Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

KEY MESSAGE: Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15 days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

Cytosolic disproportionating enzyme2 is essential for pollen germination and pollen tube elongation in rice.

Degradation of starch accumulated in pollen provides energy and cellular materials for pollen germination and pollen tube elongation. Little is known about the function of cytosolic disproportionating enzyme2 (DPE2) in rice (Oryza sativa). Here, we obtained several DPE2 knockout mutant (dpe2) lines via genomic editing and found that the mutants grew and developed normally but with greatly reduced seed-setting rates. Reciprocal crosses between dpe2 and wild-type plants demonstrated that the mutant was male sterile. In vitro and in vivo examinations revealed that the pollen of the dpe2 mutant developed and matured normally but was defective in germination and elongation. DPE2 deficiency increased maltose content in pollen, whereas it reduced the levels of starch, glucose, fructose, and adenosine triphosphate (ATP). Exogenous supply of glucose or ATP to the germination medium partially rescued the pollen germination defects of dpe2. The expression of cytosolic phosphorylase2 (Pho2) increased significantly in dpe2 pollen. Knockout of Pho2 resulted in a semi-sterile phenotype. We failed to obtain homozygous dpe2 pho2 double mutant lines. Our results demonstrate that maltose catalyzed by DPE2 to glucose is the main energy source for pollen germination and pollen tube elongation, while Pho2 might partially compensate for deficiency of DPE2.

A conserved motif is essential for the correct assembly of proglutelins and for their export from the endoplasmic reticulum in rice endosperm.

Rice glutelins are initially synthesized as 57-kDa precursors at the endoplasmic reticulum (ER) and are ultimately transported into protein storage vacuoles. However, the sequence motifs that affect proglutelin folding, assembly, and their export from the ER remain poorly defined. In this study, we characterized a mutant with nine amino acids deleted in the GluA2 protein, which resulted in specific accumulation of the GluA precursor. The deleted amino acids constitute a well-conserved sequence (LVYIIQGRG) in glutelins and all residues in this motif are necessary for ER export of GluA2. Immunoelectron microscopy and stable transgenic analyses indicated that proglutelins with deletion of this motif misassembled and aggregated through non-native intermolecular disulfide bonds, and were deposited in ER-derived protein bodies (PB-Is), resulting in conversion of PB-Is into a new type of PB. These results indicate that the conserved motif is essential for proper assembly of proglutelin. The correct assembly of proglutelins is critical for their segregation from prolamins in the ER lumen, which is essential for enabling the export of proglutelin from the ER and for the proper formation of PB-Is. We also found that the interchain disulfide bond between acidic and basic subunits is not necessary for their assembly, but it is required for proglutelin folding.

A ß-Ketoacyl-CoA Synthase Is Involved in Rice Leaf Cuticular Wax Synthesis and Requires a CER2-LIKE Protein as a Cofactor.

Cuticular waxes are complex mixtures of very-long-chain fatty acids (VLCFAs) and their derivatives, forming a natural barrier on aerial surfaces of terrestrial plants against biotic and abiotic stresses. In VLCFA biosynthesis, ß-ketoacyl-CoA synthase (KCS) is the key enzyme, catalyzing the first reaction in fatty acid elongation and determining substrate specificity. We isolated a rice (Oryza sativa) wax crystal-sparse leaf 4 (WSL4) gene using a map-based cloning strategy. WSL4 is predicted to encode a KCS, a homolog of Arabidopsis (Arabidopsis thaliana) CER6. Complementation of the mutant wsl4-1 with WSL4 genomic DNA rescued the cuticular wax-deficient phenotype, confirming the function of WSL4 The load of wax components longer than 30 carbons (C30) and C28 were reduced markedly in wsl4-1 and wsl4-2 mutants, respectively. Overexpression of WSL4 increased the cuticular wax load in rice leaves. We further isolated a cofactor of WSL4, OsCER2, a homolog of Arabidopsis CER2, by coimmunoprecipitation and confirmed their physical interaction by split-ubiquitin yeast two-hybrid experiments. Expression of WSL4 alone in elo3 yeast cells resulted in increased C24 but did not produce VLCFAs of greater length, whereas expressing OsCER2 alone showed no effect. Coexpression of WSL4 and OsCER2 in elo3 yeast cells yielded fatty acids up to C30. OsCER2 with a mutated HxxxD motif (H172E, D176A, and D176H) interrupted its interaction with WSL4 and failed to elongate VLCFAs past C24 when expressed with WSL4 in elo3 yeast cells. These results demonstrated that WSL4 was involved in VLCFA elongation beyond C22 and that elongation beyond C24 required the participation of OsCER2.

