Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus.

We isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence.

Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit.

Organic acids prevent aluminum-induced conformational changes in calmodulin.

At a molar excess of 10:1 for [citrate]/[calmodulin], citrate can prevent aluminum binding to calmodulin when present in the protein solution in micromolar concentration, as determined by fluorescence and circular dichroism spectroscopy. In contrast, citrate is only partially effective in restoring calmodulin to its native structure once the aluminum-calmodulin complex (3:1) is formed, as measured by the alpha-helix content of the protein. Considering the magnitude of the stability constant of the citrate-aluminum chelate, citrate and perhaps other carboxylic acids may protect calmodulin, and thus cells, from toxic aluminum ions.

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress.

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H(+)-transport activity were measured by quinacrine fluorescence (DeltapH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (DeltaPsi) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K(d)) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Psi(o)) of-26.0 and -13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K(d) values and the calculated intrinsic affinity constant, K(i). The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Psi(o) values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K(+)-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl(-) ions to stimulate H(+)-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.