| In vitro organogenesis refers to the regeneration processes of shoots, roots or other organs from plant tissue or callus, which is an important aspect of plant development. Up to the present, some genes involve in regulating organogenesis have been explored, but the molecular mechanism of the processes remains unclear. Preliminary evidence showed that epigenetic modification may also play important roles of the processes, however, little is known in this field, thus, systematic research is of great significance.MicroRNAs (miRNAs) are small (～21 nucleotides) noncoding RNAs that are key post-transcriptional regulators of gene expression, which belong to a kind of epigenetic modification. Most of the transcription factors with important roles in plant development are miRNAs targets. MiRNAs play crucial roles in a broad range of processes, including cell division, meristem differentiation, organ boundary establishment, leaf polarity development and so on. However, little is known about their roles in cell proliferation and shoot regeneration.In our lab, we found that the auxin receptor TIR1 and the transcription factor MYB96 have significantly different expression levels in the embryogenic and non embryogenic callus, suggesting their roles in cell proliferation and shoot regeneration. Thus, it is of great significance for elucidating their roles in these processes.In this work, we focused on the molecular mechanism of in vitro cell proliferation and shoot regeneration using Arabidopsis culture system. First, the expression profiles of miRNAs in embryogenic and non embryogenic callus were analyzed using miRNA chips, and 15 differently expressed miRNAs were detected. Furthermore, the roles of candidate miRNAs and the target genes (such as ARF10 and TIR1)in in vitro cell proliferation and shoot regeneration were explored and discussed; Second, the effects of MYB96 on cell proliferation and shoot regeneration were analyzed and the possible molecular mechanism were explored. The results provided information for understanding the molecular mechanism of in vitro cell proliferation and shoot regeneration.Additionally, the molecular mechanism of MYB96 in regulating lateral root development under drought stress was analyzed.The main process and results of this research were as follows:1. Roles of Arabidopsis miRNAs in in vitro shoot regeneration and cell proliferation1.1 Histological features of embryonic and non-embryonic calliThe embryonic calli (C1), which were yellow-green in colour, contained embryonic cell mass, somatic embryos and regenerated shoots. The non-embryonic calli (C2), which were pale green and fragile, was characterized by loosely packed, irregularly shaped cells. C2 completely lost the ability to differentiate.1.2 Analysis of miRNAs expression profiles between C1 and C21) According to the miRNA microarray data, there were 15 miRNAs differently expressed between C1 and C2. miR397, miR398, miR774, miR843 and miR859 were proved to be more abundant in C1, while miR157, miR159, miR160, miR165, miR166, miR167, miR319, miR390, miR393 and miR394 were more abundant in C2.2) Results of Stem-loop RT-PCR confirmed the microarray data.3) According to the data of Semi-quantitative RT-PCR and Real-time PCR, most miRNAs and their target genes showed opposite expression patterns in C1 and C2.1.3 Expression patterns of candidate miRNAs1) According to their expression patterns on CIM (callus induction medium), SIM (shoot induction medium) and RIM (root induction medium), four distinct groups were recognized:(1) miRNAs specifically up- or down-regulated in SIM. Examples were miR397 (which was up-regulated) and miR160a,394a,393a and 159b (which were down-regulated), suggesting their possible roles in shoot regeneration;(2) miRNAs specifically up-or down-regulated in RIM. Examples were miR159a,165a, 398,774 and 859 which were all up-regulated, suggesting their possible roles in root regeneration;(3) During CIM pre-incubation, miR166a was specifically up-regulated, suggested its possible roles in cell proliferation;(4) Those up- or down-regulated during pre-incubation on CIM, as for instance miR157a,166b and 319a/b, suggesting their possible roles in de-differentiation.2) As the SIM incubation time extended to 21 days,5 miRNAs (abundance in C1) were all significantly up-regulated. MiR398, miR843 were strongly accumulated by 21 days. MiR397 and miR859-774 were up-regulated throughout the incubation of SIM.3) According to the Real-time PCR, miR398a/b/c were strongly expressed in the stem and leaf; Similarly, miR397a and miR859-774 were expressed more strongly in the stem; miR843 was much more abundant in the stem and the shoot apex.1.4 Effects of miR160 and ARF10 on shoot regeneration and the possible molecular mechanism1) Effects on shoot regeneration The number of regenerated shoot number per explant of 35S::miR160a,35S::ARF10, mARF10 and arf10 were compared. 