| Cremastraappendiculata(D.Don)Makino is perennial,valuable and rare medicinal plant and important ornamental plant of Orchidaceae.The medicines used for drying pseudobulbs.It has the functions of clearing heat and detoxifying,moistening the lungs and relieving cough,activating blood circulation and removing blood stasis and so on.Its pseuodbulbs contain rich active molecules.Especially in recent years,it was found a variety of antitumor active components which have attracted attention and importance at domestic and overseas.However,there are serious resource problems for C.appendiculata.Under natural conditions,the seed setting rate was less than 5%,and the seed have no endosperm,and embryo consists of only a group of undifferentiated mature cells,which makes it almost impossible to have sexual reproduction in natural conditions.The pseudobulbs are almost the only reproductive organ.Regrettably,only the annual pseudobulb can produce one newbud and grow into one new pseudobulb in each year.Although the other pseudobulbs also have the potential germination and growth of buds,the buds cannot germinate since they are inhibited.So,pseudobulbs string is formed year by year.These biological characteristics results that the reproductive coefficient is extremely low.And there are few reports on the regulation mechanism of branch development of underground stems.Therefore,the regulation mechanism of the "shoot branch" was explored in this plant,which provided a new idea for the elimination of lateral bud inhibition,in order to lay a theoretical foundation for the molecular breeding and branch development of underground stems.In this paper,the three-year-old C.appendiculata was used as the material to study the morphogenesis and dormancy mechanism of the lateral bud.From the transcriptome level,gene expression level and hormone level analysis,it is revealed for the interaction between auxin,cytokinin and stigolactone to regulate the branching development.The main results are as follows:1.The microstructural analysis technique of light microscopy and scanning electron microscopy and qRT-PCR analysis were used to research the dormancy mechanism of the lateral bud.The results showed that decapitation induced lateral bud germination;Microstructural analysis found that the parenchyma cells from the base of the lateral buds changed significantly during the germination of the lateral buds(These cells gradually become rounded and full from the collapsed and wrinkle shape during the lateral bud germination.);The results of water test showed that the free water content in the lateral buds increased gradually during the germination of the lateral buds,and the bound water content decreased first and then increased slightly;qRT-PCR results showed that the expression of cell wall expansion protein genes ?EXPA1,EXPA2,EXPA6 and aquaporin genes PIP1-2 and PIP2 were up-regulated,while the expression of aquaporin gene ?TIP in the tonoplast was down-regulated.Therefore,the lateral bud dormancy of C.appendiculata may be caused by a certain factor regulating the transcription levels of the cell wall modifying protein and the aquaporin genes,thereby creating a low free water stress environment in the lateral bud,and inducing the lateral bud to enter dormancy.2.The physiological changes were discussed during the germination of lateral buds,and the role of phytohormones in regulating lateral buds break was further confirmed by exogenous hormone treatment.The results showed that the level of IAA in the lateral buds was significantly reduced after decapitation(P<0.05).After the germination of the lateral buds,the level of IAA increased slightly,but it was still significantly lower than that of the control(P<0.05).The change trend of CTK level was opposite to the change of IAA level in this process.During the germination of the lateral buds,the ABA level decreased in both the control group and the Decap group,but the Decap group decreased more rapidly.After 15 days of decapitation,it reached a significant level compared with the control(P<0.05).GA3 content was gradually increasing,and the significant level compared with the control also appeared after 15 days of decapitation(P<0.05).Applied zeatin induced lateral bud germination on 51.67% of biennial pseudobulbs and lateral bud germination on 35.00% of triennial pseudobulbs,while applied GA3 did not induce lateral bud germination.In addition,qRT-PCR analysis found that the expression of CaIPT was significantly increased after decapitation(P<0.05).These results suggest that decreased levels of IAA and increased levels of CTK are required for germination of lateral buds,and it may be that IAA regulates the level of CTK by controlling the expression of CaIPT gene,thereby controlling the growth of lateral buds.Changes of GA3 and ABA levels are associated with lateral bud growth,but their level changes may not be necessary for lateral bud germination in this plant.3.The transcriptome profiles revealed the molecular regulatory network of the shoot branching of C.appendiculata.The results showed that transcriptional sequencing yielded 597,053,172 high-quality clean reads,and assembled into 239,732 unigenes with an average length of 921 bp.Mapping with 7 public databases,179,559 unigenes(74.90%)were highly similar.GO and KEGG enrichment analysis of 5,988 DEGs obtained were significantly enriched in hormone signal transduction and hormone metabolism.This indicated that plant hormones played an important role in regulating the growth of lateral buds.Expression heat map analysis of DEGs related to branching hormones and branched transcription factors,it was found that the auxin-degrading enzyme gene(CaDAO),the key enzyme genes for cytokinin synthesis(CaIPT and CaCYP735A),the regulators that maintain viabilities of the meristem cells(CaWUS),and the positive regulators of the branch(CaWRKY71)were highly expressed at the TD2 stage,while the branch negative regulator(CaBRC1)was lowly expressed.At the TG2 stage,the auxin synthesis key enzyme gene(CaYUCCA)and the cytokinin degradation metabolic key enzyme gene(CaCKX)were highly expressed,while the CaDAO,CaIPT,CaCYP735 A and CaWRKY71 genes were low expressed.For these two stages,there were also significant differences in the expression patterns of auxin and cytokinin signaling molecules.These results indicate that auxin inhibited expression of key enzyme genes for cytokinin synthesis through its signal transduction pathway,thereby controlling cytokinin level to indirectly negative regulating lateral bud growth.4.The mechanism of auxin regulating the shoot branching of C appendiculata was studied by decapitation and auxin transport inhibitors to treat pseudobulbs strings.The HPLC detection found that the level of IAA in the lateral buds decreased rapidly after decapitation,NPA and TIBA treatment,while the level of CTK increased rapidly.The qRT-PCR detection found that the positive regulation factor CaWRKY71 was up-regulated during the lateral bud transformation,but not during the germination.The rapid expression of auxin oxidative degradation metabolic enzyme gene CaDAO might be induced by CaWRKY71 gene,which in turn reduced IAA levels.Signals with decreased auxin levels induced expression of key enzyme genes CaIPT and CaCYP735 A in cytokinin synthesis,and inhibited the expression of key enzyme genes CaCCD7 and CaCCD8 in strigolactone synthesis.Signals with increased cytokinin levels promoted CaWUS expression while inhibiting the expression of the branched negative regulator CaBRC1.At the germination stage,the bud growth signal induced the expression of CaYUCCA,a key enzyme gene for auxin synthesis.The signal of increased auxin levels promoted the expression of CaCCD7,CaCCD8 and cytokinin catabolic enzyme gene CaCKX.During this stage,although the level of cytokinin was decreased,it was still significantly higher than the control(P<0.05).The CaBRC1 regulated by cytokinin was still inhibited.Therefore,auxin indirectly controls the growth of the lateral buds via regulating the levels of cytokinin and strigolactones in this plant. |
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