MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine-tuning of cooked rice texture

Abstract

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine-tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met-723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica-type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α-amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

Keywords: Oryza sativa; MutMapPlus; amylopectin; cooked rice texture; mutant allele; starch branching enzyme.

Figure 1

Gelatinization properties and grain appearance of age1, age2, SSIIa‐NIL and their double allele lines. (a) Gelatinization of endosperm starch at various concentrations of urea solution. Urea concentration of onset gelatinization was shown by red bars. (b) Grain appearance. Images by reflected light (upper panel) and transmitted light (lower panel) are shown. Parent plants of the seeds used in this experiment were grown in the paddy field of Hokuriku Research Station (Joetsu, Japan) in 2016.

Figure 2

Expression level and enzyme activity of starch biosynthetic enzymes. (a) mRNA levels of starch biosynthetic enzyme genes in developing caryopses were analysed by qRT‐PCR. eEF‐1α was used as an internal control. The value for Nipponbare (WT) was set to 1, and relative values were shown. Asterisks indicate significant difference from background lines (Nipponbare or SSIIa‐NIL) of each line (Student's t test, *P < 0.05, **P < 0.01, ***P < 0.001). (b) Immunoblot (IB) analysis of soluble endosperm extract from developing seeds. Arrowhead on the right indicates bands corresponding to truncated BEIIb. Asterisks indicate nonspecific signals. (c) branching enzyme, starch synthase and debranching enzyme activities in soluble extract of developing endosperm were detected by activity staining after native‐PAGE. Enzymes corresponding to each activity band were indicated on the right in each panel.

Figure 3

Genetic mapping of age1. (a) and (b) Nipponbare‐type allele frequency plot of chromosome 2 generated by rough mappings. The results of age mutants‐Koshihikari (a) and age mutants‐SL09 (b) F2 populations are shown. The regions in which no polymorphisms were detected are shaded with grey boxes. (c) SNP index and Δ(SNP index) plots of the chromosome 2 generated by MutMapPlus analysis. SNP index of the mutant (M) bulk (upper panel) and the wild‐type (WT) bulk (middle panel) and Δ(SNP index) calculated by subtraction of the index of WT bulk from that of M bulk (lower panel) is shown. Red lines indicate the sliding window average of 4 Mb intervals with 50 kb increment. Candidate region confined by the Fisher's P value of <0.05 is indicated with a red box. (d) Schematic diagrams of BEIIb gene in age1 allele. Open and black boxes indicate untranslated regions and exons, respectively. Uppercase and lowercase letters indicate DNA sequence of exon and intron, respectively. Translated amino acids are shown under the DNA sequence. Substituted nucleotide and amino acid are shown by red letter.

Figure 4

Complementation of age1 by genomic fragment of BEIIb gene. (a) Schematic diagrams of constructs used for complementation. LB, left border; RB, right border; HPT, hygromycin phosphotransferase; Pro, promoter; Ter, terminator; Met, translation start site; stop, translation termination site. Restriction sites were indicated above. (b) Gelatinization of endosperm starch of complementation lines in 5.0 m urea solution. VC, vector control. (c) IB analysis using anti‐BEIIb antibody (upper panel), CBB staining (middle panel) and branching enzyme activity staining (lower panel) of soluble extract of developing endosperm. Black, red and blue arrowheads indicate bands corresponding to BEIIb, Myc‐tagged BEIIb and BEIIa, respectively. The transgenic plants were grown in a greenhouse.

Figure 5

Insertion of Tos17 in BEIIb gene in age2. (a) Gelatinization of endosperm starch of F1 seeds in 5.0 m urea solution. Lines used for each cross and evaluation of gelatinization were shown under the image. WT, G and NG indicate Nipponbare, gelatinized and not gelatinized, respectively. (b) Schematic diagrams of BEIIb gene in age2. Primers used in (c) and Figure 6 were shown by arrows. Open and black boxes indicate untranslated regions and exons, respectively. Location of Tos17 insertion is indicated above the gene. Uppercase and lowercase letters indicate DNA sequence of exon and intron, respectively. Translated amino acids were shown under the DNA sequence. (c) Alternatively spliced BEIIb transcript in age2 was detected by RT‐PCR analysis. Total RNA without reverse transcription (‐RT) was used as a template for negative control (lower panel). DNA size markers are indicated on the right.

Figure 6

DNA markers for detection of age1 and age2 alleles and SSIIa ^indica allele. (a) Primers for DNA markers. DNA sequence shown is 5′ to 3′. Allele‐specific and additional mismatch nucleotides are shown in lowercase letters. (b) Band patterns of amplified DNA. Amplicon lengths are indicated on the right.

Figure 7

Chain length distribution (CLD) patterns of amylopectin. CLD of amylopectin was examined by HPAEC‐PAD, and then the difference in the profiles between each line and Nipponbare was calculated by subtracting the ratio of a chain of given length of the sample with that of the mean value of Nipponbare. Values are the means of three replicates. The grains used for this experiment were ripened in the paddy field.

Figure 8

α‐amylase digestion properties of purified starch. Raw, gelatinized or retrograded starches were treated with α‐amylase for indicated time. The amount of reducing sugar produced by the reaction was determined. Values are the means ± SD of three replicates.

Figure 9

Stickiness and hardness of the cooked rice of the mutant lines as measured by a sensory evaluation test. Each sample was evaluated by 24‐trained panellists under two conditions, in which cooked rice was cooled for 10 m (warm) or 2 h (cooled) at room temperature. For hardness, lower scores are harder texture. Asterisks denote significant differences from Nipponbare (Student's t test, *P < 0.05, **P < 0.01, ***P < 0.001).