Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in Paris polyphylla var. Yunnanensis

Paris polyphylla var. yunnanensis (Franch.), one of the best-known medicinal plants in China, has a dehiscent anther which physiologically work in pollination, however, the dehiscent anther always closes in response to darkness every day, and watering or raining every time. To explore this frequently closing and its unkown physiology, next-generation sequencing was performed, and the transcriptome was de novo assembled. RNA-sequencing was carried out in 15 samples including seven openning samples, four closed samples owing to darkness or watering, and tissue samples (leaf, petal, calyx, and stigma) were used for control. We obtained 72.75 GB data, assembled into 79,815 unigenes. Differentially expressed unigenes (DEGs) between opened and closed anther samples were 6231 and the DEGs between anther and control samples were 2831. Comparation between the two DEGs by KEGG enrichment showed that “plant hormone signal transduction” pathway is the most significant pathway for DEGs from closing anther vs. opening anther, and expression model of DEGs in the pathway might elicit change in germination and seed dormancy. Further examination of the action of the signal pathway on physiology showed “chromatin binding” function was prominent in “DNA binding” function of annotated DEGs between opened and closed anthers, of the 215 “chromatin binding” unigenes, 120 were involved in epigenetic silencing, and 50 of the epigenetic unigenes were directly related to germination or seed dormancy, strongly correlating anther closing to epigenetic modification and seed dormancy. These results were verified that at least three auxins involved in seed dormancy showed same expression patterns occurred in abnormal closing anther and seed embryo in Paris *Bin Wang contributed equally as the first author. How to cite this paper: Cheng, X., Wang, B., Liu, L.Y., Zhao, Z., Ling, X., Zhao, F. and Wang, D.K. (2019) Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in Paris polyphylla var. Yunnanensis. Agricultural Sciences, 10, 15171533. https://doi.org/10.4236/as.2019.1012112 Received: October 28, 2019 Accepted: November 29, 2019 Published: December 2, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Paris polyphylla is a temperate genus of flowering plants belonging to the family Trilliaceae that includes 24 species distributed throughout Europe and East Asia.
Paris is native to Southwest China [1], and the Yunnan-Guizhou Plateau is considered the center of its diversity [2] [3]. As one of the most well-known medicinal plants in China, P. polyphylla var. yunnanensis is highly prized for its pain-relieving and anti-inflammatory properties [4] [5] [6]. Recent studies have shown that P. polyphylla also has anticancer activity [7] [8].
From over-collection during the past decades, wild resources have become scarce, and P. polyphylla is now considered an endangered species. In fact, its conservation is only possible through natural propagation because asexual reproduction via tissue culture remains a challenge [9]. The rate-limiting factors in the cultivation of P. polyphylla is prolonged dormancy (18 months) and slow growth from seed (3 -4 years) [10] [11]. Thus, understanding the mechanism of prolonged seed dormancy in this species is key to accelerating its propagation.
Previously, we observed that the anthers of P. polyphylla closed in the evening and reopened in the morning, with the entire dehiscence period lasting up to 20 days [12]. Indeed, alterations in darkness, lower temperature, and humidity can drive the closing and reopening of dehiscent anthers [13]. Edwards et al. [14] reported that the dehisced anthers of Lilium philadelphicum closed when it rained, and they proposed that effective anther closing is an adaptation to protect pollen during the flowering period. However, as revealed by our previous study results [15], the anther dehiscence period of P. polyphylla var. yunnanensis can last up to 23 days, but pollen viability peaks on day 1 and gradually decreases, and the receptivity of the stigma to pollen peaks on days 11 to 13. Within this short period, the occurrence of anther closure at night and during rain can shorten the pollination period and seriously affect normal fertilization. However, little is known about the molecular mechanisms underlying the sensitivity or ability of dehiscent anthers to close in response to the environment, and whether and how the genetic mechanism of the closure of anthers of P. polyphylla affects further physiological activity.
In this study, RNA-sequencing (RNA-Seq) technology was used, and the 1519 Agricultural Sciences transcriptome of P. polyphylla anther was de novo assembled. Furthermore, the differential expression of unigenes (DEGs) between closed and opened anthers was analyzed. The transcriptomic data can improve our understanding of the mechanism underlying abnormal closure of anthers in flowering plants.

Plant Material
The seeds of P. polyphylla var. yunnanensis were harvested from plants grown in

Illumina Reads Processing and de Novo Assembly
The original paired-end reads were filtered to obtain the "clean reads" for de novo assembly. This process included the removal of reads with adaptor contamination or unknown nucleotides, and low-quality reads with ambiguous sequence "N." The "clean reads" from all samples of P. polyphylla were used for the de novo assembly using Trinity software [16]. This software assembles the reads into longer transcripts to form contigs, clusters the contigs according to paired-end information and similarity among contigs, and selects the main transcripts as unigenes.

