Introduction

Rice grain chalkiness refers to the opaque spots in the endosperm which can affect the quality, specially the appearance of the rice grain (Fitzgerald et al. 2000; Yamakawa et al. 2007). Chalky grains are predisposed to rupture during polishing leading to a decrease in the proportion of edible rice (Wan et al. 2007). Chalk in rice grains directly and indirectly contributes to both the proportion of chalkiness and broken grains, which are two of the major traits that most markets base on to dictate the price of rice (Fitzgerald et al. 2009). Floury endosperm is one phenotype of chalky rice with loosely packed starch granules in the center and normal outer layer of the endosperm. Five loci of rice floury endosperm (flo) have already been reported. The loci of flo-1, flo-2, and flo-3 were located on chromosome 5, 4, and 4, respectively (Satoh and Omura 1981; Kumamaru et al. 1997; Nishio and Iida 1993). The flo4 are mutants with insertional mutations in the OsPPDKB which is an important modulator of carbon flow for starch and lipid biosynthesis during grain-filling (Kang et al. 2005). The rice SSIIIa mutants named flo5 are generated by T-DNA insertions in the coding sequence of the gene, resulting in a white-core floury endosperm (Ryoo et al. 2007). Recently, a mutant which lost the function of a gene that encodes a cell wall invertase required for carbon partitioning during early grain-filling named gif1 has been reported, and the grains of this mutant also have the floury endosperm phenotype (Wang et al. 2008). Another mutant named flo2 rice mutant has been reported, and its wild-type (WT) gene named FLO2 which is a superior conductor of the novel regulatory cascade of endosperm organogenesis may also have important roles in the response to high-temperature stress (She et al. 2010). A new floury endosperm mutant named flo(a) whose gene was located on chromosome 4 by positional cloning was reported in 2010. Preliminary results showed that the protein of FLO(a) might play a significant role in rice starch biosynthetic pathways (Qiao et al. 2010). The deleted gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1) in a rice somaclonal mutant T3612 that produces small grains with a floury endosperm was revealed by the positional cloning of the mutation (Han et al. 2011).

Starch biosynthesis enzymes in the cereal endosperm include AGPase, granule-bound starch synthase, soluble starch synthase, starch branching enzyme, starch debranching enzyme, and plastidial starch phosphorylase (Pho1) (Jeon et al. 2010; James et al. 2003). Based on the results from studies on flo serial mutants, we can know that the rice endosperm will develop abnormally if each of these essential enzymes in cereal starch biosynthesis is defective. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first step of starch biosynthesis in plants (Hannah and Greene 1998). Observations from potato, maize, barley, and rice indicate that AGPase is a rate-limiting enzyme in starch biosynthesis (Hannah and Greene 1998; Denyer et al. 2004; Giroux et al. 2003; Dickinson and Preiss 1969). It is the enzymatic site for the regulation of storage polysaccharide accumulation in plants and bacteria, which is allosterically activated or inhibited by metabolites of energy flux (Dickinso and Preiss 1969; Furlong and Preiss 1969; Haugen et al. 1976; Preiss et al. 1975; Jin et al. 2005; Dickinson and Preiss 1969). AGPase is a tetramer that comprised two small and two large subunits, each of which is encoded by distinct genes (Okita et al. 1990). In rice, the AGPase gene family consists of two SSU genes (OsAGPS1 and OsAGPS2) and four LSU genes (OsAGPL1, OsAGPL2, OsAGPL3, and OsAGPL4) (Akihiro et al. 2005; Lee et al. 2007; Ohdan et al. 2005; Jeon et al. 2010). The protein structure of AGPase in potato has also been reported (Jin et al. 2005). For cereals, it is largely extra-plastidial in the endosperm and it is functionally different from non-cereal plants (James et al. 2003). This could be due to a process that may have originated from a whole-genome-duplication event in an early ancestor of the grasses (Comparot-Moss and Denyer 2009).

Here, a new floury endosperm mutant was isolated and named flo6 (floury endosperm 6) following the phenotype of floury endosperm. We report the phenotypic and candidate gene analysis of the flo6 gene in rice. The FLO6 gene which encodes a presumed rice AGPase large subunit OsAPL2 was isolated and sequenced to ensure that it was responsible for flo6 mutation. Thus, flo6 mutant was renamed with osagpl2-3 as a new mutant of OsAPL2.

