Rice Mutants Lacking Starch Synthase I or Branching Enzyme IIb Activity Altered Starch Biosynthetic Protein Complexes

Abstract

Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60–700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700–kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

Effects of BEIIb-Deficiency on the Cluster Structure of Amylopectin and the Internal Structure of Starch Granules in Endosperm and Culm of Japonica-Type Rice

“…Is there any scenario explaining the different contribution of BEIIa to amylopectin biosynthesis either in the presence or absence of BEIIb? As already mentioned above, Crofts et al (2018) recently reported that the BEIIb protein must play an important role in the formation of several kinds of protein complexes among starch biosynthetic isozymes in rice endosperms using a japonica -type WT rice cultivar Nipponbare and EM10. For example, BEIIb forms a protein complex among BEI, SSI, SSIIa, and SSIIIa.…”

“…It is known that rice BEIIb has a very strict chain-length specificity because it synthesizes almost exclusively the DP7 and DP6 chains by attacking the DP12-14 chains ( Nakamura et al, 2010 ; Sawada et al, 2014 ). It can be also pointed out the rice endosperm BEIIb plays a key role in the formation of enzyme-enzyme complexes ( Crofts et al, 2015 , 2017 , 2018 ). These properties of BEIIb might be related to its role during the formation of cluster chains and hence BEIIb could affect profoundly the crystalline characteristics and physiochemical properties of starch granules.…”

“…We hypothesize that BEIIa was active in the culm whereas it was inactive in the endosperm in the presence of BEIIb. The transition between the active and inactive state of the BEIIa activity might be controlled by the capacity of BEIIb through its pivotal role in forming the protein-protein complex (see Crofts et al, 2017 , 2018 ), although the mechanism remains to be resolved in the future study.…”

Structure and Function of Branching Enzymes in Eukaryotes

“…Furthermore, it has been reported that one of the recombinant OsBEI, OsBEIIa, or OsBEIIb interacts with recombinant starch synthase I from rice under in vitro conditions to produce branched α-glucan (96). Alternative protein complexes are also found in rice mutants lacking BEIIb activity or starch synthase isozyme I, IIa, and/or IIIa activities (97,98). (Fig.…”

Starch Components, Starch Properties and Appearance Quality of Opaque Kernels from Rice Mutants

“…The amylose is synthesized by granule-bound starch synthase I, and the amylopectin is synthesized by the combined actions of soluble starch synthase, starch branching enzyme, and starch debranching enzyme. Mutants with a mutation or expression change of starch-synthesis-related enzyme genes can alter starch components [24,25].…”