OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)and β-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based on α-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.

Starch,the main energy storage substance,accounts for 80%-90% of the total mass in mature rice grains,and comprises two different glucose polymers:amylose and amylopectin(Thompson,2000;Du et al,2019).Starch synthesis is a complex process involving a series of biosynthetic enzymes.OsPho1mutations cause severe reductions in rice endosperm starch(Satoh et al,2008).The Pho1 reaction is freely reversible and can either synthesize or degrade synthetic glucan.Fettke et al(2010)showed that α-D-glucose-1P(G1P)is efficiently and directly bound to reserve starch granules in potato tuber cells,demonstrating that the G1P pathway acts in parallel with AGPase pathway in starch biosynthesis.The OsPho1-OsDpe1 complex uses a wider range of substrates for enhanced synthesis of larger Malto-oligosaccharides(MOS)than each enzyme,and significantly improves the substrate affinity of OsPho1 at 30 °C.OsDpe1 enablesOsPho1 to perform glycosylation reactions involving maltose and maltotriose(Hwang et al,2016).The role of OsPho1 in starch metabolism remains unclear.Therefore,we need to enrich our knowledge of OsPho1’s potential function.

Temporal and spatial expression showed thatOsPho1expression was mainly biased toward developing grains and highly expressed at 5 DAF(Fig.S1-A).A vector withGUSreporter gene driven by theOsPho1promoter was transformed into wildtype ZH11.Histochemical observation ofGUSactivity in T0positive transgenic plants corroborated thatOsPho1presented the seeds’highly expression patterns(Fig.S1-B).The protoplasts transiently transformed,and laser scanning confocal microscope showed that OsPho1-GFP is localized at chloroplast cells(Fig.S1-C).

Compared with ZH11,11 bp deletion incr-pho1-31and 4 bp deletion incr-pho1-32,18 bp deletion in bothcr-pho1-30andcr-pho1-34,and a large fragment deletion incr-pho1-41were observed.All these mutations changed the amino acid and protein sequence(Fig.S2-C).Six amino acids(Ile,Gly,Pro,Ser,Pro and Gln)were successively deleted incr-pho1-34,and a translation premature stop was resulted due to two stop codons(TGA and TAA)incr-pho1-37.SDS-PAGE and western blotting results showed that ZH11 had normal OsPho1 protein levels,butcr-pho1-30,cr-pho1-31,cr-pho1-32,cr-pho1-34,cr-pho1-37andcr-pho1-41homozygous mutants lost OsPho1 protein because of the deletion mutation(Fig.S2-D).Among the identified mutants,cr-pho1-34andcr-pho1-37were selected for further phenotypic analysis.

ZH11 showed transparent endosperm,whereas brown rice and milled rice ofcr-pho1-34andcr-pho1-37displayed whitecored and chalky endosperm,indicating the phenotypes of the different mutation types were consistent(Fig.1-A and Fig.S2-E).To eliminate the influence of the environment,T1plants were used for the co-segregated genetic analysis.During the growth process,the plants’ architectures showed no any differences at the booting stage(Fig.S3-A).These experiments illustrated thatOsPho1controls the white-core chalkiness trait.The filling rate of thecr-pho1-37mutant was slower than ZH11 from 3 to 27 DAF,resulting in a reduced fresh 1000-grain weight(Fig.S4-B).Compared with the wild-type,the grain length of the mutants increased(Fig.S4-E).The yield per plant was lower in the mutants(Fig.S4-H).No obvious differences were observed in the tiller number per plant(Fig.S4-C).

Fig.1.Phenotype identification and physicochemical characteristics analyses of Zhonghua 11(ZH11)and mutants in T1 generation.

The transection of mature endosperm was observed by the scanning electron microscopy(SEM).The starch granules observed in the middle of the ZH11 endosperm were densely packed with an irregular polyhedron shape,whereas many abnormal starch granules and small rounded granules were present in the central endosperm ofcr-pho1-34andcr-pho1-37(Fig.S5-A to -I).The developing endosperm cells at 9 DAF in the wild-type and mutants were observed by the transmission electron microscopy(TEM).The results showed that the amyloplasts were filled with polyhedral granules and formed a typically complex starch structure in the wild-type(Fig.S5-J),whereas many rounded starch granules with enlarged gaps between them in the central regions of the endosperm cells were observed in the mutants(Fig.S5-K and -L).These results indicated thatOsPho1plays an important role in the formation of compound starch granules and amyloplast development in endosperm cells.

