Allelic variation in rice Fertilization Independent Endosperm 1 contributes to grain width under high night temperature stress

Abstract

A higher minimum (night-time) temperature is considered a greater limiting factor for reduced rice yield than a similar increase in maximum (daytime) temperature. While the physiological impact of high night temperature (HNT) has been studied, the genetic and molecular basis of HNT stress response remains unexplored. We examined the phenotypic variation for mature grain size (length and width) in a diverse set of rice accessions under HNT stress. Genome-wide association analysis identified several HNT-specific loci regulating grain size as well as loci that are common for optimal and HNT stress conditions. A novel locus contributing to grain width under HNT conditions colocalized with Fie1, a component of the FIS-PRC2 complex. Our results suggest that the allelic difference controlling grain width under HNT is a result of differential transcript-level response of Fie1 in grains developing under HNT stress. We present evidence to support the role of Fie1 in grain size regulation by testing overexpression (OE) and knockout mutants under heat stress. The OE mutants were either unaltered or had a positive impact on mature grain size under HNT, while the knockouts exhibited significant grain size reduction under these conditions.

Keywords: FIS-PRC2; genome-wide association analysis; grain development; grain quality; grain size; heat stress; rice; starch.

Fig. 1

Manhattan plots of genome‐wide association results for rice mature grain length (a) and width (b) under control and high night temperature (HNT) stress conditions. The blue line indicates cutoff of significance threshold (P < 3.3 × 10⁻⁶ or −log₁₀(P) > 5.4) level and significant single nucleotide polymorphism (SNPs) are highlighted with maroon dots. The previously known major grain size genes/quantitative trait loci (QTLs) (GS3 and qSW5) under optimal (unstressed) conditions are labeled. Fie1 (chr 8:2098482, SNP position) is a candidate gene for regulating phenotypic variation of grain width under HNT conditions in rice (indicated with an arrow).

Fig. 2

Characterization of Fie1 locus as a determinant of grain width under high night temperature (HNT) stress in rice. (a) Upper panel: Fie1 gene model with the significant single nucleotide polymorphism (SNP) position in the intron (exons, rectangle; intron, line). Lower panel: box plot showing the additive effect of SNP8.2098482 under control and high night temperature (HNT) conditions; error bars represent ± SD. The SNP is significant (−log₁₀(P) = 5.65) under HNT. The x‐axis shows the allelic groups, major (CC) and minor (GG), and the y‐axis shows grain width (mm). (b) Representative mature grain images of four major (denoted as ‘M’; M1–M4) and minor (denoted as ‘m’; m1–m4) allelic group accessions under control and HNT conditions (n = 10 grains). Images were digitally extracted and scaled for comparison (bar, 1 cm). (c) Relative transcript abundance of Fie1 in the four major and minor allelic group accessions at 4 and 7 d after fertilization (DAF) under HNT conditions. Values were normalized against control for the respective time point. Error bars represent ± SD (n = 10–15 developing seeds per biological replicate; two biological and three technical replicates were used). Significant differences are depicted by asterisks (***, P < 0.001; **, P < 0.01; *, P < 0.05) based on a t‐test.

Fig. 3

Fie1 negatively regulates grain width under control conditions in rice. (a) Mature grain length, width, and single‐grain weight of wild‐type (WT), overexpression (Fie1^OE10 and Fie1^OE11) and knockout mutants (fie1^CR2 and fie1^CR3). Box plots show the median and the upper quartiles and black dots signify outliers. Significant differences (P < 0.05) are indicated by different letters based on a t‐test (n = 300–600 marked seeds from 15–20 plants). (b) Developing grain length and width of the mutants. Florets marked at the time of fertilization were collected at the respective developmental time points (4, 7 and 10 d after fertilization; DAF). Error bars represent ± SE. Significant differences are depicted by asterisks (***, P < 0.001; **, P < 0.01; *, P < 0.05) based on a t‐test (n = 15–20 marked developing grains from four plants per line).

