| Plant leaf is a main photosynthetic organ responsible for converting carbon dioxide and water into carbohydrates.It provides carbon for plant growth and atmospheric oxygen for aerobic life.However,different plants have different phyllotaxic characteristics,and the leaf order in the same plant also has different patterns at different development stages.As one of the important model plants,A.majusis is the main material for the study of international molecular biology and flower breeding,and is used to study the genetics of flower and leaf development.Interestingly,phyllotaxic shifts of A.majusis occurred during their growth and development.The research of phyllotaxis characteristics will not only lay a theory foundation of the botanical phyllotaxic development,but also provide an important reference for related gene function analysis.However,there is no effective method to identify these critical time points of leaf order transformation until now.In this study,the materials of phyllotaxy sites in different development periods were selected:1.By using the method of plant anatomy,different phyllotaxis patterns were detected by anatomic observation.We found that opposite leaf primordium was formed in the 1-6 growth cones in A.majusis,and then the double decussate phyllotaxis were produced.In the 7th section,the growth cone of 24.4% produced the opposite leaf primordia,which formed the alternate phyllotaxis,and 75.6% of the growth cones produced decussate leaf primordia,forming the double decussate phyllotaxis.In section 8,the growth cone of 16.0% produced opposite leaf primordium,forming an he double decussate phyllotaxis,and 84.0% growth cone single side produced alternate leaf primordium,forming alternate phyllotaxis.Section9 is up to sections 13 and 17,and all the growth cones produced alternate leaf regions on one side,forming alternate phyllotaxis.The results showed that the main change site was from the 7th to 8th node,where the node of the phyllotaxis order changed from the opposite to the alternate.2.According to the results of the morphological anatomy of the leaf sequence above,the leaf sequence tissues of four sites were selected.the Solexa/Illumina RNA-seq high-throughput sequencing was used to evaluate the global changes of transcriptome levels among different phyllotaxic patterns.More than 86,315,782 high quality reads from four node regions of phyllotaxic development were sequenced and assembled into 58,509 unigenes.Based on the heat map analysis,a large number ofdifferently expressed genes(DEGs)were related to phyllotaxic patterning formation,especially shown in hormone perception and signaling,carbohydrate biosynthesis and metabolism,and the related transcriptions factors(TFs).These DEGs were distributed into 119 pathways described in the KEGG database.The qRT-PCR results indicated that the selected unigenes involved into hormone signaling pathway and carbohydrate metabolizing were highly up-regulated at the connected point between the alternate phyllotaxy and the double decussate phyllotaxy.Moreover,transcription factor families with the high expression were also identified.A large number of DEGs did not align with any databases and might be novel genes in phyllotactic development.These genes will serve as an invaluable genetic resource for elucidating the molecular mechanism of phyllotactic development.3.Our transcriptome data showed that a large number of transcription factors were involved in regulating the development of A.majusis phyllotaxis.Homologous comparison found MYB transcription factor family,AmDIV in different leaf arrangements has a high expression level in the process of transformation,in order to further analyze its role in the phyllotaxis formation of A.majusis.Firstly,the homology comparison and evolutionary tree were carried out.The expression levels in different nodes and subcellular localization were analyzed.The genes are mainly expressed in the root,stem and leaf tissues of A.majusis,especially in SAM.A fusion protein expression vector was constructed to analyze the subcellular localization of AmDIV protein,and it was found that the protein was located in the nucleus.At the same time,phenotypic characteristics of AmDIV transgenic Arabidopsis were detected in comparison with wild-type plants.We observed that the transgenic plants showed shorten main root length,increased lateral root length and quantity,slow leaf development,increased leaf size,delayed flower bud differentiation and inflorescence axis,and eventually increased plant height.Moreover,it also manifested the extended vegetative period and delayed reproductive growth traits.In particular,some of the leaves in transgenic plants were distorted,and the flocculation structure was found on the leaves.IAA induction showed that the increase of the root hair of transgenic plants indicated that AmDIV might participate in the regulation of the auxin signal distribution during phyllotaxic development.In summary,this research preliminarily determines the key site points of phyllotaxic morphology changes through the anatomy of tissues and organs.The transcriptome analysis was used to explore the difference expression genes betweendifferent phyllotaxis.Based on the different expression,a key functional gene AmDIV was selected and expressed in Arabidopsis thaliana.Effects of AmDIV on phyllotaxic morphology and plant growth were also analyzed.Moreover,IAA was used to treat the transgenic plants and the morphological changes were observed.Our results indicated that AmDIV plays an important role in the phyllotaxy formation of A.majusis. |
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