Due to the abnormal starch granules observed in the endosperm ofcr-pho1mutants,the physicochemical properties of starch were also examined.The data showed no differences in the total starch content and soluble sugar content(Fig.1-B and -E),whereas the amylose contents incr-pho1-34andcr-pho1-37seeds increased by about 2.95% and 3.05%,respectively,compared with ZH11(Fig.1-C).In addition,total protein contents decreased and total lipid contents increased(Fig.1-D and -F).Due to the important role ofOsPho1in amylopectin synthesis,specifically,at the α-glucan process stage,the content of α-glucan increased by 2.68% and 12.16%,compared with ZH11(Fig.1-G).Gelatinization properties of the starch grains in ZH11 and mutants were also detected.The gel consistency of the starch grains in the mutants increased significantly compared with ZH11(Fig.1-H).The chain length distribution analysis showed that short chains with degree of polymerization(DP)values of 6-12 glucose units in mutants increased significantly compared with ZH11,whereas the proportions of intermediate chains with DP values in the range of 13 to 24 in mutants were lower(Fig.1-I).We measured rice flour starch pasting properties using a rapid visco-analyzer(RVA),the mutants maintained lower levels with peak values of 43.28% and 48.23%,and final viscosity values of 61.60%and 58.36% compared with ZH11,respectively(Fig.1-J).The thermal gelatinization temperatures of the starch from ZH11,cr-pho1-34andcr-pho1-37were analyzed via differential scanning calorimetry.The results showed that the onset(To),peak(Tp)and conclusion(Tc)temperatures of gelatinization in mutants were significantly lower than those of ZH11 by 6 °C-8 °C,with the gelatinization enthalpy of starch(ΔH)declining(Fig.1-K).Various concentrations(0-9 mol/L)of urea solution were added to powdered starch,and the gelatinization characteristics of mutantstarches had the most significant differences in 4-5 mol/L urea compared to that of ZH11.The results also indicated that the starch ofcr-pho1endosperm was more easily gelatinized in 4-5 mol/L urea solution than that of ZH11(Fig.1-L).The endosperm starches of the mutants were more difficulty to be gelatinized than those of ZH11(Fig.1-M).In summary,the physicochemical characteristics of the endosperm starches in mutants were significantly different from those in ZH11,presenting thatOsPho1plays an important role in rice starch synthesis.

In the natural field environment,expression levels of most genes in the mutants were higher than those in ZH11,potentially indicating a feedback regulation mechanism.In particular,expression levels ofAGPS2b,ISA2,ISA3andBT1were significantly increased,withpPGMexpression levels only slightly increased(Fig.S7).

RNA expression analyses of 5 DAF seeds harvested from the T0generation showed that the RNA expression levels of two transgenic plants increased compared with ZH11(Fig.S8-A).Some transgenic plant grains displayed more chalkiness,whereas ZH11 was transparent(Fig.S8-B).Both knockdown and overexpression ofOsPho1may lead to grain chalkiness.

In future research,it will be useful to investigate protein interactions further to explain the potential mechanism using a yeast two-hybrid system or testing wild-type and mutant enzymatic activities of OsPho1,pPGM and AGPase at different temperatures.

ACKNOWLEDGEMENT

This study was supported by the National Science Foundation of China(Grant No.3197150429).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

File S1.Methods.

Fig.S1.Expression at different tissues,β-glucuronidase staining and subcellular localization in rice protoplasts.

Fig.S2.Vector map of Cas9/guide RNA and clustered regularly interspaced short palindromic repeats-associated endonuclease 9(CRISPR/Cas9)-mediated editing ofOsPho1and phenotype identification.

Fig.S3.Plant architecture pictures at booting stage and co-segregated genetic analysis.

Fig.S4.Phenotype identification of the ZH11 and mutants in the T1generation.

Fig.S5.Scanning and transmission electron microscopy images of ZH11 and mutantsin the T1generation.

Fig.S6.Scanning electron microscope slitting observation of wild-type and mutants.

Fig.S7.Relative expression level of starch synthesis related genes in nature field environment.

Fig.S8.qRT-PCR analysis and brown rice phenotype of overexpression transgenic lines.

Table S1.Primers used in this study.