Miniature-inverted-repeat transposable elements contribute to phenotypic variation regulation of rice induced by space environment

Abstract

Introduction: Rice samples exposed to the space environment have generated diverse phenotypic variations. Miniature-inverted-repeat transposable elements (MITEs), often found adjacent to genes, play a significant role in regulating the plant genome. Herein, the contribution of MITEs in regulating space-mutagenic phenotypes was explored.

Methods: The space-mutagenic phenotype changes in the F3 to F5 generations of three space-mutagenic lines from the rice varieties Dongnong423 (DN423) and Dongnong (DN416) were meticulously traced. Rice leaves samples at the heading stage from three space-mutagenic lines were subjected to high coverage whole-genome bisulfite sequencing and whole-genome sequencing. These analyses were conducted to investigate the effects of MITEs related epigenetic and genetic variations on space-mutagenic phenotypes.

Results and discussion: Studies have indicated that MITEs within gene regulatory regions might contribute to the formation and differentiation of space-mutagenic phenotypes. The space environment has been shown to induce the transposable elements insertion polymorphisms of MITEs (MITEs-TIPs), with a notable preference for insertion near genes involved in stress response and phenotype regulation. The space-induced MITEs-TIPs contributed to the formation of space-mutagenic phenotype by modulating the expression of gene near the insertion site. This study underscored the pivotal role of MITEs in modulating plant phenotypic variation induced by the space environment, as well as the transgenerational stability of these phenotypic variants.

Keywords: TEs; environment stress; mites; phenotype variation; rice; space environment; space mutagenesis.

Figure 1

Acquisition of rice space-mutagenic lines and patterns of space-mutagenic phenotype change. (A) Schematic diagram of the acquisition and selection of space-mutagenic lines. The parental generation of space-mutagenic lines was carried by the SJ-10 retrievable scientific satellite. The space-mutagenic plants were identified within individual plants of the parental generation. The pedigree method was employed for mutant selection across subsequent generations. The mutant phenotypes of SA3-7 and SA6-2 were identified in the parental generation, while that of SC6-6 was identified in the F1 generation. Seeds from both space-mutagenic rice plants and corresponding ground control plants were harvested in F2-F4 generations; (B) Rice plants from different treatment groups in the F3-F5 generations of three space-mutagenic lines were cultivated in a standardized manner in the phytotron. Three plants were selected from each group for high coverage WGBS and WGS. The rice plants displayed here were all from the F3 generation; (C) The plant height in SA3-7 at F3-F5 generations; (D) The tiller number in SA6-2 at F3-F5 generations; (E) The tiller number in SC6-6 at F3-F5 generations. 15 strains per group were used as biological replicates; ** A highly statistically significant difference between the space and control samples using independent samples T-test (P < 0.01).

Figure 2

Analysis of MITEs methylation level changes within gene regulatory regions of three rice space-mutagenic lines. (A) 1. MITEs methylation modification and gene expression in gene regulatory regions under normal conditions. 2. Altered levels of siRNAs produced by MITEs in gene regulatory regions affect gene expression. 3. MITEs methylation modification in gene regulatory regions affects gene expression by affecting its binding to TFs. The thickness of the gray arrows represents the intensity of gene expression, the blue circle represents TFs that are bound to cis-acting elements, whereas the gray circle represents TFs that are bound to cis-acting elements located within MITEs; (B) The patterns of methylation level alterations on MITEs located within 2 kb upstream and downstream of genes in space-mutagenic rice samples across F3-F5 generations in SA3-7; (C) The patterns of methylation level alterations on MITEs located within 2 kb upstream and downstream of genes in space-mutagenic rice samples across F3-F5 generations in SA6-2; (D) The patterns of methylation level alterations on MITEs located within 2 kb upstream and downstream of genes in space-mutagenic rice samples across F3-F5 generations in SC6-6; Hyper and hypo represent the proportions of gene regulatory region MITEs in space-mutagenic rice samples with increased and decreased methylation levels, respectively. The heatmap above displays SD of differential methylation proportions in gene regulatory region MITEs among the three space-mutagenic samples per generation. Color intensity from light to dark represents increasing SD values. (E) The number of MITEs in gene regulatory regions that exhibit methylation level changes significantly and strongly correlated (P < 0.05, R > 0.65) with space-mutagenic phenotypes across generations in three rice space-mutagenic lines.

