Localization of transgene-derived friabilins in rice endosperm cells

Abstract

Puroindoline-a (Pina), puroindoline-b (Pinb) genes in the wheat hardness-locus region encode 15-kDa friabilin proteins, whose accumulation in the endosperm leads to grain softness texture. In wheat, the PINA and PINB friabilins are associated with starch granules in the endosperm cell, while there is no friabilin in rice. The rice endosperm structure consisting of compound starch granules is fundamentally different from that in wheat. We previously produced two different lines of transgenic rice plants with the large genomic fragment including Pina or Pinb of Aegilops tauschii. However, localization of exogenous friabilins in the rice endosperm cell still remains to be determined. In the present study, we stacked the two different transgenic rice lines. The F 4 seeds of the stacked line, in which the homozygosity of the Pina and Pinb transgenes was checked by FISH analysis, were used for histochemical analysis of the endosperm cell. Immunodetection of PINA and PINB proteins using the Durotest antibody showed that wheat-derived friabilins were localized between compound starch granules as well as between starch granules in the rice endosperm cell. This suggests that such localization of the friabilins might prevent tight interaction between the compound starch granules and between the starch granules in the rice endosperm, leading to its soft texture.

Production of bioactive wheat puroindoline proteins in Nicotiana benthamiana using a virus-based transient expression system

“…Prior studies have identified unreduced PINs at 15 kDa, and the antibody probably did not react with PINs after reduction of disulphides or denaturation for immunocytochemical analyses, but the epitope recognised by this antibody has not been mapped (Greenwell, 1992;Wiley et al, 2007). Recently, this antibody has been used for immuno-localisation of PINs in the endosperm cells of transgenic rice, which found them localised between starch granules (Fujiwara et al, 2014). correct formation of tertiary structures in these methods, while the bioactivity was lost in denaturing conditions.…”