No abstract

The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.

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Larvae of an anhydrobiotic insect, Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTpsα/β) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTpsα did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis.

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

No abstract

Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.