Figure 3

The detection of MITEs-TIPs variation in three rice space-mutagenic lines. (A) Several potential mechanisms in which MITEs transposition affects adjacent genes. Modules in the schematic diagram not specifically labeled represent the same meanings as those in Figure 2A . 1. A newly inserted MITE may provide cis-acting elements to an adjacent gene. 2. A newly inserted MITE may introduce a new epigenetic modification pattern to an adjacent gene. 3. Inserting a MITE into an intron can alter the expression of the adjacent gene; (B) A comparative analysis of MITEs-TIPs differences among different samples from F3 to F5 generations in SA3-7; (C) A comparative analysis of MITEs-TIPs differences among different samples from F3 to F5 generations in SA6-2; (D) A comparative analysis of MITEs-TIPs differences among different samples from F3 to F5 generations in SC6-6.

Figure 4

The distribution preference of space-induced MITEs-TIPs. (A) The bubble plot of GO enrichment analysis of genes near space-induced MITEs-TIPs in SA3-7; (B) The bubble plot of GO enrichment analysis of genes near space-induced MITEs-TIPs in SA6-2; (C) The bubble plot of GO enrichment analysis of genes near space-induced MITEs-TIPs in SC6-6; (D) The chromatin distribution of space-induced MITEs-TIPs in the three space-mutagenic lines; The outermost circle represents the 12 chromosomes of the rice genome, with light gray indicating heterochromatic regions and dark gray indicating euchromatic regions on each chromosome. From the inner circle to the outer circle, the chromatin distribution of space-induced MITEs-TIPs in SC6-6, SA6-2 and SA3-7 at F3-F5 generations were shown respectively. Each chromosome is divided into bins of 1,000,000 bp, with a color gradient from light pink to dark pink indicating the number of space-induced MITEs-TIPs detected in each bin.

Figure 5

Identification and verification of the key genes involved in space-mutagenic phenotype changes under the influence of MITE methylation level change. (A) The chord plot of GO enrichment analysis for genes which the methylation level of regulatory regions MITEs significantly and strongly correlated (P < 0.05, R > 0.65) with change of plant height across generations in SA3-7; (B) The chord plot of GO enrichment analysis for genes which the methylation level of regulatory regions MITEs significantly and strongly correlated with change of tiller number across generations in SA6-2; Right side: significantly enriched GO terms (P < 0.05); Left side: corresponding genes. (C) OsBRI1 expression levels and its downstream MITE methylation levels in SA3-7 across successive generations; (D) OsLHP1 expression levels and its upstream MITE methylation levels in SA6-2 across successive generations; *A statistically significant difference between the space and control samples using independent samples T-test (P < 0.05).

Figure 6

The expression levels of genes involved in phenotypic regulation near space-induced MITEs-TIPs. (A) Position relationship between OsEPFL5 and space-induced MITEs-TIPs in SA3-7; (B) Relative expression levels of OsEPFL5 in successive generations in SA3-7; (C) Position relationship between OsSLL1 and space-induced MITEs-TIPs in SA6-2; (D) Relative expression levels of OsSLL1 in successive generations in SA6-2; (E) Position relationship between OsD1 and space-induced MITEs-TIPs in SA6-2; (F) Relative expression levels of OsD1 in successive generations in SA6-2; (G) Position relationship between OsSLL1 and space-induced MITEs-TIPs in SC6-6; (H) Relative expression levels of OsSLL1 in successive generations in SC6-6; (I) Position relationship between OsD1 and space-induced MITEs-TIPs in SC6-6; (J) Relative expression levels of OsD1 in successive generations in SC6-6; *A statistically significant difference between the space and control samples using single sample T-test (P < 0.05), **A highly statistically significant difference between the space and control samples using single sample T test (P < 0.01).