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    Chloroplast genome evolution in New Zealand mycoheterotrophic Orchidaceae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Murray, Katherine Jane Hope
    The plastid genomes, or plastomes, of most photosynthetic land plants are highly similar. In contrast, those of non-photosynthetic, heterotrophic land plants are often reduced in both size and gene content. The apparent degradation of mycoheterotrophic plant plastomes has been attributed to a functionally-driven stepwise pattern of loss. However, the number of complete plastome sequences available for mycoheterotrophic plants is small and taxonomic coverage is biased. In this thesis, the plastomes of two mycoheterotrophic orchid species endemic to New Zealand, Corybas cryptanthus Hatch (Diurideae) and Danhatchia australis Garay & Christenson (Goodyerinae), as well as those of an albino and several photosynthetic representatives of Corybas are reported. Beyond increasing the number of mycoheterotrophic plastomes available for evaluating broad hypotheses about plastome evolution in non-photosynthetic plants, these data also provide insights into two little studied aspects of plastome evolution in mycoheterotrophs; intraspecific variation in the plastomes of mycoheterotrophs and the differences between mycoheterotrophs and their closest photosynthetic relatives. The plastomes of C. cryptanthus and D. australis differ in the extent to which they are degraded. Perhaps unexpectedly, the plastome of C. cryptanthus, which has close photosynthetic relatives and therefore is likely to have arisen more recently than the taxonomically isolated D. australis, is more reduced. Specifically, the plastomes of C. cryptanthus are approximately half the size and have half the gene content of the other Corybas sequenced whereas the plastome of D. australis is similar to those available for photosynthetic relatives. This contrast may reflect underlying differences between the two genera; the photosynthetic relatives of D. australis have plastomes containing NADH dehydrogenase (ndh) genes whereas those of photosynthetic Corybas have lost their ndh genes and their small single copy regions are highly reduced. These features may have predisposed the ancestor of C. cryptanthus to rapid genome degradation. Finally, observations on these results strongly suggest that plastome degradation follows, rather than precedes, the shift to mycoheterotrophy.
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    The biosynthesis of galactolipids in chloroplast envelopes : a thesis for the degree of Master of Science in Biochemistry
    (Massey University, 1979) Benny, Athol Graeme
    The procedure of Douce et al (1973) was employed for the isolation of envelopes from purified chloroplasts of spinach (Spinacia oleracea) and maize (Zea maize var. Wis. 235). Maize chloroplasts gave very low yields of envelope protein and low incorporation of radioactivity from UDP-14C-Galactose into galactolipids. However the use of spinach chloroplasts resulted in higher yields of envelope protein and high levels of a galactosyltransferase that synthesised galactolipids from endogenous lipid substrates and added UDP-14C-Galactose. The products of galactosyltransferase were identified as MGDG and DGDG by comparison with standard lipids on thin layer chromatography. The procedure for the isolation of chloroplast envelopes reported by Poincelot and Day (1973) gave a higher yield of less contaminated envelope membranes and an increased specific activity of galactosyltransferase compared to the results obtained using envelopes isolated by the method of Douce et al (1973) Total incorporation of radioactivity from 0.3 μM UDP-14C-Galactose by galactosyltransferase was dependent on the time and temperature of incubation and the nature of the incubation buffer. Maximum incorporation (about 72% of the added radioactivity) was obtained upon incubation at 30°C for 30 min, in 50 mM HEPES-NaOH at pH 8.0. MGDG was identified as the major labelled lipid (MGDG:DGDG ratio 1.7:1). Lower pH values gave higher incorporation into DGDG. A cation dependence of galactosyltransferase was observed and incorporation was stimulated by addition of Ca2+, Mg2+ or Ba2+. Maximum incorporation was obtained with 5 mM Ba2+. In contrast 5 mM Cu2+ completely inhibited incorporation. The sulphydryl nature of the chloroplast galactosyltransferase (Chang, 1970; Mudd et al 1971) was confirmed with galactosyltransferase of the chloroplast envelope. Linoleic acid at 0.72 μM completely inhibited transferase activity. The inhibition by linoleate could be partially removed by addition of about 10 mM Ca2+ or Ba2+ but 10 mM Mg2+ and BSA (30 μg per ml) were without effect. UMP, UDP and UTP at 1 mM inhibited incorporation by transferase. UDP was the most effective inhibitor and gave 50% inhibition of incorporation at about 5 μM. NADH and PPi did not significantly affect incorporation. The addition of exogenous diacylglycerol (1-palmitoyl, 2-oleoyl glycerol or 1, 2-di-linoleoyl glyerol) did not increase the incorporation of radioactive galactose into galactolipids. Incorporation was inhibited by 0.3% Triton X-100 and 6 mM sodium cholate. No radioactivity from added 14C-diacylglycerol was incorporated into MGDG by chloroplast envelopes. Preincubation of the chloroplast envelopes with phospolipase C or D reduced the total amount of radioactivity incorporated by galactosyltransferase. Transferase activity was detectable after preincubation of the envelopes with trypsin and protease. The fatty acid composition of MGDG, DGDG and DG from whole tissue, chloroplasts and chloroplast envelopes of spinach is presented. The characteristic highly unsaturated nature of the fatty acids of MGDG and DGDG is in contrast to the relatively saturated fatty acid content of DG isolated from whole tissue and chloroplasts. However, DG isolated from chloroplast envelopes contained predominantly 16:0, 18:1 and 18:3.
