| Mainly distributed in northern China and western mountains, Larix spp. is an importantconifer species with highly economic and ecological values. Somatic embryogenesis is anuseful tool to improve genetic traits of larch by molecular methods. It is also an idealexperimental material to study the mechanism behind gymnosperms embryogenesis. However,stable and efficient induction of somatic embryos is affected by various genotypes and cultureconditions, and abnormal embryos were initiated frequently in our experiments. Auxin and itspolar transport play important roles in embryogenic morphogenesis of Arabidopsis and otherplants, and it had been suggested that the HD-ZIP III transcription factors may participate inpolar auxin transport, but it is still largely unknown the functions of auxin and miR166regulation of HD-ZIP III in gymnosperm somatic embryogenesis. Based on the somaticembryogenesis of larch, we had studied the effects of polar auxin transport inhibitor1-N-naphthylphthalamic acid (NPA) and over-expression of miR166a on somatic embryodevelopment of larch, and analyzed the sequence structures and expression patterns of twoauxin receptors and four HD-ZIP III members. The following results were obtained:(1) To study polar auxin transport in somatic embryogenesis of larch, somatic embryostreated with NPA was used to mock the larch mutant in which auxin polar transport wasinhibited. As a result, after treated with10mg/L IAA and3mg/L NPA, the pro-embryogenicmass (PEM) grow more slowly, with the polarity destructed.3mg/L NPA-treatment had noeffect on the frequency of induction of somatic embryos, but the NPA-treated somatic embryosformed a cup-sharp apical embryo, cotyledons fused and programmed cell death of suspensorsdelayed. The microstructure of NPA-treated somatic embryos were observed by semithinsection, and the cells in NPA-treated embryos were not organized normally and patternformation was disrupted, indicating that the growth balance between embryo and suspensorwas changed by NPA-treatment. (2) Two homologues of auxin receptors from larch were identified and designatedLaAFB1and LaAFB2, both of which contained LRR and F-box domains. Identification of thecleavage products of LaAFB1in vivo confirmed that it was regulated by miRNA, and thecleavage sites occurred at10th or13th base of miR393. Secondary siRNAs that were derivedfrom miR393-guided cleavage of LaAFB1were predicted by bioinformatics, and they wereonly found in small RNA transcriptome of seedling, but not in somatic embryos, indicating thatmiR393-guided cleavage of LaAFB1occurs in post-embryogenesis. The expression patterns ofLaAFB1and LaAFB2in PEM, mature somatic embryos, root, stem and leaf were analyzed byqRT-PCR. The results showed that they shared similar expression pattern and both showedlower transcript levels in mature somatic embryos, which suggested that it is required todepress the auxin sensitivity for maturation and development of somatic embryos in larch.(3) Four HD-ZIP III homologues from larch were identified and designated LaHDZ31,32,33and34. They all contained HDã€START and MEKHLA domains. The occurrence of amiR165/166target sequence in all four cDNA sequences indicated they might be targets ofmiR165/166. Identification of the cleavage products of LaHDZ31and LaHDZ32in vivoconfirmed that they were regulated by miRNA. Their expression patterns during somaticembryogenesis and the effects of NPA on their expressions were investigated. The resultsshowed that the four genes had higher expression levels at mature stages than at theproliferation stage, and that NPA treatment down-regulated the expression of LaHDZ31,32and33at cotyledonary embryo stages, but had no effect on the expression of LaHDZ34. Weconcluded that these four members of Larix HD-ZIP III family might participate in polar auxintransport and the development of somatic embryos.(4) Two miR166precursor genes were obtained and designated LaMIR166a andLaMIR166b. Their expression patterns in root, stem, leaf, normal somatic embryos andNPA-treated embryos were studied, and both genes showed very differential expressionpatterns. The transcripts of LaMIR166a could be detected in all organs and developmentalstages of somatic embryos, while the LaMIR166b showed undetectable transcript levels inPEM and leaf. Taken together, miR166and their targets were co-expressed at late stages of somatic embryogenesis, but showed inverse NPA-responses. LaMIR166a was cloned into thepSuper1300+binary vector allowing over-expression under a Super promoter. Subsequently,five hygromycin-resistant embryonic cell lines were obtained by Agrobacteriumtumefaciens-mediated transformation, and they were identified to be positively transgenic byPCR amplification. Study on the phenotypes and microscopic structures of transgenic somaticembryos indicated that over-expression of LaMIR166a inhibited hypocotyl elongation, andaffected the initiation of cotyledons and morphology of stem apical meristem.From the research of auxin and miR166in somatic embryogenesis, we found that theeffects of LaMIR166a over-expressing on apical morphogenesis of somatic embryo weresimilar with that of NPA-treatment, although the developmental defects of LaMIR166aover-expressor was slighter than that of NPA-treated somatic embryos. The expression level ofWOX was up-regulated in LaMIR166a over-expressing and NPA-treated somatic embryos,indicating that the miR166and auxin might share common regulatory pathways during somaticembryogenesis of larch. These results provided a theoretical basis for further study of themolecular mechanisms behind conifer somatic embryogenesis. |
