| Members of the genus Anoectochilus Bl.are favored for their velvety leaves with bright reticulated gold variegations,as well as their excellent medicinal efficacy.Leaf variegation plays an important role in the survival and reproduction of plants.However,the anatomical basis and molecular mechanism of leaf variegations and ground colours of this genus are still unknown.In this study,wild A.roxburghii is divided into four types,namely,the common type(C),the green leaf type(G),the yellow-streaked type(Y)and the few-veined type(F).The macroscopic morphology under soil cultivation and tissue culture is observed,and it is found that their leaf variegation and ground colour characteristics remain stable under the culture conditions in this study.On this basis,leaf morpho-anatomy and transcriptome sequencing are conducted to investigate the structural basis and molecular mechanism of the leaf variegations and ground colours of four types of A.roxburghii.Meanwhile,structural basis of leaf variegations of the other eight species of the genus Anoectochilus(A.baotingensis?A.burmannicus?A.calcareus ? A.chapaensis ? A.elatus ? A.hainanensis ? A.longilobus and A.malipoensis)are analyzed.The main results of this study are as follows:1.Through vacuum water infiltration experiment and freehand section observation,it is found that the leaf variegations of four types of A.roxburghii belong to the air space type and the pigment type.The eight species of the genus Anoectochilus have similar characteristics on the transverse leaf sections across midvein,and their leaf variegation types are the same as A.roxburghii.In the variegated leaf areas of the Anoectochilus species,palisade cells just above the vascular bundles are globular other than funnelshaped,resulting in obvious intercellular air spaces which determining the brightness of variegations along the veins.Meanwhile,the globular cells have no chloroplast but contain non-photosynthetic pigments which determining the colours of variegations along the veins.In addition,the colouration of the central variegated area on the leaf of yellow-streaked type is also related to the deficiency of red pigments and chloroplast abnormality in the spongy tissue cells.2.By freehand section observation,it is found that the leaf ground colours of Anoectochilus species are mainly related to the concentration and distribution of red pigments in spongy tissue cells and the number of palisade tissue layers.The lack of red pigments results in green leaves of A.chapaensis,A.longilobus,A.malipoensis and A.roxburghii,and the increase of palisade tissue layers can also lead to green leaves of A.longilobus.3.Tissue culture method is used to obtain sterile seedlings of four types of A.roxburghii for transcriptomic sequencing.A total of 76.61 Gb clean data are obtained by transcriptomic sequencing,and then 57,394 unigenes are obtained by assembly and redundancy removal.GO and KEGG analysis of differentially expressed genes(DEGs)in the green leaf type,yellow-streaked type and few-veined type are carried out with the common type as control group(C?vs?G,C?vs?Y,C?vs?F).The results show that the DEGs in the three comparison groups may play functions in pigment metabolism.On this basis,and combined with the leaf cross-section characteristics,MapMan4 is used for further analyze.4.The DEGs of the green leaf type compared to the common type(C?vs?G),and the common DEGs of the green leaf type compared to the common type,yellowstreaked type and few-veined type(C?vs?G,Y?vs?G and F?vs?G)are analyzed by MapMan4.It is found that the causes of the emergence of the green leaf type of A.roxburghii related to abnormal metabolic pathway gene expression of pigment,which mainly manifested by the down-regulated expression of flavonoids synthesis related gene CHI and carotenoids synthetic related gene PSY,chlorophyll synthesis related genes GSA and CHLG,and up-regulated expression of chlorophyll degradation related genes HCAR and SGR.Among them,the down-regulated expression of CHI may be the most direct cause of the formation of the green leaf type.5.The DEGs of the yellow-streaked type compared to the common type(C?vs?Y)are analyzed using MapMan4.It is found that the formation of the central yellow area on leaf is mainly related to pigment metabolism and chloroplast damage,which mainly manifested by the down-regulated expression of flavonoids synthesis related genes PAL,KAT,CHS and AS,the up-regulated expression of carotenoids biosynthesis related genes PDS and Crt ISO,and the expression of genes related to chloroplast protein degradation(Deg,Spp A,Fts H)mainly up-regulated.6.The DEGs of the few-veined type compared to the common type(C?vs?F),and the common DEGs of the few-veined type compared to the common type and yellowstreaked type(C?vs?F and Y?vs?F)(F,C and Y groups all have non-green leaves)are analyzed using the MapMan4.It is found that the reason why the few-veined type of A.roxburghii produce only a few variegations along the veins may be related to photoprotection,which mainly manifested by the up-regulated expression of genes related to photosystem subunits and their assembly factors,photosynthetic acclimation and photosystem damage repair,non-photochemical quenching,cyclic electron flow and plastid movement.In this study,clear freehand slice images of the four types of A.roxburghii and the other eight members of the genus Anoectochilus are added,thus the leaf variegation type is determined.Transcriptomic sequencing is performed on four types of A.roxburghii with different leaf variegations and ground colours.The genes related to the leaf ground colours of the green leaf type,the central yellow area of yellow-streak type,as well as the leaf variegation patterns of few-veined type of A.roxburghii are screened out,which provid a basis for genetic breeding and subsequent in-depth study on the evolution of the leaf variegation characters of Anoectochilus species. |
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