Pistil Transcriptome Analysis to Disclose Genes and Gene Products Related to Aposporous Apomixis in Hypericum perforatum L.

Abstract

Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change ≥ 2 in at least one comparison, respectively. Differentially expressed genes were enriched for multiple gene ontology (GO) terms, including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated with DNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analyzed in great detail. Overall, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway. Furthermore, based on gene annotation and co-expression, we underline a putative role of hormones and key actors playing in the RNA-directed DNA methylation pathway in regulating the developmental changes occurring during aposporous apomixis in H. perforatum.

Controlling Apomixis: Shared Features and Distinct Characteristics of Gene Regulation

“…This discrepancy might potentially be explained by master regulators which control complex programs of gene activity. Transcriptional analyses to identify genes differentially expressed in sexual as compared to apomictic plants have been presented for a variety of species including Pennisetum ciliare and Pennisetum glaucum [83,84], Panicum maximum [85,86], Poa pratensis [87,88], Brachiaria brizantha [89,90], Paspalum notatum and Paspalum simplex [91][92][93][94], Eragostris curvula [95], Medicago falcata [96], Boehmeria tricuspis [97], Hypericum perforatum [62,98], Hieracium [99,100], Boechera [101][102][103], and also for Citrus [76] ( Table 2). These studies provide evidence for temporal deregulation of the gene regulatory processes governing sexual reproduction in apomicts and identify large numbers of up to hundreds of genes to be differentially expressed.…”

Apomixis Technology: Separating the Wheat from the Chaff

“…Attempts to identify components of apomixis by transcriptional profiling of reproductive organs using Differential Display PCR (DD-PCR), SuperSAGE (serial analysis of gene expression), and high-throughput sequencing on microdissected ovules revealed 1) differentially expressed genes in reproductive tissues of apomictic and sexual relatives from different plant systems (e.g., Pennisetum, [69,70]; Brachiaria, [71,72]; Panicum, [73,74]; Poa, [75], Eragrostis, [76][77][78]; Paspalum, [42,64,[79][80][81]; Hieracium, [82,83]; Hypericum [84,85]) and 2) an overall shift in gene regulation at the MMC stage and a global heterochronic gene expression between the sexual and apomeiotic ovules (e.g., Boechera, [86][87][88][89]; Paspalum [79,90,91]; Pennisetum, [92]; Hieracium, [83,93,94]; Hypericum [85,95]; Ranunculus [96,97]). Such wide-ranging de-regulation on gene expression levels between sexual and apomictic ovules affect genes encoding varied biological functions (GO classes) and regulatory pathways, including key genes of the sexual pathway, RNA-directed DNA methylation and transcription regulation, hormonal signaling, and cell cycle control (see details in the next section).…”

“…In Hypericum, a truncated gene ARI showing homology to AtARIADNE7 (encoding a ring finger E3 ligase protein involved in regulatory processes and ubiquitin-mediated protein) degradation, might have a role on protein populations altering gametophyte development [48]. In transcriptomic analyses, Galla et al [85] found evidence that differentiation of the AI cell may be related to the misregulation of RNA-directed DNA methylation (RdDM) pathways (through MEE57, CMT3, and IND2, genes involved in the maintenance of DNA methylation, and AGO9, a component of RNA silencing complexes), chromatin-remodeling proteins (through HPCHC1, a MORC-like CW-type Zinc finger protein), and hormonal homeostasis (through HPPIN8, an auxin efflux carrier involved in ovule intercellular auxin gradients).…”

The steps from sexual reproduction to apomixis

“…In the process of sexual reproduction, hormones are also important control factors involved in the differentiation of fertilized eggs and in mitosis. Some genes related to cytokinin, auxin and brassinoin are significantly upregulated during aposporous apomixis (Galla et al 2017). This shows plant hormones play important roles in apomictic reproduction.…”

“…Through these the seed production cycle would be shortened and the high cost of producing hybrid seeds would be eliminated (Bicknell and Koltunow 2004). Although apomixis is potentially important for crop breeding and has been studied extensively, the genetic regulation of apomixis remains unclear and the results obtained in studies of the apomictic mechanism are not always consistent (Galla et al 2017). Over the last few years, reviews have focused on various aspects of apomixis including the different genetic control mechanisms.…”