35S::miR160a lines produced fewer shoots in comparation with the Col-0 (1.2±0.4<5.4±0.5); 35S::ARF10 lines produced similar shoots with the Col-0 (6.4±1.22≈5.6±1.14); mARF10 regenerated significantly more shoots in comparation with the Col-0(22.3±3.98>5.8±1.3); the arf10 line regenerated fewer shoots in comparation with the Col-0 (3.8±1.14<5.63±1.41). Thus, over-expression of miR160a appeared to reduce the capacity of shoot regeneration, while that of ARF10 increased it.2) Expression patterns of ARF10 during shoot regeneration According to the GUS staining data, ARF10 was down-regulated on CIM and enhanced on SIM. After 14 days culture on SIM, ARF10 signal was detected in the regions where shoots would shortly arise or the emerging shoots, suggesting the close relationship between ARF10 and shoot regeneration.3) The possible molecular mechanism of ARF10 in regulating shoot regeneration (1) According to the analysis of Real-time PCR, WUS、CLV3、CUC1/2 were strongly up- regulated in mARF10(in vivo and in vitro), significantly higher than those in 35S::ARF10, Col-0 and 35S::miR160a. (2) Histological examination showed that WUS and CLV3 were expressed more widely in the SAM of mARF10 than those in Col-0. (3) GUS staining indicated that WUS and CLV3 emerged earlier and more widely in mARF10 root explants than those in Col-0.In conclusion, over-expression of miR160a reduced the ability of shoot regeneration, while removing the repress of miR160 on ARF10 significantly improved the ability via up-regulation of WUS, CLV3 and CUC1/2.1.5 Effects of candidate MiRNAs on cell proliferation1) Over-expression and knockdown mutants were used to examine whether the various candidate miRNAs were involved in cell proliferation. The data showed that 44% of the men1,25% of miR160a-OX and miR159b-OX formed visible outgrowths, which were higher than 16% of Col-0.2) GUS staining showed that miR159b and miR166b were localized at the sites of cell proliferation. In conclusion, miR166, miR159 and miR160 are all associated with the in vitro cell proliferation process.2. Effects of TIRl on shoot regeneration and possible molecular mechanism2.1 Expression patterns of TIR1 in vitro and in vivo1) Analysis of Real-time PCR suggested that TIRl was up-regulated during SIM incubation and required CIM pre-incubation for its up-regulation on SIM.2) GUS staining confirmed the Real-time PCR results. During the late phase of SIM incubation TIR1 signal was particularly concentrated in protuberances on the verge of shoot regeneration or the forming new shoots or leaves.3) Analysis of Real-time PCR demonstrated that TIR1 transcripts are much more abundant in the leaf than in either the root or the stem.2.2 Effects of TIR1 on shoot regeneration and cell proliferation1) Effects on shoot regeneration:The ranking of shoot regeneration ability from root and hypocotyl explants was 35S::TIR1>Col-0>tir1-1. The regenerated shoot number is 4.28±1.81>1.67±0.82>0.33±0.54 (from root explants) and 7.5±0.8> 6.7±0.65>2.3±0.33 (from hypocotyls explants). These results indicated that over-expression of TIR1 improved the shoot regeneration ability; silence of the gene significantly reduced it.2) Extended CIM culture can’t restore tir1-1’s reduced shoot regeneration ability. 3) Effects on cell proliferation:Protuberances formed from tirl-1 root explants were fewer and smaller than those from Col-0 and 35S::TIR1, suggesting lower cell proliferation ability of tirl-1 explants.2.3 The possible molecular mechanism1) According to the Real-time PCR, cytokinin related genes CDKB1;1, CKS1, IPT4, ARR15 and somatic embryogenesis related genes LEC2, FUS3 were all up-regulated in 35S::TIR1 explants, among them, ARR15, CKS1and LEC2 were significantly down-regulated in tirl-1 explants.2) LEC2 and ARR15 had similar expression patterns with TIR1 during shoot regeneration, suggesting their contribution to TIR1for shoot regneration.3. Roles of MYB96 in cell proliferation and shoots regeneration3.1 Expression patterns of MYB96 during callus formation and shoots regeneration1) According to the analysis of Real-time PCR, MYB96 were specifically down-regulated during SIM incubation. It was 1/12 of that on CIM 10 day,1/7 of