Materials and Methods

Plants Materials

The flo6 mutant was isolated from M2 generation of rice cultivar Nipponbare (Oryza sativa L. ssp. japonica) by mutagenesis with ethyl methanesulfonate (EMS). Seeds (M0) were presoaked in water for 16 h, dried, and then treated with 0.4 % EMS solution for 8 h. The treated seeds were then washed several times with distilled water. All seedlings (M1) from the treated seeds were transplanted into the paddy field, and the seeds which constitute the M2 generation were harvested at maturity (Thang et al. 2010). An F2 population of grains derived from F1 plants that were obtained from the cross between flo6 mutant and normal rice cultivar R9703 (O. sativa L. ssp. indica) was used for genetic mapping. A total of 286F2 mutant individuals were identified from the population. In addition, 1,564 grains from F1 plants derived from the cross between flo6 and WT were used for genetic analysis.

Scanning Electron Microscopic Analysis

Grain samples for scanning electron microscopic analyses were directly dried to a critical point with liquid CO2, mounted on SEM stubs, and then coated with gold palladium. The mounted specimens were observed under an SEM (Hitachi TM-1000 Tabletop Microscope).

Cooking and Nutrition Quality Analysis

The measurement of protein content (PC) and amylose content (AC), gel consistency (GC), thermodynamic characteristics (GT) of rice floury, floury rapid viscosity analysis (RVA) profile, and amino acid content were conducted according to the standard of the Ministry of Agriculture (1988), the National Standard of the People’s Republic of China (1999); Xu et al. (2004); Bao et al. (2004) and Wu et al. (2002), respectively.

DNA Extraction and PCR Analysis

The rice grains were sterilized by the method of Kato et al. (2010). Rice genomic DNA was extracted from the plants grown in 1/2 MS medium at trefoil stage. The DNA extraction and PCR reaction system for mapping were according to the method by Li et al. (2010).

Linkage Analysis and Genetic Mapping

A total of 350 markers (300 SSR markers and 50 InDel markers) well distributed on the 12 chromosomes were taken from the rice database Gramene (http://www.gramene.org/markers/microsat/) and Shen et al. (2004), respectively. The markers showing polymorphisms between Nipponbare and R9703 were selected to determine the approximate chromosomal position of flo6 gene by using the bulked segregation analysis (BSA) method developed by Michelmore et al. (1991). Genetic mapping was according to the methods of Zhao et al. (2010) and Sui et al. (2012).

Development of New InDels Markers

Thirty-four InDel markers were designed in the present experiment by the methods of Li et al. (2010) and Wang et al. (2011). However, only one marker ID1-12 (forward, 5′-TAAGAGAGGTTGAAATGGAGTC-3′; reverse, 3′-ATTTATCTCAACAGACGGTTTT-5′) which has polymorphism between Nipponbare and R9703 was used for mapping the flo6 gene.

Sequencing Analysis of the Candidate Region

The rice Gramene database (http://www.gramene.org/) was referred to for candidate gene annotation. The special attention was drawn to a putative gene, LOC_Os01g44220, for analyzing the annotation information in the candidate region. To sequence the LOC_Os01g44220, ten sets of primers were designed and synthesized (Supplementary Table 1). Specific DNA fragment were amplified from the genomic DNA of flo6 mutant and WT and then cloned into TA cloning vector pMD19-T (Takara, Dalian, China) for sequencing.

In Silicon Analysis

The following proteomics tools: ProtParam (http://web.expasy.org/protparam/), TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/), PredictProtein (http://www.predictprotein.org/), SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html), TargetP (http://www.cbs.dtu.dk/services/TargetP/), SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/), CPHmodels 3.0 server (http://www.cbs.dtu.dk/services/CPHmodels/), multiple sequence alignment tools ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/), and software SweetMollyGrace 1.3 were used to analyze the predicted protein parameters of the WT and flo6. The SNP database dbSNP BUILD 136 (http://www.ncbi.nlm.nih.gov/snp/) was used to analyze the SNP sites. SITF sequence (http://sift.bii.a-star.edu.sg/www/SIFT_seq_submit2.html) was used to predict the effects of amino acid substitutions on protein function.

