OsDPE2 Regulates Rice Panicle Morphogenesis by Modulating the Content of Starch.

Starch is a carbon sink for most plants, and its biological role changes with response to the environment and during plant development. Disproportionating Enzyme 2 (DPE2) is a 4-α-glycosyltransferase involved in starch degradation in plants at night. LAX1 plays a vital role in axillary meristem initiation in rice. Herein, results showed that Oryza sativa Disproportionating Enzyme 2 (OsDPE2) could rescue the mutant phenotype of lax1-6, LAX1 mutant. OsDPE2 encodes rice DPE2 located in the cytoplasm. In this study, OsDPE2 affected the vegetative plant development of rice via DPE2 enzyme. Additionally, OsDPE2 regulated the reproductive plant development of rice by modulating starch content in young panicles. Furthermore, haplotype OsDPE2(AQ) with higher DPE2 enzyme activity increased the panicle yield of rice. In summary, OsDPE2 can regulate vegetative and reproductive plant development of rice by modulating starch content. Furthermore, DPE2 activities of OsDPE2 haplotypes are associated with the panicle yield of rice. This study provides guidance for rice breeding to improve panicle yield traits.

Phosphorylation of DPE2 at S786 partially regulates starch degradation.

In plants, transitory starch is synthetized during the day and degraded at night to provide the continuous carbon needed for growth and development. Starch metabolism is highly coordinated, as the starch degradation rate must be coupled to the amount of starch synthetized during the day. Maltose is one of the chloroplastic products obtained from starch degradation, and maltose is exported to the cytosol where disproportionating enzyme-2 (DPE2) is responsible for its metabolism. The amount of DPE2 remained unchanged throughout the day, but its activity notably increased at the end of the day (7 p.m.), suggesting that posttranslational modification drives the mechanism underlying the regulatory activity of this enzyme. Sucrose nonfermenting-related kinase-1 (SnRK1), a protein kinase that controls the activity of several metabolic enzymes, was able to interact and phosphorylate DPE2 at three different residues localized in the α-glucanotransferase domain. This phosphorylation acts as a positive regulator of DPE2, increasing its activity. Complementation of dpe2-deficient mutants with the wild-type (WT) and S786A forms of DPE2 showed that the nonphosphorylated form of DPE2 only partially restored starch degradation, suggesting that phosphorylation at S786 is involved in enzyme regulation.

Maltose Processing and Not ß-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways in higher plants and is characterized by high water use efficiency. This mainly relies on major nocturnal CO2 fixation sustained by degradation of storage carbohydrate such as starch to provide phosphoenolpyruvate (PEP) and energy. In contrast to C3 plants where starch is mainly degraded by the hydrolytic route, different observations suggested the phosphorolytic route to be a major pathway for starch degradation in CAM plants. To elucidate the interplay and relevant contributions of the phosphorolytic and hydrolytic pathways for starch degradation in CAM, we assessed diel patterns for metabolites and enzymes implicated in both the hydrolytic route (ß-amylase, DPE1, DPE2, maltase) and the phosphorolytic route (starch phosphorylase) of starch degradation in the CAM orchid Phalaenopsis "Edessa." By comparing the catalytic enzyme activities and starch degradation rates, we showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by ß-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.

Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants.

Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and ß-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify ß-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels.

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis.

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes.