Small RNA and mRNA Sequencing Reveal the Roles of microRNAs Involved in Pomegranate Female Sterility

Abstract

Female sterility is a key factor restricting plant reproduction. Our previous studies have revealed that pomegranate female sterility mainly arose from the abnormality of ovule development. MicroRNAs (miRNAs) play important roles in ovule development. However, little is known about the roles of miRNAs in female sterility. In this study, a combined high-throughput sequencing approach was used to investigate the miRNAs and their targeted transcripts involved in female development. A total of 103 conserved and 58 novel miRNAs were identified. Comparative profiling indicated that the expression of 43 known miRNAs and 14 novel miRNAs were differentially expressed between functional male flowers (FMFs) and bisexual flowers (BFs), 30 known miRNAs and nine novel miRNAs showed significant differences among different stages of BFs, and 20 known miRNAs and 18 novel miRNAs exhibited remarkable expression differences among different stages of FMFs. Gene ontology (GO) analyses of 144 predicted targets of differentially expressed miRNAs indicated that the “reproduction process” and “floral whorl development” processes were significantly enriched. The miRNA–mRNA interaction analyses revealed six pairs of candidate miRNAs and their targets associated with female sterility. Interestingly, pg-miR166a-3p was accumulated, whereas its predicted targets (Gglean012177.1 and Gglean013966.1) were repressed in functional male flowers (FMFs), and the interaction between pg-miR166a-3p and its targets (Gglean012177.1 and Gglean013966.1) were confirmed by transient assay. A. thaliana transformed with 35S-pre-pg-miR166a-3p verified the role of pg-miR166a-3p in ovule development, which indicated pg-miR166a-3p’s potential role in pomegranate female sterility. The results provide new insights into molecular mechanisms underlying the female sterility at the miRNA level.

The emerging role of small RNAs in ovule development, a kind of magic

“…A similar approach was used to investigate the potential regulatory effects of miRNAs and lncRNAs on rice female gametophyte abortion, comparing the miRNA transcriptome in ovules of a high frequency female-sterile line (fsv1) and a wild-type line (Gui 99) during ovule development (Yang et al 2017;Liu et al 2019). These studies identified 100 known miRNAs and around 500 lncRNAs that were significantly differentially expressed in fsv1 compared to the wild-type (i.e., Gui 99); thus, it provides a pool Chen et al (2020) of ncRNAs with a potential role in rice female gametophyte (Yang et al 2017). One of the major barriers in commercial application of rice polyploids is their low fertility.…”

“… Ortiz et al ( 2019 ) Pinus taeda miRNA detection by RT-PCR Oh et al ( 2008 ) Punica granatum sRNA-seq, miRNA analysis and targets pred. Chen et al ( 2020 ) degrad. = degradome; pred.…”

Identification of miRNAs and Target Genes at Key Stages of Sexual Differentiation in Androdioecious Osmanthus fragrans

“…In GO enrichment analysis of predicted target genes, most of the target genes in the three stages were classified into the biological process of plant organ development, especially related to reproductive development ( Table S4 ). For example, the target genes encoding homeobox-leucine zipper protein (HB), regulated by the miR166 family, may affect the healing of carpels or ovule development by regulating vascular cell differentiation and the initiation of meristematic tissue, thus leading to the pistil abortion [ 39 ]. The target genes of probable indole-3-pyruvate monooxygenase (YUC) involved in auxin synthesis were regulated by the miR396 family, modulated gynoecium development or formation by influencing auxin homeostasis [ 40 , 41 , 42 ].…”

Integrated analysis of miRNAome transcriptome and degradome reveals miRNA-target modules governing floral florescence development and senescence across early- and late-flowering genotypes in tree peony

“…MiRNAs have become crucial modulator of gene expression, primarily by means of the cleavage/inhibit of target genes translation during or after transcription . MiRNAs regulate almost all the crucial biological processes of the plants' life cycle, such as growth and development (Li et al, 2019), flowering (Spanudakis and Jackson, 2014), ripening (Guo et al, 2018), postharvest senescence (Pei et al, 2013;Chen et al, 2020), and plant-environment interactions (Basso et al, 2019). MiRNAs can rapidly reprogram the expression patterns of downstream genes that strictly regulate agronomic trait, for instance, florescence (Wang et al, 2009).…”

“…Integrated analysis of the miRNAome, transcriptome, and degradome analyses can enhance the understanding of the genome wide co-expression patterns of miRNA-mRNA pairs and links the biological interactions of miRNA-target modules ( Liu et al., 2020a ; Wang et al., 2021 ). Integrated miRNA, transcriptome, and degradome-seq analysis of miRNA-mRNA involved in flowering of pepper ( Shu et al., 2021 ), floral development and abscission of yellow lupine ( Glazinska et al., 2019 ), female sterility of pomegranate ( Chen et al., 2020 ), male sterility of rice ( Sun et al., 2021 ), flower development across capsicum species ( Lopez-Ortiz et al., 2021 ), stamen development in moso bamboo ( Cheng et al., 2019 ), flowering induction in Lilium × formolongi ( Zhang et al., 2021 ), floral transition in Magnolia × soulangeana ‘Changchun’ ( Sun et al., 2021 ) has provided evidence demonstrated of regulatory pathways and gene networks of miRNAs and their targets associated with flowering. Moreover, studies have shown that miR319 - TCP , miR156 - SPL , miR159 - MYB , miR172 - AP2 and miR399 - PHO2 nodes play important roles in floral transition ( Waheed and Zeng, 2020 ).…”