ABSTRACT
KEY MESSAGE: Genome-wide identification of C2H2-ZF gene family in the poly- and mono-embryonic citrus species and validation of the positive role of CsZFP7 in sporophytic apomixis. The C2H2 zinc finger (C2H2-ZF) gene family is involved in plant vegetative and reproductive development. Although a large number of C2H2 zinc-finger proteins (C2H2-ZFPs) have been well characterized in some horticultural plants, little is known about the C2H2-ZFPs and their function in citrus. In this work, we performed a genome-wide sequence analysis and identified 97 and 101 putative C2H2-ZF gene family members in the genomes of sweet orange (C. sinensis, poly-embryonic) and pummelo (C. grandis, mono-embryonic), respectively. Phylogenetic analysis categorized citrus C2H2-ZF gene family into four clades, and their possible functions were inferred. According to the numerous regulatory elements on promoter, citrus C2H2-ZFPs can be divided into five different regulatory function types that indicate functional differentiation. RNA-seq data revealed 20 differentially expressed C2H2-ZF genes between poly-embryonic and mono-embryonic ovules at two stages of citrus nucellar embryogenesis, among them CsZFP52 specifically expressed in mono-embryonic pummelo ovules, while CsZFP7, 37, 44, 45, 67 and 68 specifically expressed in poly-embryonic sweet orange ovules. RT-qPCR further validated that CsZFP7 specifically expressed at higher levels in poly-embryonic ovules, and down-regulation of CsZFP7 in the poly-embryonic mini citrus (Fortunella hindsii) increased rate of mono-embryonic seeds compared with the wild type, indicating the regulatory potential of CsZFP7 in nucellar embryogenesis of citrus. This work provided a comprehensive analysis of C2H2-ZF gene family in citrus, including genome organization and gene structure, phylogenetic relationships, gene duplications, possible cis-elements on promoter regions and expression profiles, especially in the poly- and mono-embryogenic ovules, and suggested that CsZFP7 is involved in nucellar embryogenesis.
ABSTRACT
Citrus nucellar poly-embryony (NPE) is a mode of sporophytic apomixis 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 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 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.