Results

Characterization of the flo6 Mutants

Although no distinct morphological differences were found between the WT and the flo6 mutant plants, some obvious differences can be found in the grains. The grains of the WT had transparent endosperms (Fig. 1a, f, g), while those of the flo6 mutant had white-core endosperms (Fig. 1b, h, i). Cross-sectioning of the grains revealed that the outer part of flo6 grain as well as that of the WT was normal. However, the interior of the mutant grain was floury-white which was not as that of the WT (Fig. 1j, k). The scanning electron microscopic results showed that the starch granules of the WT grains were packed, and the edges of starch granules in the cross-sectioning were sharp (Fig. 1l, m). The starch granules in the normal sections of the flo6 mutant were similar to those of the WT (Fig. 1n), whereas those in floury endosperm were round and loosely packed and some starch granules were compounded by film coating (Fig. 1o). The morphological trails of the grains were also analyzed in present experiment (Table 1). The chalky area ratio of flo6 mutant was much higher than that of the WT. The 1,000-grain weight, grain length, grain width, and grain thickness of flo6 were lower than that of the WT. However, the length/width ratios of the flo6 mutant grain and the WT were similar. These results suggested that the flo6 mutation has significant effects on the rice grains resulting in decreased yields and reduced grain size. But the lack of significant difference in the length/width ratios between WT and flo6 mutants indicated that the mutant gene generated no effects on the shape of grains.

Fig. 1
figure 1

Phenotypes of flo6 kernels. a, b Brown rice of wild-type and flo6. c Brown rice of F2 crossed by WT and flo6. d Brown rice of indica variety R9703. e Brown rice of F2 crossed by flo6 and indica variety R9703. f, h Milled rice of WT and flo6. g, i Brown rice of WT and flo6. j, k Crossed sections of WT and flo6. l, m SEM analysis of normal sections indicated by arrow in j. n, o SEM analysis of mutant sections indicated by arrow in k. Bars a–e = 10 mm; f–k = 1 mm; l, n = 5 μm; m, o = 10 μm

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Table 1 Grain morphology data of flo6
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Cooking and Nutrition Quality of flo6 Grains

The AC of the WT was slightly lower than that of the flo6 mutant, and no significant difference between them was detected (Fig. 2a). The values of GC, GT, and RVA profile of the flo6 mutant were lower than that of the WT (Fig. 2b, e and Table 2), and the thermodynamic parameters Tp and ΔT 1/2 of the WT and flo6 mutant were also different (Table 2). PC was higher in the flo6 mutant than the WT (Fig. 2c), as was also observed in mutant flo-4 (Kang et al. 2005). The relative content analysis of 17 amino acids revealed differences between the WT and the flo6 mutant in nine of them, and all of them, except for Ser and Met, were higher in flo6 than those in the WT (Fig. 2d). These results above indicated that the mutant gene did not change the AC but had an obvious effect on GC, GT, and RVA profile of rice floury endosperm, suggesting that the WT gene may play an important role in rice cooking quality and could affect the nutritional quality of rice.

Fig. 2
figure 2

Quality traits parameter of WT and flo6. a–c AC, GC, and PC of WT and flo6 (n = 3). d Relative content of amino acids of WT and flo6 (* P < 0.05; ** P < 0.01. n = 2). e Floury RVA curve of WT and flo6

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Table 2 Thermodynamics character of rice floury of WT and flo6
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Genetic Mapping and Map-Based Cloning of flo6 Gene

To determine whether flo6 was controlled by a single gene or multiple genes, the flo6 mutant was crossed with WT plants. The indica rice R9703 (Fig. 1d) was crossed with flo6 mutant for the construction of gene mapping population (Fig. 1e). A total of 1,217 wild-type grains and 347 flo6 grains were obtained from the F1 plants. The segregation of normal grains and mutant grains (Fig. 1c) was fitted to a 3:1 segregation ratio (P = 0.89>0.01). The 3:1 segregation ratio of normal grains to mutant grains indicated that the flo6 phenotype was controlled by a single recessive gene.

In order to determine the chromosomal location of the flo6 gene, 158 molecular markers (SSR markers and InDel markers) which showed polymorphisms between Nipponbare and R9703 were selected to conduct linkage analysis by BSA (Fig. 3a). It was observed that the InDel marker R1M30 and SSR marker RM3475 which are located on the long arm of rice chromosome 1 showed close linkage with the flo6 gene (Fig. 3b, c). Using 286 flo6 individuals from the F2 mapping population, the markers R1M30 and RM3475 were mapped on the left and right of flo6 locus with genetic distances of 8.6 and 3.5 cM, respectively (Fig. 4a, b).