Analysis of Differentially Expressed Genes
We detected the differentially expressed genes either using DESeq (http://precedings.nature.com/documents/4282/version/2), when the samples fit the replication required for an analysis, or by using EBSeq [20]. Benjamini-Hochberg method [21], using the FDR, was applied to correct the P value of the original hypothesis. Here, The false discovery rate (FDR) was set to a threshold of <0.01 [22], and fold change was set to a threshold of ≥2 as the criterion [23].
The unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis was used to compare the relationship between differential unigene clusters.
X. Cheng et al.

De Novo Assembly of the Anther Transcriptome
After filtering out low-quality reads and vector sequences, we obtained 72.75 GB of data with 13,981,216 -16,999,694 paired-end reads from each sample. Of the clean reads, more than 93% had Phred-scaled mapping scores equal to the corresponding error probability of 0.01 (Q20) ( Table 2). These results showed that the quality of these sequences was reliable. Trinity [16] was used to mix the samples and assemble a consolidated unigene library that included 12,083,102 contigs, 280,777 transcripts, and 79,815 unigenes. The N50 length of transcripts was 1215 bp and the mean length of the unigenes was 1098 bp (Table 3). Bowtie analysis [17] revealed that each read had a BLAST efficiency of >62.19%.

Closure Is an Abnormal Process of the Dehiscent Anther
Dehisced anthers of P. polyphylla close every evening and reopen every morning; to study the mechanism of this closure phenomenon, the relevant samples were classified as follows: before opening in the morning (sample T1), semi opened (T2), opened (T3), closing initiation (T4), semi closed (T5), fully closed (T6), and never opened samples (T7). Furthermore, the samples treated by watering included the following: opened before watering (T8), closed after watering (T9), semi opened after watering (T10), and fully opened after watering (Table   1). To analyze the DEGs involved in the regulation of opening and closing of anthers by external stimulus, the opened and closed samples due to night, the T1, T2, and T3 samples, which represent opened samples; and the T4, T5, and T6 samples (Table 1) (Figure 1(a)).
As the anthers of P. polyphylla can close when watering, a 10-minute watering treatment was carried out. After watering, the anthers moved from a fully opened stage to closed, and were divided into opening before watering (T8), water-closing (T9, T10), and opening after watering (T11). There were DEGs between opening before or after watering (T8 and T11) and closing after watering (T9, T10   genes involved in this pathway were mostly down-regulated. In addition, the common pathway for night closing and watering-induced closing is consistently involved in oxidative phosphorylation (energy generation) and ribosomes (protein translation), but the gene cluster for opening contained fewer unigenes involved in energy pathways, meanwhile, those unigenes involved in energy metabolism during opening were down-regulated (Table 4). These results suggest that closing of dehisced anthers is an energy dissipation process that requires the synthesis of related proteins. Conversely, the opening process does not require such high levels of energy and protein synthesis. That is, the closing of dehiscent anther is an abnormal stress response process and opening is a naturally restorative process, therefore, we inferred that closure is a response to withstand adverse stress.

Abnormal Closing of Anther by External Stimulus Induces Epigenetic Silencing That Affects Seed Dormancy
In  Of the 6321 annotated DEGs, the unigenes involved in "response to external stimuli" were prominent in all biological processes (Figure 2(a)). Further analysis was carried out based on speculation that the external stimuli might transfer a signal to the nucleus, and finally induce "DNA binding" or "chromatin binding". In total, 215 unigenes were annotated to chromatin binding, the "chromatin binding" unigenes showed prominent in number (215 in 279, 77%). Further analysis showed that among the 215 unigenes, 120 (56%) unigenes (Figure 2(b)) are involved in silencing by epigenetic modification, including histone acetylation, histone H3-K9 methylation, histone lysine methylation, DNA methylation, Figure 2. Functional annotation and classification of DEGs between opening samples (including biological repetition of T3, T8, and T11) and closed samples (including biological repetition of T1, T6, T9, and T10), involving responses to night and watering, the DEGs derived from duplicate values of comparation between three samples in "opening group" and each sample in "closed group". The results showed in (a) Function of "response to external stimuli" are prominent in the DEGs; (b) There were 215 unigenes annotated to "chromatin binding" function; (c) The 120 unigenes involved in "epigenetic modification" among the 215 unigenes with "chromatin binding" function, and (d) 50 epigenetic-involved unigenes showed seed dormancy or germinationrelated function. chromatin silencing by small RNA, miRNA synthesis involved in gene silencing by miRNA, RNA splicing, histone phosphorylation, histone H3-S10 phosphorylation, methylation-dependent chromatin silencing, posttranscriptional gene silencing by RNA, covalent chromatin modification, ubiquitination, and posttranscriptional gene silencing (Figure 2(c)). Interestingly, among the unigenes involving epigenetics, 50 were annotated to seed dormancy-or germination-related function simultaneously (Figure 2(d)). Moreover, 202 unigenes were located in mitochondria; among them, nine unigenes were involved in seed dormancy, germination, and development. These results showed that epigenetic modification genes have important roles in impacting seed dormancy, germination and development.
Furthermore, the results of KEGG pathways Enrichment analysis of DEGs between opening vs. closing and between anthers vs. control (leaf, petal, calyx, and stigma), showed the "plant hormone signal transduction" pathway is the most significant in former, but not in latter (Figure 3), and the variation in the expression of DEGs involved in the pathway analysis showed that the key genes, such as GID1, are up-regulated in the signal transduction to germination while closing, indicating down regulation of germination. Meanwhile, key genes, such as PYR/PYL and PP2C, which were down-regulated, and SnRK2 which up-regulated in the signal transduction were induced seed dormancy while closing (Figure 4), indicating a closing impact to seed dormancy through the "plant hormone signal transduction" pathway.

