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The interaction between miRNAs (microRNAs) and target genes plays an important role in plant pistil development. MiRNAs related to pistils were explored in pomegranate. The differentially expressed miRNAs were screened at different developmental stages of pomegranate pistils, and their target differentially expressed mRNAs were further identified to clarify the regulatory effect of miRNAs on pistil development. In our study, 61 conserved miRNAs were identified in 30 families, including miR395, miR394, miR393, miR161, miR162, and miR168. Among them, miR156, miR157, miR159, miR160, miR164, miR165, miR166, miR167, miR169, and miR172 were involved in the development of flower organs. Eight miRNAs were randomly selected and verified for qRT-PCR analysis. The result analysis indicated that miR160, miR164, and miR172 might be positive factors in the regulation of pomegranate pistil development. MiR156 and miR166 might be involved in regulation of pomegranate pistil development as negative factors. [ABSTRACT FROM AUTHOR]

Based on the genome and small-RNA sequencing of pomegranate, miRNA167 and three target genes PgARF6 were identified in "Taishanhong" genome. Three PgARF6 genes and their corresponding protein sequences, expression patterns in pomegranate flower development and under exogenous hormones treatments were systematically analyzed in this paper. We found that PgARF6s are nuclear proteins with conserved structures. However, PgARF6s had different protein structures and expression profiles in pomegranate flower development. At the critical stages of pomegranate ovule sterility (8.1–14.0 mm), the expression levels of PgARF6s in bisexual flowers were lower than those in functional male flowers. Interestingly, PgARF6c expression level was significantly higher than PgARF6a and PgARF6b. Under the treatment of exogenous IBA and 6-BA, PgARF6s were down-regulated, and the expression of PgARF6c was significantly inhibited. PgmiR167a and PgmiR167d had the binding site on PgARF6 genes sequences, and PgARF6a has the directly targeted regulatory relationship with PgmiR167a in pomegranate. At the critical stage of ovule development (8.1–12.0 mm), exogenous IBA and 6-BA promoted the content of GA and ZR accumulation, inhibited BR accumulation. There was a strong correlation between the expression of PgARF6a and PgARF6b. Under exogenous hormone treatment, the content of ZR, BR, GA, and ABA were negatively correlated with the expressions of PgARF6 genes. However, JA was positively correlated with PgARF6a and PgARF6c under IBA treatment. Thus, our results provide new evidence for PgARF6 genes involving in ovule sterility in pomegranate flowers. [ABSTRACT FROM AUTHOR]

Backgrounds: Pomegranate is an excellent tree species with nutritional, medicinal, ornamental and ecological values. Studies have confirmed that SPL factors play an important role in floral transition and flower development. Results: Used bioinformatics methods, 15 SPL (SQUAMOSA promoter-binding protein-like) genes were identified and analyzed from the 'Taishanhong' pomegranate (P. granatum L.) genome. Phylogenetic analysis showed that PgSPLs were divided into six subfamilies (G1 ~ G6). PgSPL promoter sequences contained multiple cis-acting elements associated with abiotic stress or hormonal response. Based on the transcriptome data, expression profiles of different tissues and different developmental stages showed that PgSPL genes had distinct temporal and spatial expression characteristics. The expression analysis of miR156 in small RNA sequencing results showed that miR156 negatively regulated the expression of target genes. qRT-PCR analysis showed that the expression levels of PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 in leaves were significantly higher than those in buds and stems (p < 0.05). The expression levels of PgSPL5, PgSPL12 and PgSPL13 in flower buds were significantly higher than that in leaves and stems (p < 0.05). The full-length of coding sequence of PgSPL5 and PgSPL13 were obtained by homologous cloning technology. The full length of PgSPL5 is 1020 bp, and PgSPL13 is 489 bp, which encodes 339 and 162 amino acids, respectively. Further investigation revealed that PgSPL5 and PgSPL13 proteins were located in the nucleus. Exogenous plant growth regulator induction experiments showed that PgSPL5 was up-regulated in leaves and stems. PgSPL13 was up-regulated in leaves and down-regulated in stems. When sprayed with 6-BA, IBA and PP333 respectively, PgSPL5 and PgSPL13 were up-regulated most significantly at P2 (bud vertical diameter was 5.1 ~ 12.0 mm) stage of bisexual and functional male flowers. Conclusions: Our findings suggested that PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 played roles in leaves development of pomegranate. PgSPL5, PgSPL12 and PgSPL13 played roles in pomegranate flower development. PgSPL5 and PgSPL13 were involved in the response process of different plant hormone signal transduction in pomegranate development. This study provided a robust basis for further functional analyses of SPL genes in pomegranate. [ABSTRACT FROM AUTHOR]