Rice Chalky Grain 5 regulates natural variation for grain quality under heat stress

Abstract

Heat stress occurring during rice (Oryza sativa) grain development reduces grain quality, which often manifests as increased grain chalkiness. Although the impact of heat stress on grain yield is well-studied, the genetic basis of rice grain quality under heat stress is less explored as quantifying grain quality is less tractable than grain yield. To address this, we used an image-based colorimetric assay (Red, R; and Green, G) for genome-wide association analysis to identify genetic loci underlying the phenotypic variation in rice grains exposed to heat stress. We found the R to G pixel ratio (RG) derived from mature grain images to be effective in distinguishing chalky grains from translucent grains derived from control (28/24°C) and heat stressed (36/32°C) plants. Our analysis yielded a novel gene, rice Chalky Grain 5 (OsCG5) that regulates natural variation for grain chalkiness under heat stress. OsCG5 encodes a grain-specific, expressed protein of unknown function. Accessions with lower transcript abundance of OsCG5 exhibit higher chalkiness, which correlates with higher RG values under stress. These findings are supported by increased chalkiness of OsCG5 knock-out (KO) mutants relative to wildtype (WT) under heat stress. Grains from plants overexpressing OsCG5 are less chalky than KOs but comparable to WT under heat stress. Compared to WT and OE, KO mutants exhibit greater heat sensitivity for grain size and weight relative to controls. Collectively, these results show that the natural variation at OsCG5 may contribute towards rice grain quality under heat stress.

Keywords: GWAS - genome-wide association study; chalkiness; grain; heat stress; imaging; rice.

Figure 1

Relation between variation in the ratio of grain R and G pixel intensity (RG) values and grain chalkiness in response to heat stress (HS). (A) Light box images of control and HS-treated rice (cv. Kitaake) grains showing the difference in grain chalkiness. (B) RG values of control and HS-treated grains (C) Light-box images of 10 selected RDP1 accessions with a lower and higher percentage change for RG (HS/Control) (D) RG values of grains shown in (C) C and HS indicate control and heat stress, respectively. ** indicates the significance of a t-test with P<0.05. Scale bar=1cm.

Figure 2

Manhattan plots of genome-wide association studies for the ratio of grain R and G pixel intensity values under control and heat stress. The black dotted horizontal line represents the genome- wide significance threshold (–log10(P) > 6.5). SNPs associated with significant genes are highlighted.

Figure 3

Grain chalkiness in chalky grain 5 (OsCG5) allelic variants under heat stress (HS) is regulated by its transcript abundance (A) The structure of the OsCG5 gene. The position of guide RNA (gR) and SNP-5.23896968 are labeled in red and black symbols, respectively. (B) Promoter-GUS expression of OsCG5 in developing endosperm at 3 and 4 days after fertilization (DAF). Scale bar = 0.25cm (C) Distribution of the ratio of grain R and G pixel intensity (RG) of major and minor allelic accessions (Ma and Mi, respectively) under control and HS (D) RT-PCR based transcript estimation of OsCG5 in 2 DAF old grains of Ma and Mi under control and HS. Ma1-NSFTV 113; Ma2- NSFTV 333; Ma3- NSFTV 255; Mi1-NSFTV 19; Mi2- NSFTV 33; Mi3- NSFTV 345. Scale bar=1 cm. (E) Lightbox images of grains from Ma and Mi whose expression of OsCG5 transcript was estimated in (D) C and HS indicate control and heat stress, respectively. Significance level for t-test, *P<0.05; **P<0.01; ***P<0.001; ns, non-significant.

Figure 4

Characterization of the function of chalky grain 5 (OsCG5) in grain chalkiness using CRISPR-Cas9 knockout (KO) and Overexpression (OE) lines. (A) RT-PCR assay showing higher transcript abundance of OsCG5 in OE lines relative to WT rice (cv. Kitaake). (B) Positions of Cas9 deletions in KO#5 and KO#6 lines (1 bp and 109 bp, respectively). (C) Distribution of the ratio of grain R and G pixel intensity values in WT, KO and OE genotypes under control and heat stress (HS). The significance was estimated using two-way ANOVA. N = 7-8 plants. Scale bar=1 cm. (D) Phenotypic difference in grain chalkiness for WT, KO and OE under control and HS. Scale bar=1 cm. (E) Hyperspectral reflectance of grains from WT, KO and OE genotypes at wavelength range 650-1650 nm under control and HS. C and HS indicate control and heat stress, respectively.

Figure 5

Morphometrics differences in single grain weight (SGW) and grain width from WT, KO and OE plants under control and heat stress. (A) SGW (B) Grain width. The significance level was estimated using two-way ANOVA. N = 6-7 plants. C and HS indicate control and heat stress, respectively.