| Bromeliaceae are monocotyledon herb plants with great variety, and have high edible and ornamental value. The growth of Bromeliaceae is strict with the environmental condition; they always have the demerits of long vegetative stage and blooming in uncertain reasons. Ethylene or its alternatives treatment to induce Bromeliaceae blooming was widely applied. Moreover, the Bromeliaceae showed different responses to ethylene treatment depending on growth stage:mature plants bloomed but juvenile plants did not. Aechmea fasciata is a kind of famous ornamental bromeliads of Aechmea in Bromeliaceae, and was also stimulated for flowering with ethylene. Heretofore, yet little is understood about molecular mechanism behind ethylene induced flowering in bromeliads.A. fasciata juvenile and mature plants treated with ethrel as the experimental materials, three transcriptome librarys were constructed using high-throughput short read sequencing technology (Illumina). After the bioinformatics analysis, differentially expressed genes were screened. In addition, we tried to use an innovative approach to screen genes of interest from local A. fasciata transcriptome database we constructed. The full-length cDNA of these genes were isolated by RACE technology, and GeXP assay analysis was applied to analyze the genes expression levels in different stages treated samples of juvenile and mature plants. Finally the molecular mechanism of Bromeliaceae flower stimulation by ethylene was proposed according to experimental results, and the problem of different responses.to ethylene treatment between juvenile and mature plants have a primary explanation.The main results were listed as follows:1. In view of little genomic data of Bromeliaceae is available, massive transcriptome sequences data of A. fasciata was obtained from high-throughput sequencing. A great number of differentially expressed gene related to ethylene signal transduction pathway and floral transition were screened. RACE and semi-quantitative RT-PCR were applied and ensured the reliability of the bioinformatics analysis.2. Comparative software BLAST from NCBI was used to construct an innovative local comparative transcriptomes database. With previously known gene sequences, we screened genes and miRNAs fragments of interest by homologous alignment. This is a much easier and whole new approach to screen target genes from transcriptome library. 3. The full-length cDNA sequences of44genes were isolated by RACE technology.4. The expression analysis of genes in ethylene signal transduction pathway revealed that EIL gene and its targets ERF transcription factors could increase expression levels in1hour and then activate downstream reaction. This suggests that ethylene signal have a high transmission rate in plant. Additionally, the expression levels of AP2/EREBP family members have great diversity in change trend and time. We speculate that different members ware regulated in different ways.5. After ethylene treatment, we analyzed the expression changes of genes in five flowering pathways.(1) In autonomous pathway, expression of key gene FLD was inhibited, ethylene showed inhibition effect to this pathway. And we didn’t screened FLC, the most important gene of this pathway in the whole transcriptome.(2) In gibberellin (GA) pathway, DELLA and miR159act as suppressors of GA signaling were up-regulated, they could repress the floral meristem-identity genes LFY, SOC1and target gene GAMYB, and delay the blooming.(3) We didn’t got any genes from vernalization pathway, we hypothesized it’s relevant to the fact that low temperature is absent in Bromeliads’ tropical living environment.(4) In photoperiod pathway, expression of GI gene was inhibited, and light signal integrating gene CO had no significant change in early stage but increased in late stage, and this up-regulation is later than its downstream target gene FT’s promoting.(5) Flowering repressors TFL1-like and AP2-like genes expression changed and affect flowering induction. In summary, ethylene couldn’t affect sitimulate flowering in the fomer four pathway mentioned above, but we hypothesized the flowering repressors could regulate the flower induction of ethylene signal.6. Experimental results showed that ethylene could stimulate a FT gene which is homologous with RFT1in rice increasing and then induced flowering. This process has no specificity to development stage, juvenile and mature plants both have RFT1-like expression promoting. In addition to this, there is another FT gene which is homologous to Hd3a in rice, it increased after2d in mature plants but maintained very low level all the time in juvenile plants, this Hd3a changed after CO, also after FT’s downstream gene API. As a downstream gene of CO, This Hd3a-like gene expression tendency had good agreement with CO. we speculated this hd3a-like could be activated by CO. Double FT system in rice could give us some reference to these two FT genes in A. fasciata.7. Experimental results showed miR156and its target genes, some SPLs transcription factors, have different expression trend between juvenile plants and mature plants, which maybe the signs of phase change occurred in mature but not in juvenile plants. miR172, have an ability of phase change and flowering transition, its remarkable rise in mature plants is much earlier than juvenile plants. Three AP2-like genes as the targets of miR172, AP2-1and AP2-3were both inhibited dramaticly; AP2-2was up-regulated in juvenile plants and down-regulated in mature plants. We surmised all these differences were originated from the variance of function and position in these AP2-like genes. We hypothesized AP2-land AP2-3have the function of inhibiting FT gene like SMZ in Arabidopsis, they could depress FT in normal times but ethylene could antagonize this inhibition. AP2-2might have the same function of control phase change like TOE in Arabidopsis and GL15in maize, the decreasing in mature plants activated phase change and the increasing in juvenile inhibited phase change, so the matures bloomed but the juveniles did not. Hence, different growth stages of A. fasciata could make different responses to ethylene treatment. This phenomenon of mature plants bloomed but juvenile plants not is probably related to juvenile-to-adult phase change.8. Before ethylene treatment, RFT1-like gene didn’t express in both juvenile and mature plants. However, the repression mechanism between them might be different. In juvenile plants, RFT1-like was depressed by TFL1-like genes with high expression, and miR172’s target gene AP2-2, which also could inhibit FT expression, almost not expressed. But in mature plants, TFL1-like expressed scarcely, suppression of RFT1was performed by miR172’s target gene AP2-2with high expression.9. Two AP1-like genes have consistent expression tendency. In the early stage, they became lower in juvenile plants, but higher in mature plants and initiate bloom finally. Based on the experimental results, flowering transition probably didn’t need LFY or SOC1in A. fasciata.10. Bromeliaceae flowering induction with ethylene is a dual mechanism composed of activation and judgment. Activation is started by RTF1-like gene, and it’s a nonspecific process both in juvenile and mature plants. Flowering determination is controlled by mir172and its targets AP2-like, and cooperated with mir156and its targets SPLs. |
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