Rice fertilization-Independent Endosperm1 regulates seed size under heat stress by controlling early endosperm development

Abstract

Although heat stress reduces seed size in rice (Oryza sativa), little is known about the molecular mechanisms underlying the observed reduction in seed size and yield. To elucidate the mechanistic basis of heat sensitivity and reduced seed size, we imposed a moderate (34°C) and a high (42°C) heat stress treatment on developing rice seeds during the postfertilization stage. Both stress treatments reduced the final seed size. At a cellular level, the moderate heat stress resulted in precocious endosperm cellularization, whereas severe heat-stressed seeds failed to cellularize. Initiation of endosperm cellularization is a critical developmental transition required for normal seed development, and it is controlled by Polycomb Repressive Complex2 (PRC2) in Arabidopsis (Arabidopsis thaliana). We observed that a member of PRC2 called Fertilization-Independent Endosperm1 (OsFIE1) was sensitive to temperature changes, and its expression was negatively correlated with the duration of the syncytial stage during heat stress. Seeds from plants overexpressing OsFIE1 had reduced seed size and exhibited precocious cellularization. The DNA methylation status and a repressive histone modification of OsFIE1 were observed to be temperature sensitive. Our data suggested that the thermal sensitivity of seed enlargement could partly be caused by altered epigenetic regulation of endosperm development during the transition from the syncytial to the cellularized state.

Figure 1.

Severe heat stress reduces seed size. A, Images of developing rice seeds at 24, 48, 72, and 96 HAF under control (28°C) conditions. This stage is characterized by rapid increase in seed size. B, Seeds collected from plants exposed to severe heat stress (42°C) imposed at 24 HAF for 2 d show a decrease in the size of developing seeds. Bars = 1 mm.

Figure 2.

Morphometric analysis of mature rice seeds obtained from plants grown under control and moderate heat stress. A, Length. B, Width. C, Weight. Length, width, and weight were significantly (an asterisk indicates P = 0.0001, n = 50) reduced by a short duration of heat stress. The data are presented as means ± sds.

Figure 3.

Histological analysis of early seed development under control and severe and moderate heat stress conditions. Seeds were harvested at 24, 48, 72, and 96 HAF for control and severe stress samples. For moderate stress, seeds were collected at 48- and 72-HAF time points. A to D, Seeds from control condition plants (28°C). Endosperm (ed) cellularization was initiated by 72 HAF and completed by 96 HAF. E to G, Sections were obtained from plants exposed to a severe heat stress of 42°C initiated at 24 HAF for 2 d. Cellularization was delayed under severe heat stress, and the central vacuole (cv) was visible at 96 HAF. H and I, Under moderate heat stress (34°C), seeds exhibited early cellularization, and the endosperm was cellularized by 72 HAF. rms, Radical microtubule system. Bars = 200 µm.

Figure 4.

Expression-based heat map of the rice PRC2 genes in response to moderate heat stress. The heat map was generated from the microarray expression values for seed samples collected at 48 and 72 HAF under control and moderate heat stress conditions. The six rice PRC2 members in the cluster are OsFIE1 (Os08g04290), OsFIE2 (Os08g04270), OsSET1 (Os03g19480), OsCLF (Os06g16390), OsEMF2a (Os04g08034), and OsEMF2b (Os09g13630). Log-transformed (base 2) expression values were scaled between −1 and 1. Higher expression values are represented in red, and lower values are represented in blue. CLF, Curly Leaf; EMF, Embryonic flower.

Figure 5.

Gene expression of rice OsFIE1 during early seed development. A, OsFIE1 relative expression under control (28°C) and severe (42°C) and moderate (34°C) stress exhibits transcript abundance shift in response to temperature. Unfertilized ovules sample (0 HAF) was used as the baseline for relative expression. B, Gene expression of OsFIE1 at 48 HAF in seeds developing in a range of temperatures (28°C to –40°C). The 28°C seed sample was used as the baseline for relative expression of OsFIE1. The data are presented as means ± sds.

Figure 6.

Effect of OsFIE1 overexpression on mature seed size and the duration of the syncytial stage of the endosperm. A, Comparison between mature seed of rice wild type (WT) and three independent transgenic plants overexpressing OsFIE1. B, Length. C, Width. D, Weight. Controls for seed size and weight measurements are seeds obtained from the wild-type cv Kitaake plants grown under the same conditions and times as transgenic lines (n > 30 for all lines). E, Relative gene expression of OsFIE1 in seeds of transgenic lines and the wild type. F, Section from control seeds (28°C) harvested at 48 HAF. G, Sections from OE-OsFIE1 seeds harvested at 48 HAF. Bar in F = 200 µm. Bar in G = 100 µm.

Figure 7.

DNA and histone methylation analysis of OsFIE1. A, McrBC-based assay for DNA methylation level of OsFIE1 during early seed development under control and moderate heat-stressed (34°C) conditions at 48, 72, and 96 HAF; 7 DAF (OsFIE1 highly expressed) and leaf tissue (OsFIE1 not expressed) are positive and negative controls, respectively. McrBC digests methylated DNA. Top is from the primer pair targeting known methylated sites, and in the bottom, primers are controls from an unmethylated region of OsFIE1. B, ChIP analyses of OsFIE1 under control (28°C; gray bars) and moderate stress (34°C; white bars) for enrichment of silencing histone modification H3K9me2. The level of H3K9me2 is expressed as the percentage of input DNA used for the ChIP assays. Values are means ± sds of three biological replicates.

Figure 8.

Expression of rice CMT3 in developing seeds. A, CMT3 expression was measured during early seed development. The gene expression levels are relative to the 0-HAF sample; 24 and 48 HAF correspond to syncytial stages of endosperm development when the nuclei divide rapidly before cellularization. B, Expression of rice CMT3 at the 48-HAF time point in seeds developing under a range of temperatures with 2°C increments. The data are presented as means ± sds from three independent biological replicates.

Figure 9.

Gene expression of syncytial stage-specific MADS-box genes (OsMADS82, OsMADS87, and AGL36) and cellularization stage-specific SSII. A to D, Gene expression levels for syncytial and cellularization stage-specific genes in control and moderately heat-stressed seeds at 48 HAF. The stress was imposed at 24 HAF. E to H, Gene expression was measured in wild-type (WT) seeds and OsFIE1 overexpression seeds. Wild-type seeds were sampled at 24 and 48 HAF to compare with the 48-HAF OE-OsFIE1 seeds for measuring expression of MADs-box genes and ssII. The data are presented as means ± sds.

Figure 10.

Silencing histone modification (H3K27me3) levels of syncytial stage-specific type I MADS-box genes under moderate heat stress. The rice MADS-box genes assayed are OsMADS82 (A), OsMADS87 (B), and AGL36 (C). Level of H3K27me3 marks is presented as a percentage of input DNA.