Fig. 4

Grain width in Fie1 knockouts in rice is sensitive to high night temperature (HNT). (a) Upper panel: pictogram illustrating heat stress regime. Florets were marked at the time of fertilization, and 1 d after fertilization (DAF) plants were subjected to either HNT (30 ± 1°C : 28 ± 1°C, , light : dark) until maturity, high day‐night temperature (HDNT; 36 ± 1°C : 32 ± 1°C, , light : dark) until 10 DAF and moved back to control (30 ± 1°C : 23 ± 1°C, light : dark) or constantly kept under control conditions. Plants were harvested at physiological maturity and the florets marked at the time of fertilization were considered for downstream analysis. (b) Mature grain length (top panel), width (middle panel), and single‐grain weight (bottom panel) of wild‐type (WT), overexpression (fie1^OE10 and fie1^OE11), and knockout mutants (fie1^CR2 and fie1^CR3) under control (blue colored), HNT (orange), and HDNT (red) conditions. Box plots show the median and the upper quartiles and black dots signify outliers (5^th/95^th percentile). For statistics, a t‐test was used to compare HNT and HDNT with control (n = 300–600 marked grains from 15–20 plants per plant line per treatment). ***, P < 0.001; **, P < 0.01; * P < 0.05. (c) Representative mature seed images of WT and mutants under control, HNT and HDNT conditions. Images were digitally extracted and scaled for comparison (scale: 1 cm).

Fig. 5

The outer epidermal surface of rice mature seeds from wild‐type (WT), overexpression (fie1^OE10 and fie1^OE11) and knockout mutants (fie1^CR2 and fie1^CR3) subjected to control, high night temperature (HNT), and high day‐night temperature (HDNT) stress (bar, 200 µm). Double‐headed white arrows indicate length and width of a representative single‐cell in a scanning electron microscopy image.

Fig. 6

Developing grains of Fie1 knockouts are sensitive to high night temperature (HNT) in rice. (a) Pictogram illustrating heat stress regime. Florets were marked at the time of fertilization, and at 1 d after fertilization (DAF) plants were subjected to either HNT (30 ± 1°C : 28 ± 1°C, light : dark), high day‐night temperature (HDNT; 36 ± 1°C : 32 ± 1°C, light : dark) or constantly kept under control conditions (30 ± 1°C : 23 ± 1°C, light : dark). The marked florets were harvested at 4, 7 and 10 DAF (marked with black dots) for downstream analysis. (b) Relative transcript abundance of Fie1 in the wild‐type (WT) at 4, 7 and 10 DAF under HNT and HDNT conditions. Values were normalized against control for the respective time point. Error bars represent ± SD. For statistics, a t‐test was used: ***, P < 0.001; *, P < 0.05. (c) Top panel: representative images of developing grain (4, 7 and 10 DAF) of WT, overexpression (fie1^OE10 and fie1^OE11) and knockout mutants (fie1^CR2 and fie1^CR3) under control, HNT, and HDNT conditions. Images were digitally extracted and scaled for comparison (bar, 1 cm). Developing grain length (middle panel) and width (bottom panel) of the mutants. Error bars represent ± SE. For statistics, a t‐test was used to compare HNT and HDNT to the control (n = 15–20 marked developing seeds from four plants per treatment per line). ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig. 7

Misregulation of Fie1 alters starch quality under heat stress in rice. (a) Representative light‐box images of 50 mature grains from wild‐type (WT), overexpression (fie1^OE10 and fie1^OE11) and knockout mutants (fie1^CR2 and fie1^CR3) subjected to control, high night temperature (HNT) and high day‐night temperature (HDNT) during grain development (bar, 1 cm). (b) Cross‐sections of mature grain of WT and mutants subjected to control, HNT and HDNT conditions observed via scanning electron microscopy (bar, 20 µm).