Maize seed development is initiated by double fertilization. Two male gametes combined to the haploid egg cell and the dikaryotic central cell, respectively, to generate a diploid embryo and a triploid endosperm [1
]. Mature maize seeds are a composite of maternally derived tissues (pericarp, placenta, and pedicel), endosperm and embryo that provides an exquisite experimental system for developmental analyses. The endosperm accounts for 85% of the kernel mass at maturity, and is of prime agronomic importance. The endosperm function as an absorptive structure that supports embryo development and seedling germination in angiosperms [3
]. Typical of grasses, the maize embryo is developmentally precocious. The mature embryo inherits the genetic information for the next plant generation. Recent evidence also indicates that endosperm plays a critical role in the regulation of seed development through interaction with the embryo and the seed coat [4
]. Differentiation of the endosperm and embryo occurs side by side, within distinct developmental compartments and the embryo and endosperm interact extensively throughout their development. Thus, the maize kernel offers a unique opportunity to study developmental signaling between the embryo, endosperm, and maternal tissues. Elucidation of the genetic regulatory mechanisms involved in maize seed development will facilitate the design of strategies to improve yield and quality.
The transcriptome is the overall set of transcripts. Analysis of transcriptome dynamics can be used to determine aids the function of unannotated genes, identify genes that act as critical network hubs, and interpret the cellular processes associated with development. In maize, high-throughput RNA sequencing (RNA-seq) has provided insights into the programs controlling the development of different organ systems including leaves, shoot apical meristems, and the endosperm, among others, as well as the general development of whole seed [6
]. Furthermore, a study that took a global view of transcriptome dynamics over the majority of seed development stages has been reported [10
]. In another study, coupled laser-capture microdissection (LCM) and RNA-Seq were used to comprehensively identify the mRNA populations present in each of the main maize endosperm cell types, as well as the embryo and four maternal compartments of the kernel at 8 days after pollination (DAP). Specifically accumulated mRNAs in each of the compartments and co-expressed gene modules were detected [11
]. The above efforts have been made on studying the role of mRNA and the gene regulatory networks during seed development in maize. However, seed development is a tightly regulated process, which requires exquisite control over gene expression. Thus, additional studies that utilize recent advancements in biology are required.
Non-coding RNAs have recently emerged as versatile master regulators of biological functions. Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs wih lengths ranging from 200 nt to 100 kb. They play an important role in the regulation of gene expression, and act by acting as competing endogenous RNAs (ceRNAs) [12
]. Plant lncRNAs are transcribed by different RNA polymerases and have diverse structural features. They are also important regulators of gene expression in various biological processes [15
]. Their regulation occurs through a large complex network that involves mRNAs, micro RNAs (miRNAs), and proteins in animals [16
], and they have multi-faceted biological functions that vary based on location, binding site, and mode of action. Recently, lincRNAs (long intergenic noncoding RNAs) have also been shown to function as miRNA targets or decoys in plants [17
]. However, their main functions remain unclear. miRNAs are small endogenous ncRNAs of 18–24 nucleotides in length that originate from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and various biological processes. In addition to the conventional miRNA function, a reversed miRNA logic exists, in which coding and noncoding RNA targets can crosstalk through their ability to compete for miRNA binding [18
]. On the basis of this hypothesis, ceRNAs have recently been discovered, adding to the complexity of miRNA-mediated gene regulation [19
]. CeRNAs are RNAs that share miRNA recognition elements (MREs), thereby competing for miRNA binding sites and regulating each other. Several studies that analyzed lots of reports of tissues and mammalian cells have shown that the combined effects of multiple ceRNAs can have a major impact on gene expression and cellular phenotypes [20
]. However, few ceRNA interactions have been found in plants.
LncRNAs acting as potential ceRNAs can compete for the same MREs and regulate protein expression. CeRNAs are implicated in the development of some cancers. A disruption in the delicate ceRNA network can lead to tumor formation [23
]. However, no studies on ceRNA involvement in plant development have been published to date. Understanding miRNA mediated lncRNA and mRNA crosstalk can provide significant insight into gene regulatory networks and their implications for seed development.