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    Lipid biosynthesis in isolated chloroplasts : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
    (Massey University, 1979) McKee, Joseph William Adair
    Two notable features of previous work on lipid biosynthesis by isolated chloroplasts have been:- (a) The inability of chloroplasts to incorporate more than small amounts of acetate into the main constituent fatty acids of the chloroplast lipids, namely linoleic (18:2) and linolenic (18:3) acids. (b) The poor incorporation of fatty acids synthesized into galactolipids, which are the main chloroplast lipids. Both of these aspects of lipid biosynthesis were investigated using chloroplasts isolated from spinach, maize and sweetcorn. Initial attempts to improve the synthesis of polyunsaturated fatty acids from [1-14C]acetate were not successful. Consequently the main object of the investigation was directed towards increasing the incorporation of long chain fatty acids into galactolipids in the hope that increased galactolipid synthesis might also lead to increased desaturation of oleate to linoleate and linolenate. Factors affecting the rates of acetate incorporation into lipids by spinach, maize and sweetcorn chloroplasts were investigated. Optimum concentrations of acetate, ATP and CoA were found to be about 0.5mM-acetate (spinach somewhat higher at 0.75mM-acetate), 0.5mM-ATP and 0.25mM-CoA under the incubation conditions used in the present study. Acetate concentration had a major effect on the rate of incorporation; optimisation of ATP and CoA concentrations gave only small enhancements of acetate incorporation. The effect of divalent cations was also investigated for spinach chloroplasts. Optimum Mg++ was 3.0mM; addition of 1mM-Mn++ in the presence of 1mM-Mg++ gave a comparable stimulation of acetate incorporation. Acetate incorporation by spinach chloroplasts was also enhanced by the addition of Triton X-100, sn-glycerol-3-phosphate and UDP-galactose. Maximum incorporation rates obtained for maize and sweetcorn chloroplasts were 20-30nmol of acetate/mg chlorophyll/h which are up to 10-fold higher than previously reported rates for maize. Rates of up to 500nmol of acetate/mg chlorophyll/h were obtained for spinach chloroplasts which compare favourably with the rates obtained by other workers using chloroplasts isolated from younger leaf tissue. Oleic and palmitic acids with small amounts of stearic acid were the main fatty acids synthesized from acetate by isolated chloroplasts from all three sources. Little synthesis of linoleic and linolenic acids was achieved and changes in acetate, ATP and CoA concentrations had no significant effect on the synthesis of polyunsaturated fatty acids from acetate. Triton X-100 and divalent metal ion concentrations also had little effect on the synthesis of polyunsaturated fatty acids by spinach chloroplasts. The synthesis of diglycerides (DG) by isolated chloroplasts from spinach, maize and sweetcorn was enhanced by the adaition of sn-glycerol-3-phosphate (G-3-P). Synthesis of monogalactosyldiglyceride (MGDG) was enhanced by the addition of UDP-galactose particularly if G-3-P was also present. Triton X-100 greatly enhanced the synthesis of DG and also (in the presence of UDP-galactose) MGDG by spinach chloroplasts. Spinach chloroplasts gave higher rates of DG and KGDG synthesis than either maize or sweetcorn chloroplasts. The synthesis of MGDG from DG by spinach chloroplasts was investigated by double-labelling experiments, using [1(3)-3H]sn-glycerol-3-phosphate and [1-14C]acetate, fatty acid analysis and positional distribution of the incorporated fatty acids. The synthesis of MGDG was shown to occur without prior modification of the fatty acid composition of the DG. It was evident from the incorporation of oleate and palmitate into DG (and subsequently into MGDG) and from the positional distribution of these two fatty acids that a specific acylation of G-3-P occured synthesizing mainly 1-oleoyl, 2-palmitoyl-sn-glycerol. The effects of altering the proportions of oleate and palmitate synthesized on the relative amounts of these fatty acids incorporated into DG (and MGDG) were investigated. The results suggested that palmitate was incorporated into position 2 first followed by oleate into position 1. If there was more palmitate than oleate synthesized some palmitate could be also incorporated into position 1. The rates of DG synthesis calculated from [1 (3)-3H]-sn-glycerol-3-phosphate incorporation were conniderably greater than those calculated from [1-14c]acetate incorporation indicating that a considerable dilution of the label from [1-14c]acetate had occurred and that a major proportion of the fatty acid carbon had come from an alternative source. Bicarbonate, present in the reaction medium, was found to be utilized by spinach chloroplasts for the synthesis of fatty acids and lipids. Thus bicarbonate was probably the alternative source of fatty acid carbon. The fatty acids and lipids synthesized by spinach chloroplasts from exogenous acetate and bicarbonate were very similar. Although high rates of DG and MGDG synthesis have been achieved in the course of the present study by the addition of appropriate metabolites, stimulation of synthesis of these lipids did not alter the rates of synthesis of linoleic and linolenic acids from acetate. Other attempts to increase polyunsaturated fatty acid synthesis from acetate by isolated chloroplasts were also unsuccessful. The use of chloroplasts isolated from developing maize leaf sections had little effect on the rates of linoleic and linolenic acids synthesized from acetate. The addition of a 100,000 X g particulate preparation from leaf homogenate to isolated maize and spinach chloroplasts though stimulating overall incorporation of acetate, gave only minor increases in the proportion of linoleic and linolenic acids synthesized. The stimulation of phosphatidylcholine synthesis by the particulate fraction, in the presence of isolated chloroplasts, failed to result in any dramatic increases in the proportions of polyunsaturated fatty acids syntheisized. These findings are discussed in relation to the current understanding of fatty acid and lipid synthesis and recent in vivo and in vitro studies of plant lipid synthesis.