDP-Gal is not a significant direct precursor of UDP-GalA. The ³H:¹⁴C ratios of the AIR (alcohol insoluble residue)-derived monosaccharides indicate that UDP-GlcA, UDP-GalA, UDP-Xyl, UDP-Ara and UDP-Api, like UDP-Rha, arose mainly from the UDP-Glc ‘core’ metabolite. The ³H:¹⁴C ratio kinetics of UDP-GalA, UDP-GlcA, UDP-Xyl and UDP-Ara are very distinct from the ³H:¹⁴C ratio kinetics of Glc 6-P but they are similar to the isotope ratio kinetics of UDP-Glc. The ³H:¹⁴C ratios of the AIR-derived monosaccharides and ³H:¹⁴C ratios of the intermediary metabolites give strong evidence that UDP-GalA and UDP-GlcA are predominantly formed by the UDP-Glc dehydrogenase pathway and not the <i>myo-</i>inositol pathway. The ³H:¹⁴C ratios kinetics of UDP-Glc are approximately equal to those of Glc 1-P. It is observed that the ³H:¹⁴C ratio of AIR-derived GalA residue is greater than for Man and Fuc. As UDP-GalA was determined to arise predominantly from UDP-Glc, GDP-Man and GDP-Fuc cannot also stem from Glc 1-P. The predominant pathway of GDP-Man synthesis must be via Gla 6-P or Fru 6-P. The ³H:¹⁴C ratio of Rib was similar to those of GalA, Ara, Xyl and Api. A pathway is known to exist that may convert to UDP-Xyl to the RNA precursor Rib 5-P. The results suggest that this is the predominant pathway of Rib synthesis for RNA.

No description available.

Investigations of plant mitochondrial protein import have been very limited and few reports exist in the literature. The work presented in this thesis describes the development of an <i>in vitro Zea mays</i> mitochondrial protein import system, which has enabled the import of plant nuclear-encoded mitochondrial proteins to be examined. This <i>Z.mays</i> import system was characterised and partially optimised with the <i>Nicotiana plumbaginifolia</i> manganese superoxide dismutase (MnSOD). Results suggest that the energy requirements for protein import into plant mitochondria are similar to those of <i>Saccharomyces cerevisiae</i> and <i>Neurospora crassa</i>, requiring both an energised inner mitochondrial membrane and ATP. The inclusion of 1,10-phenanthroline inhibited the processing, but not the import of MnSOD and indicated that the processing activity within <i>Z.mays</i> mitochondria was dependent upon the presence of metal ions. The plant mitochondrial processing protease may therefore be similar to the characterised <i>S.cerevisiae</i> and <i>N.crassa</i> matrix processing protease. The import of the <i>Z.mays</i> adenine nucleotide translocator (ANT) protein was then investigated. Unlike the ANT of <i>S.cerevisiae</i> and <i>N. crassa</i>, this plant ANT was synthesised as a precursor protein, which was processed upon import into mitochondria isolated from both <i>Z.mays</i> and <i>Solanum tuberosum</i>. The subsequent isolation of an <i>S.tuberosum</i> ANT cDNA clone <i>PANT-1</i>, enabled the import of a second plant ANT to be investigated. Results corroborated the findings previously obtained with the <i>Z.mays</i> ANT and it was therefore concluded that the import of plant ANT proteins are distinctly different from those of <i>S.cerevisiae</i> and <i>N.crassa</i>.

Xyloglucan hydrogen-bonds to cellulose and has been proposed to form load-bearing cross-links between adjacent microfibrils in the primary cell walls of plant cells. Such cross-links may regulate wall extensibility and allow control of cell expansion. Changes in relative molecular mass (M<SUB>r</SUB>) of xyloglucan have been correlated with wall extensibility in several studies. Endotransglycosylation of xyloglucan may cut and reform cross-links, enabling limited cell expansion through temporary loosening of the cell wall. An enzyme capable of catalysing such a reaction, xyloglucan endotransglycosylase (XET), has been reported previously. A pulse-chase radiolabelling approach was used to follow the changes in M<SUB>r</SUB> of a pool of radiolabelled wall-bound xyloglucan in the cell walls of suspension-cultured rose cells as the cells aged. This was done in rapidly-expanding cells and in slowly-expanding cells to study the effects of expansion rate on xyloglucan M<SUB>r</SUB>. Radiolabelled xyloglucan extracted from the cell walls of rapidly-expanding cultures had a M<SUB>r</SUB> consistently ~80000 lower than radiolabelled xyloglucan extracted from slowly-expanding cells. The relationship between xyloglucan M<SUB>r</SUB> and expansion rate is discussed. The radiolabelled xyloglucan in the cell wall extracts of both cultures decreased in M<SUB>r</SUB> by ~40000 during the 7-day observation period, with 20-30% of the radiolabelled xyloglucan being lost from the cell wall. A similar amount of radiolabelled xyloglucan, with a mean M<SUB>r</SUB> of ~39000, accumulated in the culture medium at the same time as the loss from the cell wall. This observation is proposed to be due to trimming of loose (<I>i.e.</I> not directly bound to microfibrils) sections of wall-bound xyloglucan from the cell wall and subsequent sloughing into the culture medium. The possible effects of trimming of xyloglucan on the extensibility of the cell wall are discussed. Protoplasts isolated from suspension-cultured rose cells were used to determine the M<SUB>r</SUB> of newly-secreted xyloglucan.