Fig. 3
figure 3

Results of BSA and identification of recombinants in F2 population. a The results of BSA of five polymorphism markers in chromosome 1, 1~5 represent markers RM581, RM23, R1M7, RM493, and R1M30, respectively. W wild-type pool and M mutant type pool. b The In/Del marker R1M30 is linked to the flo6 locus in primary mapping and without recombinants in the ten individuals of F2 population. P1 flo6 mutant and P2 indica rice R9703, 1~10 represent mutant individuals from F2 population. c The SSR marker RM3475 is linked to flo6 locus and used for identification of recombinants in F2 population. Asterisk recombinants

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Fig. 4
figure 4

Map-based cloning of the flo6 gene. a Primary gene mapping of the flo6 locus. b Genetic linkage map derived from F2 population with 286 individual mutants. Above the vertical line are the names of the marks and below are the numbers of recombinants. Numbers between vertical lines are genetic distances of the flo6 locus with marks. c The construction of candidate gene LOC_Os01g44220 in MSU database (IRGSP locus: Os01g0633100, NCBI GeneBank: AK071497) that encodes glucose-1-phosphate adenylyltransferase large subunit is suspected to be the flo6 gene. Black boxes and gray boxes indicate untranslated region (UTR) and open reading frame (ORF), respectively. d Mutation site of DNA and protein sequence

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For further mapping of the flo6 loci, 32 InDel markers were developed in the region restricted by markers R1M30 and RM3475 based on the differences of genomic DNA sequences between Nipponbare (japonica cultivar) and 93-11 (indica cultivar). However, only one InDel marker named ID1-12 which showed polymorphism between flo6 mutant and R9703 was used for further mapping of the flo6 gene. The results from genotyping of 286 flo6 individuals of F2 mapping population showed that the InDel marker ID1-12 was located between flo6 locus and RM3475, away from flo6 with a genetic distance of 1.3 cM (Fig. 4b). Thus, the flo6 was mapped in an interval restricted by R1M30 and ID1-12 with a physical distance of 1,055 kb.

Attempts were made to develop polymorphic SSR or InDel markers in the 1,055-kb region. However, no polymorphic markers were found due to poor polymorphisms between genomic DNA of Nipponbare and R9703. It was thus not necessary to analyze all candidate genes in this large region since the average physical distance per centimorgan is observed to be 244 kb for the rice genome (Chen et al. 2002). Therefore, we analyzed the annotated genes approximately 500 kb upstream of ID1-12 in the rice Gramene database (http://www.gramene.org/) based on the 1.3 cM genetic distance at which ID1-12 was away from the flo6 gene.

There are 86 open reading frames (ORFs) in this region (Supplementary Table 2). Only one is a relation gene of starch synthesis, and 18 of the 86 ORFs are annotation as hypothetical protein. We will choose the relation gene of starch synthesis for sequencing, first by analyzing the information of annotation of the presumption genes. If no mutation in the locus that we chose, then we will analyze the other ORFs by the methods of bioinformatics or construct a new population of gene mapping to narrow the candidate region. An annotated gene, LOC_Os01g44220 which encodes putative ADP-glucose pyrophosphrylase large subunit named OsAPL2, was considered the optimal candidate of Flo6. The reasons is that the starch granules of the endosperm in the homozygous OsAPL2 defective mutants were smaller in size and rounder in shape when compared to those from the wild-type endosperm (Lee et al. 2007). The function of OsAPL2 is starch synthesis which is expressed at the late stages of seed development (Akihiro et al. 2005). Thus, the locus LOC_Os01g44220 was therefore amplified from the genomic DNA of different plants of the flo6 mutant and the WT by PCR and then sequenced (Fig. 4c). Results from DNA sequencing revealed a single nucleotide mutation (G→A) of the tenth exon in ORF region of LOC_Os01g44220 which led to a single amino acid substitution from acidic amino acid Glu (E) to basic amino acid Lys (K) accordingly (Fig. 4d). We analyzed the SNP sites of nucleotide sequence of the candidate gene OsAGPL2 (NCBI accession NP_001043654, definition Os01g0633100); there are 95 SNP sites in this locus and the mutation site of (G→A) does not belong to them.

