GBSSs Interaction Protein OsGBP Regulates Rice Starch Synthesis

Granular-bound starch synthase enzymes ? and ?(GBSS?,GBSS?)are responsible for amylose synthesis in endosperm and leaves of rice,respectively.Amylose synthesis is a complex process,the regulatory mechanism still not been elucidated very clear.To understand whether and how GBSS? is regulated at the protein level,we screened a rice c DNA library(10 DAF)for GBSS?-interacting proteins by yeast two-hybrid.Several positive clones were obtained.One of the proteins containing the CBM48(Carbohydrate binding model)domain,Os02g0135900,was interesting for its potential domain was able to bind to carbohydrates such as starch.After gene knockout(CRISPR-Cas9),two homozygous mutants of different mutations were obtained,and were named osgbp-1 and osgbp-2.The mutants were used for indepth investigation,the main results are as follows:1.Two homozygous mutants osgbp-1 and osgbp-2(Zhonghua11 background)were obtained using the CRISPR/Cas9 gene editing system.gene.1 bp and 2 bp were deleted in the second exon of the Os GBP respectively,resulting in the protein frameshift and premature termination.2.The osgbp mutants showed an opaque in the belly of endosperm,and the starch granules in the belly of the endosperm were round and loosely arranged.Transmission electron microscopy showed that the single starch granules in compound starch granules of endosperm(10 DAF)became smaller and the arrangement is very loose.Compared with the wild type,the total starch and amylose content decreased significantly,the ratio of medium and short chain(DP6-14)of amylopectin increased,the medium and long chain(DP15-60)decreased,the fatty acid content increased,the gel consistency and the viscosity reduced;osgbp starch was more susceptible to gelatinization in 7-9 M urea compared to wild type.Compared to the wild type,the mutants 1000-grain weight decreased significantly.The results showed that the loss function of Os GBP had an important effect on seed development and physicochemical properties of starch in endosperm.3.The leaves of osgbp were brown instead of blue after iodine staining(end of day).Compared with wild type,the amylose content of the mutant leaves could not be detected and the total starch content decreased significantly.The results of transmission electron microscopy showed that few starch granules were found in the chloroplast of osgbp.Above all Os GBP may play an important role in the synthesis of starch in leaves.4.Os GBP was a constitutively expressed gene,it mainly expressed in roots,stems,leaves,panicles and different developmental stages endosperm,and higher expression was found in leaves and developing endosperm.Tissue expression analysis and GUS staining results can confirm with each other.The subcellular localization of Os GBP protein showed that it was localized on the plastid(chloroplast)and could colocalized with GBSS? and GBSS?,suggesting that Os GBP may be involved in the amylose synthesis.5.The C-terminus of the Os GBP protein contains a CBM48 domain.starch binding experiments demonstrate that purified Os GBP could bind to starch by its CBM48 domain protein in vitro.6.By extracting the starch granule-bound protein from the endosperm and leaves,the gel was stained by silver showed that the protein abundance of GBSS? and GBSS? on starch granules of osgbp endosperm and leaf was decreased,and GBSS? was extremely significant;After extracting the total protein in the leaves and endosperm,and performing the supernatant and precipitation fractionation,it was found that there was a large amount of free GBSS? and GBSS? in the mutant supernatant,but no GBSS? band was detected in the leaf pellet(including starch),but some GBSS? was still present in the seed pellet.The above results indicate that Os GBP can help GBSS bind to starch granules and mainly play a role in the leaves,there may be other compensatory pathways in the endosperm.