The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition.

Trehalose serves as a primary circulatory sugar in insects which is crucial in energy metabolism and stress recovery. It is hydrolyzed into two glucose molecules by trehalase. Silencing or inhibiting trehalase results in reduced fitness, developmental defects, and insect mortality. Despite its importance, the molecular response of insects to trehalase inhibition is not known. Here, we performed transcriptomic analyses of Helicoverpa armigera treated with validamycin A (VA), a trehalase inhibitor. VA ingestion resulted in increased mortality, developmental delay, and reduced ex vivo trehalase activity. Pathway enrichment and gene ontology analyses suggest that key genes involved in carbohydrate, protein, fatty acid, and mitochondria-related metabolisms are deregulated. The activation of protein and fat degradation may be necessary to fulfil energy requirements, evidenced by the dysregulated expression of critical genes in these metabolisms. Co-expression analysis supports the notion that trehalase inhibition leads to putative interaction with key regulators of other pathways. Metabolomics correlates with transcriptomics to show reduced levels of key energy metabolites. VA generates an energy-deficient condition, and insects activate alternate pathways to facilitate the energy demand. Overall, this study provides insights into the molecular mechanisms underlying the response of insects to trehalase inhibition and highlights potential targets for insect control.

Validamycin A Inhibited FB1 Biosynthesis by the Target FvNth in Fusarium verticillioides.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

Membrane-bound trehalase enhances cadmium tolerance by regulating cell apoptosis in Neocaridina denticulata sinensis.

Trehalase gene is mainly expressed in the digestive circulatory system for regulating energy metabolism and chitin synthesis in insects, but it is significantly expressed in gill for immunomodulation in shrimp. However, its function in regulating immunity, particularly metal resistance in crustaceans has yet to be elucidated. In this study, one Tre2 gene (NdTre2) was isolated from Neocaridina denticulata sinensis. It could bind to Cd2+ and inhibit its toxicity. Spatiotemporal expression analysis showed that the expression of NdTre2 was highest in the gill and significantly reduced at 12 h after Cd2+ stimulation. The transcriptomic analysis of the gill after NdTre2 knockdown showed that the expression of genes synthetizing 20E was up-regulated and the increased 20E could further induce apoptosis by activating the intrinsic mitochondrial pathway, exogenous death receptor-ligand pathway, and MAPK pathway. In vitro, overexpressing NdTre2 enhanced the tolerance of E. coli in Cd2+ environment. In summary, these results indicate that NdTre2 plays an essential role in regulating immunity and chitin metabolism in N. denticulata sinensis.

Exploration of novel trehalases from cold-adapted Variovorax sp. PAMC28711: Functional characterization.

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) µmolmg-1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) µmolmg-1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.

Identification and RNAi-based function analysis of trehalase family genes in Frankliniella occidentalis (Pergande).

BACKGROUND: Insects utilize trehalases (TREs) to regulate energy metabolism and chitin biosynthesis, which are essential for their growth, development, and reproduction. TREs can therefore be used as potential targets for future insecticide development. However, the roles of TREs in Frankliniella occidentalis (Pergande), a serious widespread agricultural pest, remain unclear. RESULTS: Three TRE genes were identified in F. occidentalis and cloned, and their functions were then investigated via feeding RNA interference (RNAi) and virus-induced gene silencing (VIGS) assays. The results showed that silencing FoTRE1-1 or FoTRE1-2 significantly decreased expression levels of FoGFAT, FoPGM, FoUAP, and FoCHS, which are members of the chitin biosynthesis pathway. Silencing FoTRE1-1 or FoTRE2 significantly down-regulated FoPFK and FoPK, which are members of the energy metabolism pathway. These changes resulted in 2-fold decreases in glucose and glycogen content, 2-fold increases in trehalose content, and 1.5- to 2.0-fold decreases in chitinase activity. Furthermore, knocking down FoTRE1-1 or FoTRE1-2 resulted in deformed nymphs and pupae as a result of hindered molting. The VIGS assay for the three FoTREs revealed that FoTRE1-1 or FoTRE2 caused shortened ovarioles, and reduced egg-laying and hatching rates. CONCLUSION: The results suggest that FoTRE1-1 and FoTRE1-2 play important roles in the growth and development of F. occidentalis, while FoTRE1-1 and FoTRE2 are essential for its reproduction. These three genes could be candidate targets for RNAi-based management and control of this destructive agricultural pest. © 2024 Society of Chemical Industry.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.

Effects of exogenous trehalose on filling characteristics and sugar component content of wheat under high temperature stress during the filling period.

