Epigenetic modification of ESP, encoding a putative long noncoding RNA, affects panicle architecture in rice
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Epigenetic variants broaden phenotypic diversity in eukaryotes. Epialleles may also provide a new genetic source for crop breeding, but very few epialleles related to agricultural traits have been identified in rice. Here, we identified Epi-sp, a gain-of-function epiallele of the rice ESP (Epigenetic Short Panicle, Os01g0356951), which encodes a putative long noncoding RNA. The Epi-sp plants show a dense and short panicle phenotype, an agronomically important phenotypes that is inherited in a semidominant manner. We did not find any nucleotide sequence variation in ESP. Instead, we found hypomethylation in the transcriptional termination region (TTR) of ESP gene, which caused ectopic expression of ESP in Epi-sp plants. Bisulfite analysis revealed that the methylation status of 26 CGs and 13 CHGs within a continuous 313-bp region is essential for the regulation of ESP expression. Thus, our work identified a unique rice epiallele and demonstrated that epigenetic modification of ESP is associated with the regulation of panicle architecture in rice.
KeywordsRice lncRNA Epigenetic Panicle Pseudogene
Epigenetic Short Panicle
long noncoding RNA
shoot apical meristem
transcriptional termination region
In higher plants, epigenetic variation provides a mechanism for phenotypic variation in the absence of DNA mutations. Although a number of epigenetic mutants were identified in multiple plant species, including Arabidopsis (Jacobsen and Meyerowitz 1997; Soppe et al. 2000), Linaria vulgaris (Cubas et al. 1999), Solanum lycopersicum (Manning et al. 2006) and Cucumis melo (Martin et al. 2009), examples of epigenetic diversity in rice remain limited. The first epigenetic mutants identified in rice is the Epi-d1, that shows a metastable dwarf phenotype, often producing both dwarf and normal tillers on the same plant (Miura et al. 2009). Metastable phenotype of epi-d1 is due to metastable expression of DWARF1 (D1) gene, which is associated with DNA methylation in the D1 promoter region. Other epigenetic mutants of rice such as Epi-df (Zhang et al. 2012), Epi-RAV6 (Zhang et al. 2015), also show DNA hypermethylation or hypomethylation in the promoter of the corresponding genes, resulting in the suppression or induction of their transcription.
Previous studies have shown that epigenetic modifications can affect various aspects of plant growth and development including plant height, seed size, floral development and disease resistance in rice (Miura et al. 2009; Zhang et al. 2012; Zhang et al. 2015; Deng et al. 2017). Panicle architecture, an important agronomic trait of rice, is mainly determined by the number and length of the primary and secondary branches. Although a number of key genes controlling panicle architecture such as DEP2 (Li et al. 2010) and SP1 (Li et al. 2009) have been cloned and characterized in recent years, whether epigenetic modification is involved in the regulation of panicle architecture remain unclear. Here, we identified a natural epiallele of rice ESP and found that plants carrying this epiallele show reduced panicle size, as well as hypomethylation in the ESP transcriptional termination region (TTR) and ectopic expression of ESP. Our work revealed that epigenetic modification of ESP plays an important role in the regulation of panicle architecture in rice.
The heterozygous Epi-sp (+/−) mutant showed a significant reduction in plant height (97.4 ± 3.4 cm), compared to the wild-type plants (106.7 ± 3.1 cm), resulting from significantly shortened panicles and branches in the mutant culms (Fig. 1B and Additional file 1: Fig. S1). But there were no significant differences in the number of primary and secondary branches and grains morphology between wild type and mutant plants (Additional file 1: Figure S1). The overall appearance of the mutant panicle is more compact compared to the wild type. By contrast, the normal tillers have normal features (Fig. 1a). Interestingly, the heterozygous Epi-sp (+/−) mutant was often chimeric, producing both dwarf (Epi-sp) and normal tillers (Epi-normal) on the same plant (Fig. 1a). In addition, we found chimeric features also in the panicles (Fig. 1b). This strongly suggests that the Epi-sp mutant phenotypes are not genetically fixed and can vary epigenetically in the developing plant. For the homozygous mutant, it exhibited a dwarf phenotype (24.8 ± 2.8 cm) with a defect in shoot apical meristem (SAM) and could not set seeds in paddy field (Fig. 1c-e).
To determine the cause of the metastable phenotype of Epi-sp, we performed expression analysis of the ESP gene (Os01g0356951). Although ESP was highly expressed in short panicles of chimeric plants, the ESP transcript was not detected in normal panicles of these same plants (Fig. 2c). We also examined ESP expression in the progenies of selfed independent chimeric Epi-sp plants and found that the ESP gene was highly expressed in Epi-sp but not in Epi-normal plants (the progeny from normal panicle of chimeric plant) (Fig. 2d). These results suggest that the epigenetic phenomenon in Epi-sp is associated with the misexpression of ESP gene.
