| Currently, in vitro organ induction is one of the hot topics in stem cell research,which is defined as shoots, roots and other organs induced from explants including root, hypocotyl, cotyledon and seeds during appropriate in vitro culture. The in vitro culture system for organ induction is a proven technique that mainly is consists of callus induction and shoot differentiation. Based on the system, numerous of genes related with callus induction and shoots differentiation have been revealed. However,the mechanism of shoot regeneration is still obscured, especially the epigenetic regulation in the process of in vitro organ induction needs further explore.MicroRNAs (miRNAs) are no-coding small RNAs that regulate plant biological process including the morphogenesis and adversity stress by their post-transcriptional repression of target genes. So far, several studies have revealed functions of miRNAs in shoot regeneration that initiates further studies of miRNAs in shoot regeneration.Thus, in order to reveal the regulatory mechanism of miRNAs in shoot regeneration,more functional miRNAs remain to be explored.Based on this, some differentially expressed miRNAs (miR159 and miR165/6) are selected by analyzing the expression profiles of miRNAs between the embryonic and non-embryogenic callus in Arabidopsis. In this article, functions of miR159 in shoot regeneration were analyzed by using mir159ab mutants and indentified its target genes in the regulation of callus induction and shoot regeneration. Considering the explants used in shoot regeneration are roots, thus, we further explore functions of miR159 in root meristem and primary root growth. Also, AGO proteins are required for miRNAs functionate. AGO 10 can compete with AGO1 for binding miR165/6 and thus regulate its function. Here, we indentified functions of miR165/6 and its regulator(AGO 10) in shoot regeneration and revealed its mechanism in regulating shoot regeneration.The main results are as follows:1) A comparison of callus forming ability on CIM (Callus Induced Medium) of root explants sampled from mir159ab (the double mutant), mMYB65 (the gain-function mutant) and myb33myb65myb101 (the triple mutant) showed after 7 days, the former two both generated more calli than the wild type (WT), while the latter formed fewer.Expression of MYB33, MYB65 and MYB101 all largely increased in the cultures of mirl59ab on CIM medium. Repression of miR159 on MYB65 was further showed by detecting GFP signals of pMYB65:159-GFP in cultures of mir159ab and WT during CIM periods. The conclusion was that miR159 inhibits callus induction by repressing its target genes (MYB33, MYB65 and MYB101).2) To test function of miR159 in shoot regeneration, mir159ab was employed for shoot regeneration phenotype analysis. Dramaticlly, the number of shoots generated from mir159ab explants decreased and expression of MYB33, MYB65 and MYB101 all increased in the cultures of mir159ab on SIM (Shoot Induced Medium). Further, a comparison of shoot regeneration ability of mMYB33, mMYB65, mMYB101 (the gain-function mutant) and myb33myb65myb101 (the triple mutant) from root explants on SIM, showed explants obtained from mMYB33, mMYB65 and mMYB101 lines regenerated less shoots than did those obtained from WT, while explants of myb33myb65myb101 produced unaltered shoots. Subsequently, repression of miR159 on MYB65 was further showed by detecting GFP signals of pMYB65:159-GFP in cultures of mir159ab and WT. It was visible that the aboundance of MYB65 transcript highly accumulated in the proliferative cells generated from explants of mir159ab but only weakly accumulated in those cells generated from WT explants. To explore the effects of miR159 on signals of auxin and cytokinin in the explants cultured on SIM,pDR5:GFP and pTCSn: GFP were employed as sensor for detecting signals of auxin and cytokinin in explants of WT and mMYB65. GFP activity of pDR5:GFP increased while the activity of pTCSn:GFP decreased in the cultures of explants sampled from mMYB65 lines. Additionally, expression of WUS, CLV3 and STM decreased in the cultures of mMYB65 lines by Realtime PCR. Further,weak WUS transcript in the protuberant cell generated from mMYB65 lines was shown by detecting the GUS activity of pWUS:GUS in the cultures of mMYB65 lines on SIM. Collectively, the above results demonstrate that miR159 promotes shoot regeneration by repressing its target genes (MYB33, MYB65 and MYB101), which in turn enhances the cytokinin signals and the accumulation of WUS expression while suppresses the auxin signals during shoot regeneration.3) To explore the effects of miR159 on primary growth, the primary root length and root meristem zone size were compared between WT and mirl 59ab, which showed an enhanced growth of the primary root and root meristem in mir159ab along with high accumulation of MYB33, MYB65 and MYB101 in the primary root of mir159ab. In the roots of transgenic plants harboring pMYB65:159-GFP, GFP activity was largely suppressed, whereas the level of GFP in the cross line (mir159ab×pMYB65:159-GFP)was deposited throughout apical meristem zone. To determine whether MYB65 regulates cell cycle progression and expression of root apical stem cell related genes,we analyzed the expression of CYCB1;1, PIN1, WOX5, PLT1 and SCR by detecting GUS and GFP activities of pCYCB1;1:GUS, pPIN1:PINl-GFP, pWOX5:GFP,pPLT1:PLT1-GFP and pSCR:GFP in mMYB65 lines. It showed MYB65 can promote cell cycle progression by directly binding to CYCB1;1 promotor. The data suggest that miR159 acts as a key repressor of the primary root's growth, acting through its repression of MYB65 and consequent blocking of the cell cycle.4) Function of AGO 10 in shoot regeneration was analyzed by using the liquid culture system. A comparison of shoot regeneration ability in LSIM (Liquid Shoot Induced Medium) of seed explants from ago 10 (the knock off line), 3 5S:AGO10 in WT (the over-expression line) and 35S:AGO10 in ago 10 (the phenotype rescue line), showed explants of ago 10 regenerated more shoots than did those of WT, while explants of the over-expression line produced fewer, the rescue line rescued the shoot regeneration phenotype of the ago 10 mutant, demonstrating that AGO 10 represses shoot regeneration. AGO 10 expressed in the induced protuberance and the zone where pro-SAM generated by detecting GUS and GFP activities in the cultures of pAGO10:GUS and pAGO10:GFP in LSIM. Basing on the detection of GFP activity of U2:MIR165/6-GFP and ago10×pU2:MIR165/6-GFP during shoot regeneration, we found the GFP activity was weak in the cultures of ago10×pU2:MIR165/6-GFP,suggesting the abundance of miR165/6 largely increased in ago 10 mutant.Subsequently, AGOIO specially expressed in the cells where pro-SAM initiated and repressed the expression of miR165/6 thereby increased HD-ZIP? gene transcripts there by RNA in situ hybridization. Further,a comparison of shoot regeneration ability of MIM165/6 (target mimics of miR165/6) and men1 (the gain-function mutant)in LSIM, showed MIM165/6 lines regenerated less shoots than WT, while explants of men1 produced more shoots, demonstrating miR165/6 is a promoter in shoot regeneration. Further, the number of shoot generated from ago10×MIM165/6 was clearly less than that in ago10 but still higher than that in MIM165/6, suggesting AGO10 inhibits shoot regeneration probably partly functions by repressing miR165/166.To sum up, the function of epigenetic regulators (miR159, miR16516 and its regulator AGO10) in shoot regeneration was indentified and the mechanism was preliminarily revealed in this article. Thereby, it provides a foundation for further revealing the epigenetic mechanism in shoot regeneration. |
PDF Full Text Request