Verification of Anther Closure Involved DEGs Are Correlated to Seed Germination
To verify whether frequent abnormal closing of anthers affects biological processes that follow pollination and fertilization, such as seed dormancy and germination, the seed dormancy-related unigenes obtained from the mesodermal transcriptome of P. polyphylla var. yunnanensis [11] were compared with unigenes involved in anther closure (night closing and water-induced closing). The results revealed the significantly DEGs owning to anther closing including in the all type of seed dormancy-related unigenes found in embryo of seed, including phytohormone related, seed maturation related, cell wall growth related, circadian rhythms, flavonol biosynthesis related, cytochrome P450 and others. Among them, at leat four genes differential expression in anther closing showed similar expression pattern in embryo of seed (Table 5). interestingly, the four genes including three phytohormone related genes and one circadian rhythm gene. The three phytohormone related genes indicate similar expression pattern with showed in "plant hormone signal transduction" (Figure 4), where gibberellins up-regulated, abscisic acid down-regulated and brassinosteroid up-regulated, all which impact on seed dormancy or germination.
Furthermore, as a contrast, genes involved in the regulatory network related to the seed germination rate in Arabidopsis were used to retrieve in the "up-regulated" DEGs between closing and opening, finally, the retrieved unigenes X. Cheng et al. control (leaf, petal, calyx, and stigma). The arrow pointed to icons lie in the upper right corner showed "plant hormone signal transduction" pathway is the most significant one when closing anther compared with dehiscent anther; in contrast, the most significant one was Oxidative phosphorylation when dehiscent anthers compared with other tissues such as leaf, petal, calyx, and stigma. Note: The transverse axis is enrichment factor, which indicates the ratio of the DEGs to the proportion of the pathway entry in all the commented genes to the pathway entry. The enrichment factor is larger, indicating that the enrichment degree of the DEGs in the pathway is more obvious. The longitudinal axis is −log10 (Q-value), in which Q-value is the p ≥ value after multiple hypothesis test, so the larger the −log10 (Q-value), the more significant the enrichment of DEGs in the pathway. So, the closer the entry is to the upper right corner, the greater the reference value is.  TIP2-1, voltage-dependent anion-selective channels, and chloride intracellular channel exc-4 were among the unigenes that were up-regulated during water-closing. Hormone-related genes up-regulated during anther closure were found to be related primarily to abscisic acid (ABA), jasmonic acid (JA), and gibberellin (GA), with concomitant strong up-regulation of transmembrane proteins, kinases, receptors, and molecules related to hormonal signal transduction. Indeed, these up-regulated genes are in regulatory pathways that can inhibit seed germination and prolong seed dormancy. For example, inhibition of germination via ABA and related signal transduction genes may involve strong up-regulation of superoxide dismutase and reduction of reactive oxygen species to inhibit germination. Up-regulation of cell wall-modifying enzymes and aquaporins can potentially inhibit seed germination. In addition, water-closing functions via increased expression of gibberellin 3-oxidase (GA3OX) and dihydroflavonol-4-reductase (DFR) can inhibit germination; moreover, water deprivation Figure 5. The up-regulated unigenes involved in anther-closing forming a regulatory network show impact from "light, temperature, and water" to seed germination and dormancy.
can inhibit germination via aquaporins. Furthermore, these networks also comprised many chromatin remodeling proteins including histone-lysine N-methyltransferase (HLN), a small nuclear ribonucleoprotein (SNR), histone deacetylase (HD), and others. It is verified that up-regulation of these genes, especially chromatin remodeling genes, enable epigenetic modifications that can affect cellular processes following anther germination.