To determine whether loss of DNA methylation in the transcriptional termination region (TTR) directly causes the ectopic expression of ESP in Epi-sp, we treated the wild-type seeds with 5-aza-2′ deoxycytidine (5-aza-dC), an inhibitor of DNA methylation (Chang and Pikaard 2005), to assess its effect on the expression of ESP. Transcript levels of ESP were measured in 7-d-old seedlings with or without 5-aza-dC treatment (Additional file 1: Figure S3) and found that treatment with 5-aza-dC up-regulated ESP expression (Fig. 3c). These results indicated that the DNA methylation in the 3′ end downstream sequence of ESP plays an essential role in the regulation of ESP expression. Promoter methylation is a general mechanism for suppressing gene expression in eukaryotes. Interestingly, methylation in the transcriptional termination region (TTR) is also highly correlated with gene expression, even higher than that of promoters, especially for CG methylation in rice (Li et al. 2012). In fact, the TTR of ESP gene is a CpG island, containing 29 CpG dinucleotide repeats (Additional file 1: Figure S4). Consistently, our study showed that ESP TTR hypomethylation caused induced expression of ESP in Epi-sp plants.
To further determine the conserved effect of the DNA methylation on ESP expression in cultivated rice accessions, we measured ESP transcript levels in four cultivated rice strains (cv MH63, 9311, ZS97 and Nipponbare). Compared with the high expression of ESP observed in Epi-sp, we observed little or no ESP expression in seedlings in all the cultivated rice strains tested (Fig. 4b). Like the pattern of wild-type cv Zhonghua11, the other four cultivated rice accessions also showed higher transcriptional levels of ESP in the seedlings treated with 5-aza-dC (Fig. 4b and Additional file 1: Figure S3). Thus, it is tempting to speculate that the occurrence of ESP predates the divergence of indica and japonica rice and the epigenetic regulation of ESP expression is evolutionarily conserved in cultivated rice.
Pseudogenes are usually characterized by a combination of homology to a known gene and loss of some functionality (Mighell et al. 2000). Compared to YUCCA7 gene, ESP shares very high sequence identity with OsYUCCA7, but has extremely low protein coding probability and gene expression level in cultivated rice (Fig. 4b). Phylogenetic analyses revealed that YUCCA7-like genes are well evolutionally conserved in AA-, BB-, and FF-genome wild rice, but ESP is absent in BB-genome (Oryza punctata) and FF-genome (Oryza brachyantha) wild rice (Fig. 4a). Thus, it is highly likely that ESP is the pseudogene of YUCCA7 gene. Based on the findings mentioned above, we proposed that the ESP might be a pseudogene-derived antisense lncRNA gene.
Despite pseudogenes being considered as nonfunctional genomic locus, some pseudogenes may be functional, similar to other kinds of noncoding DNA, which can perform regulatory functions to their parent genes (Mighell et al. 2000). To explore the effect of ESP on its parent gene, we assessed the relationship between ESP and OsYUCCA7 expression in young panicle (3-5 cm) by qRT-PCR. Expression analysis revealed that the expression level of OsYUCCA7 in heterozygous Epi-sp (+/−) mutant was similar to that in WT (Fig. 5c, d). The result suggested that ESP overexpression probably had little effect on OsYUCCA7 transcription. However, whether ESP regulates the level of OsYUCCA7 protein production remains to be elucidated.
In this study, Epi-sp mutant shows low levels of DNA CG and CHG methylation in the TTR of ESP gene (Fig. 3b), causing ectopic ESP expression (Fig. 2b) and a dense and short panicle architecture in rice (Fig. 1). It is important to point out that, although the mechanism for the spontaneous hypomethylation of ESP TTR remains unknown, the TTR of ESP gene displays the characteristic of CpG island (Additional file 1: Figure S4). Since there is no CHH methylation in the TTR of ESP (Fig. 3b), CG and CHG hypermethylation in this region is likely not established by the RNA-directed DNA methylation (RdDM) pathway. Consequently, hypomethylation of this region in the Epi-sp mutant is probably not due to loss of small interfering RNAs (siRNAs). Due to the mechanistic nature of maintenance methyltransferases, TTR hypomethylation is also likely not established by lack of OsMET1 and OsCMT3 activities in a locus-specific manner. Therefore, we speculate the epiallele is probably induced by some sort of aberrant active DNA demethylation activity.
In this study, we report the identification and characterization of the rice Epi-sp mutant that displays dense and short panicle phenotype. Morphological analysis showed that the dense and short panicle phenotype in Epi-sp mutants resulted from the decrease in the length of the rachis, primary and secondary branches. The map-based cloning of ESP revealed that it encodes a putative pseudogene-derived lncRNA. Further studies to clarify the molecular mechanism of ESP should help shed light on the developmental processes underlying the formation of panicle in rice.
We thank Dr. Haitao Zhu (South China Agricultural University, China) for assistance with the map-based cloning of the ESP gene.
This work was supported by the National Natural Science Foundation of China (grant nos. 30900884 and 31671594 to XQ. Zhang); by Guangxi Key Laboratory of Rice Genetics and Breeding Open Foundation (No.2018–05-Z06-KF02); by Natural Science Foundation of Guangdong Province, China (grant nos. 2014A030313457 and 2015A020209118 to XQ. Zhang).
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The datasets supporting the conclusions of this article are included within the article and its additional files.
XL and SL performed the experiments. SK and HD conducted fieldwork and managed the experimental materials. XMX designed the experiments and analyzed the data; TFH supervised and complemented the writing; XQZ conceived the project and wrote the article with contributions of all the authors. All authors read and approved the final manuscript.
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