COI1 affects myrosinase activity and controls the expression of two flower-specific myrosinase-binding protein homologues in Arabidopsis.

Two cDNA clones homologous to myrosinase-binding proteins (MBPs) were identified by differential display in Arabidopsis thaliana (L.) Heynh. The cDNAs (MBP1 and MBP2) correspond to two open-reading frames found in a gene cluster of seven putative MBP genes located on chromosome 1. The predicted proteins MBP1 and MBP2 are similar to lectins and plant aggregating factors. In addition. MBP2 contains a region of high content of proline and alanine residues, commonly found in arabinogalactan proteins and hydroxyproline-rich glycoproteins. Transcripts corresponding to MBP1 and MBP2 genes are exclusively and abundantly expressed in flowers but are not detected in male-sterile flowers of coi1 plants, insensitive to jasmonic acid. Northern analysis and in situ hybridization revealed that MBP mRNAs are present in higher levels in immature flowers and are localized in several floral organs, including the ovary, ovules, style, anthers and filament. Transcripts of the Arabidopsis myrosinase gene TGG1 show a pattern of expression similar to that observed for the MBP genes during flower development; however, they are also abundant in green tissues and are only partially affected by COI1. Crude preparations of soluble proteins from leaf and flower extracts of wild-type Arabidopsis showed myrosinase activity when sinigrin was used as substrate. In contrast, coi1 plants showed significantly reduced myrosinase activities in both leaves and flowers. The results show that COI1 controls MBP expression in flowers and significantly affects the expression and activity of myrosinase in Arabidopsis.

Nature of the anchors of membrane-bound aminopeptidase, amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells.

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.

Structure and regulation of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase in maize.

The lysine-oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca2+, high salt concentration, organic solvents and Mg2+. The Ca2+-dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125-kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time-dependent and protease-concentration-dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65-kDa (LOR) and 57-kDa (SDH) domains. The SDH-containing polypeptides inhibited the enzymatic activity of LOR-containing polypeptides. When separated from the SDH domain by limited proteolysis and ion-exchange chromatography, the LOR domain retained its Ca2+ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.