Taking the heat-sensitive wheat variety 'Fanmai 5' (FM5) and the heat-tolerant variety 'Huaimai 33' (HM33), which were screened out in the previous experiments, as experimental materials, we conducted a field experiment with passive heat-enhancing shelters to simulate post-flowering high-temperature environment (average temperature increase of 5.13 ℃) during 2021-2022. During the filling period, we analyzed the effects of exogenous trehalose (10, 15 and 20 mmol·L-1) on the filling characteristics and sugar fraction under high temperature, with no spraying at ordinary temperature as control (CK). The results showed that treating without spraying exogenous trehalose at high temperature (H) significantly reduced wheat grain yield and grain weight during the filling period, and spraying exogenous trehalose alleviated the reduction of grain yield and grain weight at the filling stage under high temperature stress. Compared with the H treatment, grain yield and grain weight of HM33 and FM5 wheat varie-ties increased by 3.5%, 6.7% and 4.2%, 5.4%, respectively. High temperature stress significantly increased the trehalose content and trehalase (THL) activity in flag leaves of both wheat varieties, and decreased the fructose and glucose contents. Spraying exogenous trehalose increased the contents of trehalose, fructose, and glucose in flag leaves, and decreased the trehalase activity in flag leaves compared with H treatment, which could improve the glucose metabolism capacity of wheat at filling stage. The increasing effect of FM5 was higher than that of HM33. High temperature stress significantly reduced starch content of flag leaves and grains, while spraying exogenous trehalose alleviated the decrease of starch content of flag leaves and grains under high temperature stress, which was profit able for the substance accumulation of wheat grains under high temperature stress. Under the conditions of this experiment, spraying 15 mmol·L-1 trehalose at flowering stage was the best treatment for the two wheat varieties.

Steroid hormone-dependent changes in trehalose physiology in the silkworm, Bombyx mori.

Holometabolous insects undergo metamorphosis to reconstruct their body to the adult form during pupal period. Since pupae cannot take any diets from the outside because of a hard pupal cuticle, those insects stock up on nutrients sufficient for successful metamorphosis during larval feeding period. Among those nutrients, carbohydrates are stored as glycogen or trehalose, which is the major blood sugar in insects. The hemolymph trehalose is constantly high during the feeding period but suddenly decreases at the beginning of the prepupal period. It is believed that trehalase, which is a trehalose-hydrolyzing enzyme, becomes highly active to reduce hemolymph trehalose level during prepupal period. This change in the hemolymph trehalose level has been interpreted as the physiological shift from storage to utilization of trehalose at that stage. Although this shift in trehalose physiology is indispensable for energy production required for successful metamorphosis, little is known on the regulatory mechanisms of trehalose metabolism in accordance with developmental progress. Here, we show that ecdysone, an insect steroid hormone, plays essential roles in the regulation of soluble trehalase activity and its distribution in the midgut of silkworm, Bombyx mori. In the end of larval period, soluble trehalase was highly activated in the midgut lumen. This activation was disappeared in the absence of ecdysone and also restored by ecdysone administration. Our present results suggest that ecdysone is essentially required for the changes in the function of the midgut on trehalose physiology as development progresses.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Glycosylated quantum dots as fluorometric nanoprobes for trehalase.

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.

Bombyxin-stimulated ecdysteroidogenesis in relation to sugar transporter/trehalase expressions in Bombyx prothoracic glands.

Our previous studies showed that bombyxin stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) during a long-term incubation period in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. In the present study, we further investigated the downstream signaling cascade in bombyxin-stimulated PGs. Our results showed that upon treatment with bombyxin, expression levels of the sugar transport 1 (St1) and St4 genes and trehalase 1 (Treh1) gene, but not ecdysteroid biosynthesis genes were greatly enhanced compared to the controls. Treatment with LY294002 (an inhibitor of PI3K) reduced the enhanced St1 and Treh1 expression levels, clearly indicating the involvement of PI3K. Treatment with 1 mM of mpV(pic) (a potent inhibitor of protein phosphotyrosine phosphatase and activator of insulin receptor (InR) kinase) also stimulated expression levels of the St1 and Treh1 genes, thus further confirming the involvement of the InR. Determining Treh enzyme activity showed that bombyxin treatment stimulated Treh enzyme activity in time- and PI3K-dependent manners. Validamycin A (a Treh inhibitor) blocked bombyxin-stimulated Treh enzyme activity and partly decreased bombyxin-stimulated ecdysteroidogenesis. A specific sugar transport inhibitor (cytochalasin B) and a glycolysis inhibitor (2-deoxy-D-glucose (2-DG)) also reduced bombyxin-stimulated ecdysteroidogenesis. Taken together, these results indicated that increased expressions of Sts and Treh1 and enhanced Treh enzyme activity downstream of InR/PI3K are involved in bombyxin-stimulated ecdysteroidogenesis in B. mori PGs.