Discussion
Transcriptome sequencing has been used in several plant species and is considered an effective method to identify novel genes [19]. In the present study, we sequenced different anther stages of P. polyphylla using an Illumina paired-end sequencing platform and obtained DEGs between closed and opened anthers.
After sequence annotation, we found that: 1) closure of dehiscent anthers is an abnormal process in response to external factors, such as night (darkness or decreased temperature) and water uptake, 2) this process consumes a large amount of energy, and 3) it is accompanied by abundant protein synthesis. Then closure is an abnormal process of the dehiscent anther.
As the opposite direction of dehiscence, the closing anther of P. polyphylla involves a high level of protein biosynthesis, including several types of cell wallmodifying enzymes and structural proteins, whose expression are either up-regulated or down-regulated. This indicates that the cellular mechanism of abnormal anther closure in P. polyphylla involves changes in the secondary wall. Meanwhile, the KEGG enrichment analysis revealed that the pathway "planthormone signal transduction" is the most significant, and the DEGs of the pathways revealed that gibberellins, brassinosteroid were up-regulated, while abscisic acid signal transduction was down-regulated. The cellular mechanism of anther dehiscence mainly involves changes in the endothecium, membranous tissue, and stomium that lead to thickening of the secondary inner wall of the anther [24]. Another wall thickening occurs due to the dehydration of pollen grains and stomium cells [25]. After the entry of K + into the pollen grain, the regulation of osmotic potential induces turgidity and extrusion of anther stomium tissue, thus, accelerating anther dehiscence [24] [26] [27]. In addition, plant hormones including jasmonic acid (JA), ethylene, and other growth factors, are important compounds that regulate anther dehiscence, and are important factors inducing responses in anther tissue cells to water deprivation [28]. Then, anther closing shows similar mechanism with dehiscence, in cell wall modification, hormones and ion flux, but they are different in detail, especially in type of hormones and ion flux. Indeed, gibberellins, brassinosteroid were up-regulated, while abscisic acid was down-regulated as DEGs occur in closing anther.
All the changes when closing owing to cell wall modification and cell elongation hormones [29] might impact germination potential, ultimately improving seed dormancy. But what change in anther which transfers to seeds is unclear.
The epigenetic-related changes results from the involvement of mitochondriarelated genes which exclude energy production, and all types of epigenetic silencing including phosphorylation, methylation, acetylation, ubiquitination, miRNA synthesis involved in gene silencing, and mRNA splicing, should look as a hint to think over transferring of epigenetic marks. Importantly, some of these epigenetic-related unigenes are involved in regulation of seed dormancy, germination, and development. In the reckoning model refer to Arabidopsis, an regulatory network could formatted change from external stimuli to anther closing, and finally impact on seed dormancy, in which unigenes implicated in anther closure of P. polyphylla similarly include chromatin remodeling proteins, such as HLN, SNR, and HD. The HLN protein can form methylated histones, such as H3K9me2, which serves as a marker of gene expression regulation; SNR mediates the splicing of pre-mRNA by binding to the loop I region of U1-snRNA; HD is responsible for the deacetylation of lysine residues in the N terminal of the core histones (H2A, H2B, H3, and H4).
Further comparation analysis showed some known seed dormancy related genes in anther cloure occur similar expression pattern in embryo of seed. Importantly, these genes associate with regulation of three key hormone signal transduction pathways to seed dormancy. However, many questions still remain unclear, for example, if the epigenetic-related genes actually change the seed dormancy related genes? How the seed dormancy related genes expression changed in closing stage and finally keep the same expression pattern in seed?
How many genes expression change in closure finally transfer to seed?

Conclusion
Seed embryo of Paris polyphylla will stop at the globular stage for about 120 days after fertilization and seed germination requires 18 months for embryo development and release from dormancy, this long period of dormancy become the biggest obstacle for seedling. Although finer details of this mechanism for prolong dormancy should be elucidated by further studies, in the present study, the information from transcriptome of anther closing indicate that abnormal closing stimulated by night and water can lead to changes in a batch of epigenetic genes, and the epigenetic modification become the utmost cause for impacting seed germination, especially by change expression of seed dormancy related unigenes in abnormal closure and keep the same expression pattern in seed embryo.

Funding
This study was supported by the National Natural Science Foundation of China (31760256 to SH; 31260037 and 31760403 to SH) and Project of Talent introduction in Kunming university (code: YJL17003).