Function And Regulatory Mechanism Of MiR159And MiR172in Floral Development In Gloxinia (Sinningia Speciosa)

MicroRNAs are endogenous20～24nt noncoding RNAs that regulate their target genes via post-transcriptional repression or complementary sequences degradation. In Arabidopsis, about200miRNA have been identified. Among numerous miRNAs, only three families are involved in flowering time control:miR156, miR172and miR159, which play crucial roles in flowering pathway. Current knowledge about miRNA regulation of flowering time control in non-model plant, especially in ornament plants is still largely unknown. The aim of this study is to investigate whether genetic modification of miR159and miR172expression can offer an effective approach for regulation of flowering characteristics in gloxinia.1Function of miR159in flower developmentIdentification and sequence analysis of SsGAMYB. To obtain the full length of GAMYB in gloxinia, the fragments with conserved MYB domain were cloned by rapid amplification of cDNA ends (RACE). A putative complementary site of miR159a existed in the region (964～985bp) and the cleavage site was detect by RNA ligase-mediated5’rapid amplification (RLM5’-RACE). In gloxinia, mature miR159a highly accumulated in young leaves and sepals, and lowly expressed in stamens. SsGAMYB was highly expressed in stamens and carpals, while its expression level was almost undetectable in sepals. Thus, the different expression patterns revealed that miR159negatively regulated SsGAMYB in gloxinia.Transgenic gloxinia was generated using Agrobacterium-mediated transformation. We used the AtmiR159a precursor to overexpress miR159a based on the conserved function in plants. In addition, we carried out target mimicry to repress the activity of the endogenous miR159a. The transgenic plantlets were detected by reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers. Distinct bands of expected size were obtained from thirty-two plantlets of AtmiR159a overexpressing35S:miR159a transgenic lines and twenty-eight transgenic plants of MIM159overexpressing35S:MIM159lines. Altered flowering time in transgenic gloxinia. Compared with wild-type gloxinia, under short-day conditions, overexpression of miR159delayed the timing of flowering to37(±4.93) days whereas suppression of miR159accelerated flowering to20(±3.5) days. Mature miR159a level was significantly increased in35S:miR159a lines and decreased in35S:MIM159lines. While a low level of SsGAMYB was observed in35S:miR159a lines, whereas the SsGAMYB level was significantly elevated in35S:MIM159lines. These results revealed that the alteration of miR159can regulate the mRNA level of SsGAMYB and effect flowering time in transgenic lines.Expression of flowering genes downstream SsGAMYB in transgenic gloxinia. Quantitative RT-PCR analysis of flower buds showed that overexpression of miR159resulted in a significant decline in SsLFY transcript level in35S:miR159a lines, while suppression of miR159caused an increase in SsLFY in35S:MIM159lines under SD conditions. Meanwhile, the mRNA levels of SsAG, SsAP1and SsAP3were decreased dramatically in35S:miR159a lines but increased in35S:MIM159lines. These results revealed that alteration of miR159in gloxinia can affect the expression level of LFY and regulate the flowering time.Effects of altered LFY expression on floral organ development. Phenotype observation indicated that the floral morphology of35S:miR159a appeared relatively normal compared to the wild-type. Nonetheless, abnormal morphology appeared in a few of35S:MIM159lines (3of28lines). In the flower bud stage, sepals were converted partly into petals in the35S:MIM159lines, disturbing the normal development of sepals. Quantitative RT-PCR analysis showed that AP3expression level was significantly higher in abnormal sepals, while API transcript level was notablely higher and AP3was drastically lower in abnormal petals compared to wild-type flowers. It revealed that the conversion of petals and sepals might be caused by disturbed expression levels of API and AP3between the first and second whorls.2Function of miR172in flower developmentIdentification partial cDNA of SsAP2. Based on the conserved domain of AP2, degenerate primers are designed according to homologous genes in Arabidopsis, tomato and grape to amplify partial cDNA from gloxinia. Phylogenetic analysis showed that the polypeptide of SsAP2shared significant similarity with those of Arabidopsis, tomato and grape.Generation of transgenic gloxinia lines that overexpress or suppress miR172a. Transgenic gloxinia was generated using Agrobacterium-mediated transformation. After four fortnights of in vivo culture, twenty-four plantlets of35S:miR172a transgenic lines and twenty-one transgenic plants of35S:MIM172were obtained.Phenotype of transgenic gloxinias. Compared with wild-type gloxinia, under long-day conditions, overexpression of miR172a accelerated flowering to47(±2.16) days whereas suppression of miR172delayed the timing of flowering to7(±4.28) days. Mature miR172level was significantly increased and SsAP2was dramatically lower in35S:miR172a lines. While a low level of mature miR172and an elevated level of SsAP2were observed in35S.MIM172lines. These results revealed that miR172mediated cleavage of SsAP2can effect flowering time in transgenic lines.Morphological changes in transgenic gloxinias. Nonetheless, abnormal morphology appeared in a few of35S:miR172a lines (2of241ines). In the flower bud stage, sepals were converted partly into petals in the35S:miR172a lines and petals were rolled downward in35S:MIM172lines with a reduced plant height. It suggested that alteration of SsAP2activity disrupted the formation of floral organs.Our results suggested that regulation of GAMYB and flowering genes downstream by miR159is a functionally effective pathway in triggering flowering under short days. While manipulating expression level of miR172completely affected the expression of SsAP2and controlled the flowering time. Thus, manipulation of miRNAs involved in flowering time provides an applicable approach for commercial ornamental plants breeding.