C-6 fluorinated casuarines as highly potent and selective amyloglucosidase inhibitors: Synthesis and structure-activity relationship study.

A series of C-6 fluorinated casuarine derivatives have been synthesized via organocatalytic stereoselective α-fluorination of iminosugar-based aldehydes or direct nucleophilic fluorination of polyhydroxylated pyrrolizidines. Glycosidase assays against various glycosidases allowed systematic structure-activity relationship (SAR) study using molecular docking calculations. Introduction of fluorine atom(s) at C-6 position removed the trehalase and maltase inhibitory activities of all casuarine derivatives, and greatly increased their specificity towards amyloglucosidase. Inhibition of the fluorinated casuarines depended on the configuration of C-6 fluorine, of which 6-deoxy-6-epi-6-fluoro-casuarine (24) was found approximately 40-fold potent than its parent compound 6-epi-casuarine (2) as a potent and specific inhibitor of amyloglucosidase. Molecular docking calculations showed that replacement of the C-6 hydroxyls by fluorine atom(s) removed the original interactions with trehalase, but helped to reinforce the binding with amyloglucosidase via newly established fluorine related hydrogen bonding or untypical anion-π interactions. To further investigate the quantitative SARs of casuarine derivatives, the CoMFA and CoMSIA models on amyloglucosidase were established, indicating the dominating effect of electrostatic field in amyloglucosidase inhibition. The 3D-QSAR models were validated to be reliable and can be used for further optimization of casuarine-related iminosugars, as well as design and development of anti-diabetic and immunomodulatory drugs.

In silico analysis and a comparative genomics approach to predict pathogenic trehalase genes in the complete genome of Antarctica Shigella sp. PAMC28760.

Although four Shigella species (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii) have been reported, S. sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world's driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic (treA) and cytoplasmic (treF) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes (treF and treA). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S. sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes (treF and treA) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

Rediscovery of 4-Trehalosamine as a Biologically Stable, Mass-Producible, and Chemically Modifiable Trehalose Analog.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed. For further analysis, 4-trehalosamine is selected as it shows high producibility. Compared with trehalose, 4-trehalosamine exhibits better or comparable protective activities and a high buffer capacity around the neutral pH. Another advantage of 4-trehalosamine is its chemical modifiability: simple reactions produce its various derivatives. Labeled probes and detergents are synthesized in one-pot reactions to exemplify the feasibility of their production, and their utility is confirmed for their respective applications. The labeled probes are used for mycobacterial staining. Although the derivative detergents can be effectively used in membrane protein research, long-chain detergents show 1000-3000-fold stronger autophagy-inducing activity in cultured cells than trehalose and are expected to become a drug lead and research reagent. These results indicate that 4-trehalosamine is a useful trehalose substitute for various purposes and a material to produce new useful derivative substances.

Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

The vital hormone 20-hydroxyecdysone controls ATP production by upregulating binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera.

Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.

Two trehalase isoforms, produced from a single transcript, regulate drought stress tolerance in Arabidopsis thaliana.

KEY MESSAGE: Alternative translation initiation of the unique Arabidopsis trehalase gene allows for the production of two isoforms with different subcellular localization, providing enzyme access to both intra- and extra-cellular trehalose. The trehalose-hydrolyzing enzyme trehalase mediates drought stress tolerance in Arabidopsis thaliana by controlling ABA-induced stomatal closure. We now report the existence of two trehalase isoforms, produced from a single transcript by alternative translation initiation. The longer full-length N-glycosylated isoform (AtTRE1L) localizes in the plasma membrane with the catalytic domain in the apoplast. The shorter isoform (AtTRE1S) lacks the transmembrane domain and localizes in the cytoplasm and nucleus. The two isoforms can physically interact and this interaction affects localization of AtTRE1S. Consistent with their role in plant drought stress tolerance, both isoforms are activated by AtCPK10, a stress-induced calcium-dependent guard cell protein kinase. Transgenic plants expressing either isoform indicate that both can mediate ABA-induced stomatal closure in response to drought stress but that the short (cytoplasmic/nuclear) isoform, enriched in those conditions, is significantly more effective.