Active-type starch synthase (SS) IIa from indica rice partially complements the sugary-1 phenotype in japonica rice endosperm

Abstract

Key message Introduction of higher SSIIa activity to mild-type isa1 mutant by crossing results in restoration of crystallinity, starch granule structure, and production of plump seeds. Abstract Isoamylase 1 (ISA1) removes improper α-1, 6 glycosidic branches of amylopectin generated by starch branching enzymes and is essential for the formation of proper amylopectin structure. Rice isa1 (sug-1) mutants in japonica cultivar with less-active starch synthase IIa (SSIIa) and low granule-bound SSI (GBSSI) expression display wrinkled seed phenotype by accumulating water-soluble phytoglycogen instead of insoluble amylopectin. Expression of active SSIIa in transgenic rice produced with a severe-type isa1 mutant accumulated some insoluble glucan with weak B-type crystallinity at the periphery of seeds but their seeds remained wrinkled. To see whether introduction of high levels of SSIIa and/or GBSSI can restore the grain filling of the mild-type sug-1 mutant (EM653), new rice lines (SS2a gbss1 L isa1, ss2a L GBSS1 isa1, and SS2a GBSS1 isa1) were generated by crossing japonica isa1 mutant (ss2a L gbss1 L isa1) with wild type indica rice (SS2a GBSS1 ISA1). The results showed that SS2a gbss1 L isa1 and SS2a GBSS1 isa1 lines generated chalky plump seeds accumulating insoluble amylopectin-like glucans with an increase in DP 13-35, while ss2a L GBSS1 isa1 generated wrinkly seeds and accumulated soluble glucans enriched with DP < 13. Scanning electron microscopic observation of cross-section of the seeds showed that SS2a gbss1 L isa1 and SS2a GBSS1 isa1 produced wild type-like polygonal starch granules. These starches showed the A-type crystallinity comparable to the wild type, while the japonica isa1 mutant and the transgenic rice do not show any or little crystallinity, respectively. These results indicate that introduction of higher SSIIa activity can mostly complements the mild-type sug-1 phenotype.

Molecular Hydrogen Increases Quantitative and Qualitative Traits of Rice Grain in Field Trials

“…The following are available online at https://www.mdpi.com/article/10 .3390/plants10112331/s1, Table S1: The sequences of primers for qPCR, References [65][66][67][68][69][70][71][72][73][74][75] are cited in the Supplementary Materials.…”

Improving Agricultural Traits While Maintaining High Resistant Starch Content in Rice

“…Typical japonica rice cultivars exhibit low SSIIa and low GBSSI activities, resulting in shorter amylopectin branches and lower amylose content, compared with typical indica rice cultivars (Isshiki et al 1998 ; Nakamura et al 2005 ). Introduction of gene(s) encoding active-type SSIIa ( SS2a allele) and high GBSSI proteins ( GBSS1 allele) into japonica ss3a ss4b or isa1 mutant has been shown to elongate the amylopectin branches and increase amylose content and seed weight (Itoh et al 2017 ; Crofts et al 2021a , b ). Therefore, introduction or deletion of specific starch biosynthetic genes can alter the starch structure and increase the seed weight.…”

FLOURY ENDOSPERM 6 mutations enhance the sugary phenotype caused by the loss of ISOAMYLASE1 in barley

“…PUL hydrolyzes α-1,6 linkages of amylopectin and pullulan, unlike ISA1 hydrolyzes amylopectin and glycogen but not pullulan (Nakamura 1996). We used proteins from developing rice grains as a positive control, and activity bands from rice were assigned according to the previous reports (Fujita et al 2009;Crofts et al 2022). The DBE activity staining detected ISA1, PUL, and plastidial phosphorylase (PHO1) bands from rice grains (Fig.…”