Here, we performed high-throughput sequencing analysis to determine the expression profiles of lncRNAs and mRNAs during embryo and endosperm differentiation stages. We systematically identified novel and seed-specific lncRNAs, and then the differential expressions of representative lncRNAs were further confirmed using quantitative real-time polymerase chain reaction (qRT-RCR). The potential function of lncRNAs and their target genes were also predicted and analyzed. Subsequently, we determined the comprehensive functional landscape of the lncRNA-miRNA-mRNA ceRNA networks in maize seed development for the first time using bioinformatics approaches, and acquired mRNA associated pathways and gene ontology data. The result provides new insights into the regulatory mechanism of lncRNAs in seed development.
The conventional view of gene regulation focused on protein-coding genes until the discovery of numerous non-coding RNAs including lncRNAs and miRNA. A substantial number of lncRNAs exist in mammals and plants, and they play important functional roles in human disease, plant development, and other biological processes [41
]. However, a comprehensive analysis of lncRNA expression in maize seed development has not yet been performed until now. 9 DAP, 15DAP, and 20DAP represented three typical time-points in maize kernel development [43
]. The endosperm already completed differentiation, with the aleurone, transfer cell and starchy endosperm cells at 9 DAP [44
]. Additional cell types start to differentiate in the embryo at 9 DAP [45
]. At around 15 DAP, embryo-surrounding region disappears together with the suspensor and synthesis of endosperm starch and storage proteins reach peaks [43
]. Programmed cell death occurs from 20 DAP [43
]. This study is the first report on the expression of lncRNA during the different developmental stage of embryos and endosperm in maize. To date, systematic searches for lncRNAs have been conducted in 13 maize tissues, including 25 DAP embryo and 25 DAP endosperm [46
]. In this study, we identified 753 reliably expressed lncRNAs and found that they share similar features with those identified in the other 13 previously tested maize tissues. LncRNAs are shorter and are expressed at significantly lower levels than protein-coding transcripts [41
]. In addition, many lncRNAs are expressed in a tissue-specific manner, suggesting that lncRNAs expression is biologically and evolutionally controlled.
MiRNAs mediate communication between transcripts during the development of different tissues through MREs. In this study, we constructed a ceRNA network to predict the function of lncRNAs. The lncRNAs and mRNAs in ceRNA network exhibited dynamic expression and regulation patterns during the developmental processes, suggesting that ceRNA interactions also mediate the coordination of different functions during seed development.
From the lncRNA-miRNA-mRNA co-expression network, we found a total of 23 miRNAs belonging to 9 miRNA families were co-expressed with 7 lncRNAs and 69 mRNAs, forming five complex networks (Figure 5
). The nine families comprised miR156, miR166, miR167, miR171, miR396, miR398, miR408, miR444, and miR827. The miR156/SPL module is highly conserved among the phylogenetically distinct plant species, and plays important roles in regulating plant fitness, biomass, and yield [47
]. Osmotic stress reduced miR167a, which targets IAR3, then the miR167/ARF module affects auxin conjugation to coordinate growth and patterning in plants [48
]. MiR171 is involved in GA and auxin homeostasis by targeting GRAS family members in tomato [49
]. The miR396c-OsGRF4-OsGIF1 regulatory module plays an important role in grain size determination and has implications for rice yield improvement [50
]. MiR444a plays multiple roles in the rice NO3-signaling pathway that affects nitrate-dependent root growth, nitrate accumulation, and phosphate-starvation responses [51
]. The miR827/NLA module plays an important role in phosphate transport activity [52
]. In addition to protein and second messengers, small regulatory RNAs also play a role in signal transduction. In this study, we investigated the role of ncRNAs regulation in seed development. GO analyses were performed to further annotate the biological functions of ceRNAs in the lncRNA-ceRNA network. We noticed that a significant number of GO terms were related to signal transduction (Figure 6
A). This phenomenon is very interesting for the important roles of co-expressed miRNAs and their target genes in seed development. However, no combination of lncRNAs and seed development has been made before.
In summary, the application of ceRNA network analysis to transcriptomes obtained during tissue development provides a novel approach for understanding gene functionality, and give us new insights on non-coding RNA regulatory in seed development.