Integration of a phenolic-acid recovery step in the CaCCO process for efficient fermentable-sugar recovery from rice straw.

An advanced sugar-platform bioprocess for lignocellulosic feedstocks by adding a phenolic-acid (PA: p-coumaric acid and ferulic acid) recovery step to the CaCCO process was designed. For efficient PA extraction, pretreatment was 95°C for 2h, producing a yield of 7.30 g/kg-dry rice straw (65.2% of total ester-linked PAs) with insignificant effects on saccharification. PAs were readily recovered in solution during the repeated washings of solids, and the glucose yield, after 72-h saccharification of the washed solids, was significantly improved from 65.9% to 70.3-72.7%, suggesting the removal of potential enzyme inhibitors. The promotion of xylose yield was insignificant, probably due to 13.1-17.8% loss of xylose residues after washing(s). This new bioprocess, termed the SRB (simultaneous recovery of by-products)-CaCCO process, would effectively produce fermentable sugars and other valuables from feedstocks, strengthening the platform in both economic and environmental terms.

Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis.

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on ß-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.

An improved CARV process for bioethanol production from a mixture of sugar beet mash and potato mash.

A mixed mash of sugar beet roots and potato tubers with a sugar concentration of 23.7% w/v was used as a feedstock for bioethanol production. Enzymatic digestion successfully reduced the viscosity of the mixture, enabling subsequent heat pretreatment for liquefaction/sterilization. An energy-consuming thick juice preparation from sugar beet for concentration and sterilization was omitted in this new process.

Septum formation in amyloplasts produces compound granules in the rice endosperm and is regulated by plastid division proteins.

Storage tissues such as seed endosperm and tubers store starch in the form of granules in the amyloplast. In the rice (Oryza sativa) endosperm, each amyloplast produces compound granules consisting of several dozen polyhedral, sharp-edged and easily separable granules; whereas in other cereals, including wheat (Triticum aestivum), barley (Hordeum vulgare) and maize (Zea mays), each amyloplast synthesizes one granule. Despite extensive studies on mutants of starch synthesis in cereals, the molecular mechanisms involved in compound granule synthesis in rice have remained elusive. In this study, we expressed green fluorescent protein (GFP) fused to rice Brittle1 (BT1), an inner envelope membrane protein, to characterize dividing amyloplasts in the rice endosperm. Confocal microscopic analyses revealed that a septum-like structure, or cross-wall, containing BT1-GFP divides granules in the amyloplast. Plastid division proteins including FtsZ, Min and PDV2 play significant roles not only in amyloplast division, but also in septum synthesis, suggesting that amyloplast division and septum synthesis are related processes that share common factors. We propose that successive septum syntheses which create sections inside the amyloplast and de novo granule synthesis in each section are primarily responsible for the synthesis of compound granules.

Amyloplast division progresses simultaneously at multiple sites in the endosperm of rice.

The amyloplast, a form of differentiated plastid, proliferates in sink tissues, where it synthesizes and stores starch granules. Little is known about the molecular mechanism for amyloplast division and development. The rice (Oryza sativa) endosperm provides an excellent model system for studying molecular mechanisms involved in amyloplast division and starch synthesis. We compared amyloplast division processes in the endosperm of wild type and a mutant of ARC5, a member of the dynamin superfamily. Plant growth and fertility of arc5 were not significantly different from the wild type. Unlike binary fission of chloroplast in the leaf, small amyloplasts in the endosperm of wild type divide simultaneously at multiple sites, generating a beads-on-a-string structure. In addition, large amyloplasts divide by budding-type division, giving rise to small amyloplasts attached to their surfaces. ARC5 and FtsZ2-1 fused to fluorescent proteins were targeted to the constriction sites in dividing amyloplasts. Both the loss of function of ARC5 and overexpression of ARC5 fusion proteins in the endosperm did not produce spherical amyloplasts with increased diameter, but produced either fused amyloplasts with thick connections or pleomorphic types, suggesting that proper stoichiometry between ARC5 and other components in the amyloplast division machinery is necessary for the completion of the late stage of amyloplast division. The size distribution of starch granules purified from arc5 was shifted to small and the starch gelatinization peak temperature was significantly higher than for wild-type starch, suggesting that amyloplast division processes have a significant effect on starch synthesis.

Propanil and swep inhibit 4-coumarate:CoA ligase activity in vitro.

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) in the phenylpropanoid pathway in plants has attracted interest as a novel target for developing effective plant growth inhibitors (PGIs). In a previous study in which the 4CL inhibitory activity of 28 existing herbicides was investigated using an optimized in vitro screening assay, 4CL activity was found to be strongly inhibited by propanil and swep at 100 microM. Here, further experimental evidence is provided to substantiate the previous result. Using 4-coumaric acid as substrate, tobacco 4CL activity was inhibited by propanil or swep in a concentration-dependent manner, with 50% inhibition concentrations (I(50)) of 39.6 and 6 microM respectively. These herbicides also exhibited uncompetitive inhibition towards 4-coumaric acid. Furthermore, 4CLs from several plant species were inhibited by the herbicides within a range from 1 to 50 microM. It is proposed that these herbicides have another site of action as a result of the inhibition of 4CL in the phenylpropanoid pathway, and this enzyme represents a new target site for the development of PGI.

An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase.

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) exists only in plants and plays an important role in the phenylpropanoid pathway. Identification of inhibitors targeting 4CL provides a novel approach for developing effective plant growth inhibitors (PGIs). The full-length gene of tobacco 4CL (Nt4CL1) was cloned and expressed in Escherichia coli Cast & Chalm. The recombinant 4CL protein was extracted and purified by several purification steps including gel-filtration and anion-exchange chromatography. 4CL activity assay was miniaturized and optimized using a 96-well microplate and a reader. Among 28 existing herbicides, propanil and swep strongly inhibited in vitro 4CL enzyme activity, and they were selected for further studies. The process of this assay can be developed into a high-throughput screening system of PGI targeting 4CL in the phenylpropanoid pathway.