Plastidial Disproportionating Enzyme Participates in Starch Synthesis in Rice Endosperm by Transferring Maltooligosyl Groups from Amylose and Amylopectin to Amylopectin.

Plastidial disproportionating enzyme1 (DPE1), an α-1,4-d-glucanotransferase, has been thought to be involved in storage starch synthesis in cereal crops. However, the precise function of DPE1 remains to be established. We present here the functional identification of DPE1 in storage starch synthesis in rice (Oryza sativa) by endosperm-specific gene overexpression and suppression. DPE1 overexpression decreased amylose content and resulted in small and tightly packed starch granules, whereas DPE1 suppression increased amylose content and formed heterogeneous-sized, spherical, and loosely packed starch granules. Chains with degree of polymerization (DP) of 6 to 10 and 23 to 38 were increased, while chains with DP of 11 to 22 were decreased in amylopectin from DPE1-overexpressing seeds. By contrast, chains with DP of 6 to 8 and 16 to 36 were decreased, while chains with DP of 9 to 15 were increased in amylopectin from DPE1-suppressed seeds. Changes in DPE1 gene expression also resulted in modifications in the thermal and pasting features of endosperm starch granules. In vitro analyses revealed that recombinant DPE1 can break down amylose into maltooligosaccharides in the presence of Glc, while it can transfer maltooligosyl groups from maltooligosaccharide to amylopectin or transfer maltooligosyl groups within and among amylopectin molecules in the absence of Glc. Moreover, a metabolic flow of maltooligosyl groups from amylose to amylopectin was clearly identifiable when comparing DPE1-overexpressing lines with DPE1-suppressed lines. These findings demonstrate that DPE1 participates substantially in starch synthesis in rice endosperm by transferring maltooligosyl groups from amylose and amylopectin to amylopectin.

Non-syndromic tooth agenesis in two Chinese families associated with novel missense mutations in the TNF domain of EDA (ectodysplasin A).

Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA) gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.

A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy.

Directional and controllable degradation of extracellular matrix mediated by the uPA-uPA receptor (uPAR) system is ubiquitously implicated in tumor establishment, invasion and metastasis. Targeting the excessive activation of this system as well as the proliferation of the tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt the tumor development. In this study, we created a fusion protein (ALV), comprising the aminoterminal fragment (ATF) of urokinase and VAS, the antiangiogenic functional domain of vasostatin. The antitumor activity of this hybrid molecule was evaluated with both in vitro and in vivo experiments. Cell adhesion and motility assays demonstrated that ALV, owing to its ATF moiety, could interact with uPAR on the tumor cell surface with high affinity and specificity, and thereby might competitively inhibit the plasmin activation by localized urokinase and contribute to the suppression of tumor invasion. These results and speculations were validated by zymography assay and Matrigel invasion assay. In addition, ALV exhibited an improved inhibitory efficacy against endothelial cell (EC) proliferation and capillary vessel formation in a 3D angiogenesis model, proving that ATF and VAS, when fused into a chimeric molecule, cooperatively inhibited angiogenesis by targeting both the interaction of uPA and uPAR on cell surface (by ATF) and EC proliferation (mainly by VAS). Animal model confirmed that, at the same molar dose, ALV produced significantly higher therapeutic benefit than VAS and ATF in terms of tumor growth delay and mice survival prolongation. Conclusively coupling VAS with the uPAR ligand ATF resulted in an improved antineoplastic activity, which may show a novel avenue for the design of tumor therapeutic drugs.

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus).

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum.

Expression and purification of intact and functional soybean (Glycine max) seed ferritin complex in Escherichia coli.

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by Ni2+ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.

A novel genotyping strategy based on allele-specific inverse PCR for rapid and reliable identification of conditional FADD knockout mice.

The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice.

A novel ferritin gene, SferH-5, reveals heterogeneity of the 26.5-kDa subunit of soybean (Glycine max) seed ferritin.

A novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously. All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex, H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.