Differences of Protein Parameter and Multiple Sequence Alignment

In order to elucidate effect of the single nucleotide mutation (G→A) on the Flo6 gene, some protein prediction parameters of the WT and the flo6 mutants were analyzed using proteomic tools. The results showed both differences between the physical–chemical parameters and between the secondary and tertiary protein structures of the WT and the flo6 (Supplementary Table 3 and 4; Supplementary Fig. 1). The multiple sequence alignment of OsAPL1, OsAPL2, OsAPL3, Escherichia coli, Solamun tuberosum_L, and Zea mays_L2 were data-mined using the ClustalW2 program. The mutant site is close to the functional domain which interacts with ADP-Glc (Fig. 5). The substitution from acidic amino acid Glu (E) to basic amino acid Lys (K) brings a change in total electric charge around this functional domain (Supplementary Table 3).

Fig. 5
figure 5

Comparison of the predicted amino acid sequences of AGPases large submits. Multiple sequence alignment of AGPase large subunits was data-mined using the ClustalW2 program. The sequences were obtained from the NCBI database: OsAPL1 (AK069296), OsAPL2 (AK071497), OsAPL3 (AK100910), E. coli (AAY18580.1), S. tuberosum_L (P23509.2), and Zea mays_L2 (NP_001105717.1). The residues interacting with ADP-Glc were represented by black box (Jin et al. 2005). The mutant site was represented by black arrow

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Discussion

In cereals, the principal storage reserve is starch, which constitutes approximately 78 % of the dry weight of the grain, and one key regulatory step that controls the flux of carbon into starch is catalyzed by AGPase (Nagai et al. 2009; Singh and Juliano 1977). The overexpression of AGPase enhances seed weight and starch content (Li et al. 2011). Expression of a cytoplasmic-localized AGPase mutant gene from E. coli in rice endosperm resulted in enhanced starch synthesis and, in turn, higher seed weights (Nagai et al. 2009). Furthermore, the lesion of OsAGPL2 causes a shrunken endosperm due to a remarkable reduction in starch synthesis (Lee et al. 2007) and the direct effect is the formation of floury endosperm in rice. The increased ratio of chalk in rice grains could be linked with the rising global temperatures (Yamakawa et al. 2008; 2007). A common unfavorable effect of chalky or floury endosperm on grains is the decrease of 1,000-grain weight, and thus reducing its yield. Finding the gene related to chalk or floury endosperm was an effective approach towards solving this problem.

In the present study, a floury endosperm mutant (flo6) was isolated from the M2 population of the japonica cultivar Nipponbare. The morphology, cooking, and nutrition quality of the flo6 mutant grains were analyzed. A map-based cloning strategy was used to identify the flo6 gene. The results showed that the flo6 gene was located within a genetic distance of 1.3 cM away from the InDel marker ID1-12 on the long arm of chromosome 1. It is believed that, in the region of approximately 500 kb, locus LOC_Os01g44220 which encodes ADP-glucose pyrophosphrylase large subunit might be the mutant gene. Sequencing results revealed that a single nucleotide mutation (G→A) at the tenth exon of locus LOC_Os01g44220.4 lead to an amino acid substitution (E→K) in the protein sequence. The interior of the endosperm in the flo phenotype was floury-white while the outer portion was normal, suggesting that starch accumulation was abnormal in the early stages of ripening but recovered later on in development (Kang et al. 2005). OsAGPL2 functions mainly during the middle and late stages of endosperm development and accompanies a huge increase in the demand for starch synthesis (Akihiro et al. 2005; Ohdan et al. 2005; Lee et al. 2007). We analyzed the effects of amino acid substitutions on protein function of OsAGPL2 by the methods of Ng and Henikoff (Ng and Henikoff 2003, 2006). The results indicate that the single amino acid substitution of E→K on the site of E326 of the protein of OsAGPL2 will affect the protein function. A possible explanation was that the mutant effect was stronger than the normal catalytic effect of OsAGPL2 at the early stages of endosperm development, but this phenomenon reversed due to the huge increase the amount of OsAGPL2 at the middle and late stages, finally resulting in the mutant phenotype of floury endosperm. Based on the above results, we consider that the flo6 is a new mutant of OsAGPL2, and it was renamed osagpl2-3.