A Functional Study Of WOX And PLT Genes In Regulating The Development Of The Meristem

Seed plant genomes have evolved over a long period of time to form the complex molecular regulatory networks and functions they now have,and the bryophyte Marchantia polymorpha L,one of the earliest land plant lineages,has a genome that retains more ancestral characteristics.One of the keys to plant adaptation to the terrestrial environment after landing was the evolution of multiple dividing surfaces in meristems with stem cells,which led to the development of three-dimensional structures of the plant body.A reservoir of stem cells niches exists in each apical meristem zone,and these stem cells are maintained a relatively constant size,providing a continuous source of cells for plants growth and development.Our current understanding of the molecular mechanisms that maintain stem cell properties comes almost exclusively from studies of seed plants,such as the Arabidopsis shoot apical meristem maintenance factor WUS and the root apical meristem maintenance factor WOX5/PLT.In contrast,it is not clear how plant stem cells have evolved,and whether the regulatory mechanisms of their fate maintenance are conserved.During the gametophyte generation,Marchantia polymorpha undergo dichotomous branching,an ancestral characteristic of growth derived from the meristem,in which stem cells divide and develop to form two equivalent body axes,regulated by unknown molecular mechanisms.In this study,we used molecular biology,genetics and bioinformatics methods to elucidate the molecular mechanism of stem cell maintenance in Marchantia polymorpha.Phylogenetic analysis revealed that the genome of Marchantia polymorpha has one WOX homolog,Mp WOX,and two PLT homologs,Mp AP2L1 and Mp AP2L2.It was shown that Mp WOX is not involved in the maintenance of stem cell characteristics of meristem,and its mutant meristem is not different from the wild type,except that the thallus area is smaller than that of the wild type,the growth rate is slower.The phenotype was more pronounced when treated with mannitol.We also investigated the function of Mp AP2L1,which possesses four classical eu ANT motifs and is closer to the PLT protein of seed plants,compared with Mp AP2L2.The phenotype of the knockout mutant also demonstrated this,so we renamed Mp AP2L1 as Mp PLTa.Compared to the wild type,Mpplta exhibited a severe developmental defect,with the apical notch failing to maintain stem cell properties,forming expanded tissues here and were unable to undergo normal dichotomous branching.Furthermore,Mpplta could not grow normal gemma cups,there is gemmae formation in the expanded tissue,so the loss of Mp PLTa function does not affect asexual reproduction.GUS staining showed that Mp PLTa was specifically expressed in the apical notch and to a lesser extent in the midrib.By analyzing RNA-seq combined with Ch IP-seq,we obtained a series of downstream target genes of Mp PLTa,including a GRAS protein family gene,Mp GRAS9,which has an evolutionary homology relationship with Arabidopsis SHR.It has been shown in Arabidopsis that At SHR is regulated by At PLT2.In summary,Mp WOX does not affect the development of meristem,but may be involved in cell division of thalli.Mp PLTa is a key determinant of stem cell fate in meristems,suggesting that the role of PLT proteins could be conserved in nonvascular and vascular plants.In addition,there may be some conservation of the regulatory network of Mp PLTa.