Physiological And Molecular Basis Of Plant Shape And Flower Color Formation In Musella Lasiocarpa

Musella lasiocarpa,belonging to the genus Musella in Musaceae,is a large tufted herb of a single genus endemic to China.It has high ornamental value and economic value because of its tall and straight plant shape,beautiful inflorescence and long flowering period of more than half a year However,research on the physiological and molecular mechanism of the plant architecture and colors of flowers of M.lasiocarpa is not yet developed both at home and abroad.This research focused on the M.lasiocarpa clone YN01 featuring long stalk,yellow bract and strong suckering and germination ability and the M.lasiocarpa clone RD05 featuring dwarf plant,red bract and very weak suckering and germination ability.Hormonal levels in different parts of these two clones and the metabolome of pigment in their bract were measured,which,combined with transcriptome analysis,serves for the study of the physiological and molecular basis for the plant architecture and colors of flowers of the two.In the meantime,key genes related to the above ornamental characters were screened out for cloning and expression analysis,for the purpose of providing theoretical and data support for the formulation of M.lasiocarpa breeding strategy that accumulates its good traits and the improvement of industrialized development technology.This paper is also expected to serve as additional reference for research on the physiological and molecular mechanism related to plant ornamental characters.Main conclusions in this paper are as follows:（1）It was determined that the main color-presenting material in the red part of M.lasiocarpa bract consists of seven anthocyanins,of which five was cyanidins,one was peonidin and another one was delphinidin;for the yellow part,the main color-presenting material consisted of flavonoids and flavonols,with carotenoids in an assistant role.Through metabolomics analysis,237 flavonoids were detected in the bract of the yellow-flowered M.lasiocarpa YN01 and the red-flowered M.lasiocarpa RD05.Among them,there were 98 differential flavonoids between the two clones,of which 79 showed significantly up-regulated content in RD05.In the bract of these two clones,a total of 58 carotenoid compounds were detected;the total carotenoid content in YN01 bract was higher than that in RD05 bract.（2）As for the hormone level,it was determined that the main cause of M.lasiocarpa RD05dwarfing was GA content much too low and ABA content excessively high.Using high performance liquid chromatography mass spectrometry（HPLC-MS/MS）,the endogenous hormone content in the bract,leaf,rachis,sucker point and root tip of YN01 and RD05 were determined.In the rachis of the dwarf M.lasiocarpa RD05,the content of GA₃ was significantly lower than that in YN01,but the content of ABA was significantly higher than that in YN01.BL content in the rachis of the dwarf RD05 was 18.67 times that of YN01.It was speculated that the BL signal transduction pathway in RD05 was blocked,for which BL of high content could not function normally to promote growth.SL inhibited RD05’s tillering ability,which was the main cause of little or no sucker in RD05.The contents of IAA,ZT and SL in the root tip in RD05 were significantly higher than that in YN01.ZT promoted tillering while IAA and SL inhibited it.Referring to the nearly no sucker phenotype of RD05,it was concluded that ZT might be unable to promote tillering for its blocked signal transduction pathway or other unknown reasons;meanwhile,high contents of IAA and SL strongly inhibited the tillering capacity of RD05,where IAA played an indirect role,and SL,as the second messenger of IAA,inhibited tillering directly.（3）A transcriptome sequencing was first given to M.lasiocarpa,which,combined with results of the flavonoid metabolome profiling,leaded to the primary differential genes related to flavonoid（anthocyanin）synthesis:C4H and 4CL.After transcriptome sequencing and analysis of M.lasiocarpa,all Unigenes obtained were aligned with six major public protein databases respectively,namely Pfam,NR,Swiss Prot,COG,GO and KEGG.A total of 66,471 Unigenes were annotated to at least one database,accounting for 47.69%of the total.By Qvalue≤0.05,six significant difference enrichment pathways were screened out in total,in which two were related to pigment synthesis,namely the flavonoid synthesis pathway and the carotenoid synthesis pathway.Structural genes and transcription factors related to pigment synthesis were also screened and analyzed.According to results of the flavonoid metabolome profiling,the speculative diagram of flavonoid synthesis pathway and the network diagram of genetic-metabolic interaction were drawn.The results showed that in the flavonoid synthesis pathway of M.lasiocarpa,genes such as C4H,4CL,F3H and F3’5’H played a positive role in promoting flavonoid synthesis,especially C4H and 4CL genes,which were key genes for the synthesis of flavonoids and anthocyanins in the bract of red-flowered M.lasiocarpa.（4）Two key genes related to plant shape,i.e.Ml CYP734A6（Genbank registration No.MW013148）and Ml CCD8b（Genbank registration No.MW013147）were successfully cloned.Based on results of the transcriptomics differential gene screening of M.lasiocarpa,the inactive gene Ml CYP734A6 in BL metabolic pathway and the key gene Ml CCD8b in SL synthesis pathway were cloned.Using q RT-PCR,the pattern of expression of the two genes in different parts were analyzed.Ml CYP734A6 expression were found in all tissues of M.lasiocarpa,with the lowest expression in the leaf and the highest in the rachis and root tip.Ml CCD8b showed consistent tissue-specific expression in these two clones,i.e.the highest expression in the rachis,followed by the sucker point,root tip and leaf,and the lowest or no expression in the bract;there was no significant correlation between the expression level of MLCYP734A6 and BL content in different tissues of the two clones.The content of SL in root tip of two clones was consistent with the expression level of MLCCD8b gene.