Transcriptomes and DNA methylomes in apomictic cells delineate nucellar embryogenesis initiation in citrus

Abstract

Citrus nucellar poly-embryony (NPE) is a mode of sporophytic apomixis (SA) that asexual embryos formed in the seed through adventitious embryogenesis from the somatic nucellar cells. NPE allows clonal propagation of rootstocks, but it impedes citrus cross breeding. To understand the cellular processes involved in NPE initiation, we profiled the transcriptomes and DNA methylomes in laser microdissection (LMD) captured citrus apomictic cells. In apomictic cells, ribosome biogenesis and protein degradation were activated, whereas auxin polar transport was repressed. Reactive oxygen species (ROS) accumulated in the poly-embryonic ovules, and response to oxidative stress was provoked. The global DNA methylation level, especially that of CHH context, was decreased, whereas the methylation level of the NPE-controlling key gene CitRWP was increased. A C2H2 domain-containing transcription factor (TF) gene and CitRWP co-expressed specifically in apomictic cells may coordinate to initiate NPE. The activated embryogenic development and callose deposition processes indicated embryogenic fate of nucellar embryo initial (NEI) cells. In our working model for citrus NPE initiation, DNA hyper-methylation may activate transcription of CitRWP, which increases C2H2 expression and ROS accumulation, triggers epigenetic regulation, and regulates cell fate transition and NEI cell identity in the apomictic cells.

Structural variation and parallel evolution of apomixis in citrus during domestication and diversification

“…Analysis of FhRWP and its homologs across Citrinae revealed a striking pattern of MITE insertions in the 5’ promoter regions of these genes. Apomictic accessions in Citrus and Fortunella contain two or three MITE insertions in the RWP promoter, respectively, while sexually reproducing Citrinae individuals lack MITE insertion [ 6 , 53 ]. These MITE insertions correlate with RWP gene expression levels in the nucellus of apomicts [ 36 ] and act as apparent binding targets of the transcriptional regulator FhARID , which may mediate gene expression and embryogenic induction.…”

Shaping inheritance: how distinct reproductive strategies influence DNA methylation memory in plants

“…In addition to the impact of epigenetic perturbations on apomixis, at least two loci were reported to be hypomethylated in apomictic female organs compared to sexual gametophytes of the grass Cenchrus ciliaris [36], suggesting that DNA methylation loss might be more severe during development of apomictic embryo sacs than gametogenesis. Nonetheless, methylome dynamics of adventitious embryos are reminiscent to the one observed during early development of sexual embryos [24], with early nucellarderived embryos showing global CHH hypomethylation compared to surrounding maternal tissues [37].…”

Apomixis: genetic basis and controlling genes

“…Another mechanism influencing alteration of DNA methylation patterns should also be considered. Recently, a DNA methylome analysis revealed hypermethylation in the promoter of CitRWP in polyembryonic citrus, which contains a MITE insertion, while hypomethylation was detected in the promoter of CitRWP in monoembryonic citrus without a MITE insertion [ 89 ]. This result suggests that the MITE insertion may be related to the hypermethylation of CitRWP , which might activate gene transcription and further enable cells in the ovules of polyembryonic citrus to switch to an apomictic pathway.…”

“…Transcriptome comparison of apomictic Boechera , Hieracium , and Hypericum with related sexual lines revealed changes in siRNA synthesis and RNA-directed DNA methylation (RdDM)-related gene expression [ 90 – 92 ]. DNA methylation analysis showed that the overall methylation level of gametophyte-apomictic C. ciliaris [ 93 ] and sporophyte-apomictic citrus [ 89 ] was lower than that of sexually reproducing plants, suggesting that hypomethylation is related to apomictic reproduction, while in the gametophyte-apomictic Paspalum treatment with the DNA methylation inhibitor 5-azacytidine significantly reduced parthenogenesis frequency, suggesting that a high DNA methylation level may maintain apomictic reproduction [ 94 ].…”

“…The specific expression of CitRWP in citrus ovules may enable the nucellar cells to acquire an embryonic fate. Additionally, a C2H2 zinc-finger domain-containing transcription factor gene ( CitZFP ), which is homologous to the dandelion parthenogenesis gene ( PAR ), is specifically expressed in apomictic cells [ 89 ]. This gene may be another candidate gene for engineering sporophytic apomixis.…”