that on RIM 10 day.2) GUS staining showed that MYB96 was localized at the sites of cell proliferation during CIM incubation, and particularly concentrated in protuberances on the verge of shoot regeneration or the forming new shoots during SIM incubation. These results suggested its roles in in vitro cell proliferation and shoot regeneration.3.2 Effects of MYB96 on cell proliferation, shoots and roots regenerationAnalysis of in vitro cultures of Col-0、myb96-1d (over-expression mutant)、myb96-1 (knock-down mutant) suggested that abilities of cell proliferation and shoot, root regeneration were reduced in myb96-1d.3.3 The molecular mechanism of MYB96 regulating in vitro cell proliferation, shoot and root regneration1) According to the GUS staining or fluorescence observation of pCYCB1;1::GUS, myb96-1dXpCYCB1;1::GUS, DR5::GUS, myb96-1dXDR5::GUS, myb96-1dXpSCR::GFP and pSCR::GFP. Expression of CYCB1;1, DR5 and SCR were down-regulated in myb96-1d in vivo and in vitro. 2) According to the analysis of Semi-quantitative RT-PCR, STM, WUS and CLVS were down-regulated in myb96-1d during shoot regeneration; PIN4 was down-regulated in myb96-1d during root regeneration.3) According to the ChIP analysis, MYB96 could directly regulate the expression of WUS, CLV3 and PIN4.Therefore, down-regulation of these genes contributed to the reduced abilities of cell proliferation and shoot, root regeneration in myb96-1d explants, at least partly.4. MYB96 regulates lateral root development under drought stress4.1 Phenotypes of myb96-1dThe myb96-1d exhibited dwarfed growth with altered leaf morphology and reduced lateral root number.4.2 Expression patterns of MYB961) According to the analysis of subcellular localization of MYB96, the green fluorescence was detected in the nucleus.2) According to the analysis of Real-time PCR, MYB96 gene was expressed at a high level in the leaves and flowers, at a relatively lower level in the roots.3) According to the GUS staining, its expression was detected at a high level in the leaves, hypocotyls, lateral root primordial and the guard cells.4) According to the analysis of Real-time PCR, MYB96 was induced significantly by drought and ABA, and also induced moderately by IAA.4.3 MYB96 enhances ABA-mediated drought resistance1) Drought stress analysis showed that myb961-d enhanced the drought tolerance, while myb96-1 was susceptible to drought. After re-watering, the survival rates of myb96-1d, Col-0 and myb96-1 was 90%> 45% >15%.These results suggested that MYB96 may regulate drought tolerance of Arabidopsis.2) Re-expression of MYB96 in myb96-1 fully restored the drought tolerance to Col-0 verifying the roles of MYB96 in drought resistance.3) The ranking of stomatal apertures under ABA was myb96-1d<Col-0< myb96-1, indicating that MYB96 enhanced drought resistance by reducing stomatal opening.4) The RD22 expression was elevated about 12-fold in the myb96-1d mutant but only 1/4 in the myb96-1mutant of that in Col-0. In addition, levels of RD22 expression induced by drought were significantly diminished in the myb96-1. The effect of drought on the expression of MYB96 and RD22 was significantly reduced in the ABA-deficient aba3-1 mutant. These data suggested that that MYB96 enhances ABA-mediated drought resistance via RD224.4 MYB96 regulates lateral root development1) Seeds of Col-0, myb96-1d and myb96-1were germinated and grown on 1/2MS supplemented with various concentrations of ABA (0,0.5,1.0,2.0,3.0 and 5.0μM).The lateral roots were reduced more severe in the myb96-1d mutant than in wild-type and myb96-1,demonstrating that MYB96 was an important component of ABA signaling pathways in regulating lateral root formation.2) With IAA treatments, MYB96 GUS activity was extended throughout the whole root system, mainly in the vasculature of the root.3) The myb96-1d reduction lateral root phenotype could not be fully recovered by exogenous auxin.4) The possible molecular mechanism:(1) According to the analysis of Real-time PCR, the expression levels of GH3.3, GH3.5/WES1, GH3.6/DFL1 and ARF17 were up-regulated by over 2-fold in the myb96-1d than that in Col-0 and myb96-1. All these genes were related with lateral root development. Thus, over-expression MYB96 reduced lateral root number by up-regulating these genes.(2) According to the analysis of Real-time PCR, GH3.3, GH3.5/WES1, GH3.6/DFL1 were also induced by exogenous ABA, suggesting that MYB96-mediated ABA signals regulated lateral root development via GH3 genes under drought stress. |
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