The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined . This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase . The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase . This fraction also had NADH diaphorase activity and contained cytochromes b5 and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1 .12 g/cma and sedimented separately from the ribosomes ; electron micrographs showed that it was comprised of smooth membranes . When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1 .16 g/cma and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum . Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes .

SUMMARYThe rangeland grass, Bouteloua gracilis was inoculated with its mycorrhizal symbiont, Glomus fasciculatus, to determine the influence of vesicular-arhuscular mycorrhizae on water status, stomatal behaviour and photosynthesis as well as gross plant morphology, biomass and phosphorus content. Mycorrhizal infection increased transpiration rates by over 100% with 50 to 70 °o lower leaf resistances to water vapour diffusion. Leaf xylem pressure was not different between mycorrhizal and non-mycorrhizal plants indicating that whole-plant resistance to water transport was reduced by more than 50 %. Photosynthetic rates under saturating light conditions increased 68% with infection as a consequence of a 33 % reduction in stomatal resistance and a 67 % reduction in mesophyll resistance to CO2 uptake. Mycorrhizal infection did not affect biomass or gross plant morphology after 30 weeks of growth, but increased chlorophyll and phosphate concentrations by 28 °o and 70 % respectively. These physiological changes indicate that mycorrhizae may substantially alter survival ability of Bouteloua gracilis.

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Bouteloua gracilis was grown in defined, axenic culture with and without vesicular–arbuscular (VA) mycorrhizae. Leaves and roots of mycorrhizal and nonmycorrhizal plants were harvested and assayed for cytokinin content using a soybean callus tissue bioassay. Total cytokinin activity was 57 and 111% greater in leaves and roots, respectively, in mycorrhizal over control plants. Cytokinin activities, separated using paper chromatography with water saturated n-butanol as a solvent, doubled in roots and leaves at Rf values of 0.3 and 0.9 and increased 9-fold in roots at an Rf value of 0.1 with infection. This appears to be the first demonstration of altered cytokinin levels in plants resulting from mycorrhizal infection.

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate, 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346, 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

Degenerate oligonucleotide primers corresponding to conserved regions flanking the active-site domain of 1-aminocyclopropane-l-carboxylate (ACC) synthase (EC 4.4.1.14) were used for the polymerase chain reaction (PCR) to amplify DNA fragments from mRNA isolated from tomato fruit and tomato suspension cell culture. Antibodies raised against two conserved peptide sequences (TNPSNPLGTT and SLSKDLGLPGFRVG) were used to screen for positive colonies, after the PCR products were cloned into a Bluescript plasmid and expressed in Escherichia coli. Four distinct cDNA fragments encoding ACC synthase homologs were isolated. While pBTAS1 and pBTAS4 were obtained from fruit mRNA, cell culture mRNA yielded three sequences, pBTAS1, pBTAS2, and pBTAS3. Sequencing ofthese gene fragments revealed that pBTAS1 and pBTAS4 were identical to those full-length sequences previously reported by Van Der Straeten et al. [Van

Bouteloua gracilis (H.B.K.) Lag ex Steud (blue grama) was grown in a defined medium with and without the vesicular–arbuscular mycorrhizal fungus Glomus fasciculatus for 50 days. Levels of gibberellin-like substances (GA) and a substance like abscisic acid (ABA) of mycorrhizal and nonmycorrhizal plants were measured using the barley half-seed bioassay and UV detection of peaks from a μ-Bondapak–NH2 anion exchange high-performance liquid chromatograph column, respectively. Infection by mycorrhizal fungi resulted in significantly increased GA activity in the leaves and a tendency for decreased activity in the roots. ABA concentration decreased in leaves of infected plants but remained unchanged in roots. Increased levels of GA with reduced ABA in the leaves may alter substantially the physiology of B. gracilis.

The intracellular location of several enzymes concerned with phospholipid metabolism was investigated by examining their distribution in organelles separated on sucrose gradients from total homogenates of castor bean (Ricinus communis var. Hale) endosperm. The enzymes phosphatidic acid phosphatase, CDP-diglyceride-inositol transferase, and phosphatidylethanolamine-L-serine phosphatidyl transferase were all primarily or exclusively confined to membranes of the endoplasmic reticulum. These results and those reported previously on lecithin synthesis establish a major role of the endoplasmic reticulum in phospholipid and membrane synthesis in plant tissues.