Hiromi Aoki

A simple Gateway-assisted construction system of TALEN genes for plant genome editing.

TALEN is an artificial nuclease being applied for sequence-specific genome editing. For the plant genome editing, a pair of TALEN genes is expressed in the cells, and a binary plasmid for Agrobacterium-mediated transformation should be assembled. We developed a novel procedure using the Gateway-assisted plasmids, named Emerald–Gateway TALEN system. We constructed entry vectors, pPlat plasmids, for construction of a desired TALEN gene using Platinum Gate TALEN kit. We also created destination plasmid, pDual35SGw1301, which allowed two TALEN genes to both DNA strands to recruit using Gateway technology. Resultant TALEN genes were evaluated by the single-strand annealing (SSA) assay in E. coli cells. By this assay, the TALENs recognized the corresponding targets in the divided luciferase gene, and induced a specific recombination to generate an active luciferase gene. Using the TALEN genes constructed, we created a transformant potato cells in which a site-specific mutation occurred at the target site of the GBSS gene. This suggested that our system worked effectively and was applicable as a convenient tool for the plant genome editing.

Arabidopsis thaliana FLO2 is Involved in Efficiency of Photoassimilate Translocation, Which is Associated with Leaf Growth and Aging, Yield of Seeds and Seed Quality

FLO2, FLOURY ENDOSPERM 2, is highly conserved in higher plants, and rice FLO2 has been predicted to be involved in regulation of accumulation of storage compounds. We analyzed the function of Arabidopsis thaliana FLO2 (AtFLO2) because A. thaliana set structurally different seeds from those of rice. Although the flo2 mutant of A. thaliana showed normal germination, inflorescence and morphogenesis of flowers, peculiar phenotypes on leaves and siliques were observed, suggesting that this gene played important roles during both the vegetative and reproductive stages. The mutant leaves showed a decrease in chloroplast numbers, and increased total biomass with faster growth. When grown in high light intensity conditions, it was observed that aging events were induced. The flo2 mutant showed depressed transportation of photoassimilates into the sink organs. In the reproductive stage, the flo2 mutant had significantly smaller size siliques, causing a reduced yield of seeds. These seeds were structurally weak, and the quality of seeds was significantly lowered, with reduction of accumulation of storage compounds by seeds. A positron-emitting tracer imaging system (PETIS) analysis detected a decreased amount of photoassimilate transport in the flo2 mutant. Therefore, it was presumed that the phenotypes of the flo2 mutant were caused by reduced performance of translocation or transportation of the photoassimilates. Our observation suggests that AtFLO2 is strongly involved in regulation of translocation and transport of assimilates, and contributes greatly to quality control of the various processes involving substance supply or transfer, such as photoassimilation, leaf enlargement, yield of seeds in a silique and accumulation of seed storage compounds.