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. 2023 Mar 13:14:1138523.
doi: 10.3389/fpls.2023.1138523. eCollection 2023.

Generation of new rice germplasms with low amylose content by CRISPR/CAS9-targeted mutagenesis of the FLOURY ENDOSPERM 2 gene

Xiaohong Song  1   2 , Zhihui Chen  2   3 , Xi Du  2   3 , Bin Li  1   2 , Yunyan Fei  2   3 , Yajun Tao  2   3 , Fangquan Wang  2   3 , Yang Xu  2   3 , Wenqi Li  2   3 , Jun Wang  2   3 , Guohua Liang  2   3 , Yong Zhou  2   3 , Xiaoli Tan  1 , Yulong Li  1 , Jie Yang  1   2   3
Affiliations

Generation of new rice germplasms with low amylose content by CRISPR/CAS9-targeted mutagenesis of the FLOURY ENDOSPERM 2 gene

Xiaohong Song et al. Front Plant Sci. .

Abstract

FLOURY ENDOSPERM 2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein located in the nucleus, is considered to be a regulatory protein that controls the biosynthesis of seed storage substances. The diversity of flo2 allele is attributable for the variations in grain appearance, amylose content (AC), and physicochemical properties, influencing the eating and cooking quality (ECQ) of rice. In this study, we used CRISPR/Cas9 to introduce loss-of-function mutations into the FLOURY ENDOSPERM 2 gene in Suken118 (SK118), a widely cultivated elite japonica rice variety in Jiangsu, China. Physiochemical analyses of the flo2 mutants were congruent with previous studies, exhibiting lowered AC and viscosity, risen gel consistency (GC) and gelatinization temperature (GT) values, which were all instrumental to the improvement of ECQ. However, the wrinkled opaque appearance and the decrease in grain width, grain thickness and grain weight imply trade-offs in grain yield. Despite the ex-ante estimation for low yielding, the superior ECQ in these novel genotypes generated by using genome editing approach may have the potential for formulating high value specialty food.

Keywords: CRISPR/Cas9; FLOURY ENDOSPERM 2; amylose content (AC); eating and cooking quality (ECQ); genome editing; japonica rice.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
CRISPR/Cas9-induced mutations in the FLOURY ENDOSPERM 2 genes. (A) Schematic of the FLOURY ENDOSPERM 2 gene structures and target sites. Exons and introns are indicated with black rectangles and black lines, respectively. The spacer and PAM sequences were marked in black and orange. (B) The expression vector of pYLCRISPR/Cas9-Flo2. The expression of Cas9 is driven by the maize ubiquitin promoter (OsUBQ); the expression of the sgRNA scaffold is driven by the rice OsU3 small nuclear RNA promoter; the expression of hygromycin (HPT) is driven by CaMV35S promoter. Tnos, the terminator; LB and RB, left border and right border, respectively. (C) Potential miss analysis. Red fonts indicate different bases.
Figure 2
Figure 2
Phenotype and amino acid variations assays. (A) Phenotypes of flo2 mutant lines (d291-2 and d291-3) in SK118 background. Bar = 5 cm. (B) Amylose content (%). Data are given as the mean ± S.D. (n ≥ 3). Student’s t test: *P < 0.05; **P < 0.01; ns, no significance. (C) Amino acid variations of the FLO2 protein in the flo2 mutant. Wild-type, SK118. Homozygous mutations identified at the target sites of d291-2 and d291-3 mutant lines in the T1 generation. Amino acid variations were indicated in red box, with the number representing the order in the proteins. The ellipsis dots represent premature stop codons.
Figure 3
Figure 3
Knockout of FLOURY ENDOSPERM 2 gene has multiple effects on agronomic traits. (A) Grain phenotypes of flo2 mutant lines (d291-2 and d291-3) in SK118 background. Bar = 1 cm. (B) grain length (mm). (C) grain width (mm). (D) grain thickness (mm). (E) plant height(cm). (F) panicle length (cm). (G) grain numbers per panicle. (H) tiller numbers per plant. (I) 1000-grain weight (G). Data are given as the mean ± S.D. (n ≥ 20). Student’s t test: **P < 0.01; ns, not significance.
Figure 4
Figure 4
Improvement of ECQ and taste value of flo2 transgenic rice. (A) Gel consistency (mm). (B) RVA spectra of rice flours. (C) Total protein content of the rice flours (%). (D) Gelatinization temperature (GT) of rice flours from SK118, d291-2 and d291-3. Data are given as the mean ± S.D. (n ≥ 3). Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 5
Figure 5
Appearance phenotype analysis of flo2 mutants rice grain. (A) Appearance of polished rice from flo2 mutant lines (d291-2 and d291-3) in SK118 background. Bar = 1 cm. (B) Transverse sections of rice grains. Bar = 300 μm. (C) Rice grains stained with KI-I2. Bar = 300 μm.
Figure 6
Figure 6
Morphology of starch granules in endosperm cells of flo2 mutants. (A) Scanning electron microscopy images of the transverse section of flo2 mutants and wild-type. 25×, Bars = 200μm. 500×, Bars = 20 μm. 1000×, Bars = 10 μm.
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