Genome-Wide Identification Of PIN And PILS Gene Families In Areca Catechu And Functional Study Of AcPIN6 In Brace Root Architecture

Areca catechu L.is a tall perennial evergreen tree of the genus Palmetto in the palm family.It is a typical tropical plant and a traditional southern medicine in China.The root system of Areca catechu is an advental root system without seed roots.The root system consists of crown roots and brace roots.Plants adapt to the environment by optimizing the size and structure of their vegetative organs,including roots,shoots,and leaves.Indole-3-acetic acid(IAA),the most prevalent form of the plant hormone auxin,plays a crucial regulatory role in nearly all areas of plant growth and development.The accurate distribution of IAA in roots is critical for the construction of root systems in plants.The PIN and PILS families of auxin transporters control intracellular and intercellular transport of IAA,initiating or inhibiting the construction of plant organs.Areca catechu is a widely distributed tropical monocotyledonous plant,and its auxin related phenotype is quite different from that of model plants.Therefore,it is of great significance to analyze the relationship between PIN and PILS families and root configuration of Areca catechu.The evolution of PIN and PILS families and the potential role of AcPIN6 in brace roots of Areca catechu were studied through bioinformatics and molecular biology.The results are as follows:1.In this study,a total of 12 PIN genes and 8 PILS genes were identified from the whole genome of Areca catechu through bioinformatics.The genes from the PIN family were dispersed unevenly over seven chromosomes,and the genes from the PILS family8 were dispersed unevenly across six chromosomes.The number of PIN and PILS genes distributed on chromosome 13 was the largest,with 5 genes.The number of copies of PIN1,PIN3,PIN5,PIN9,PILS2,PILS6 and PILS7 genes in Palmiaceae were higher than those in Arabidopsis thaliana.The PIN3 and PIN10 subfamilies were exclusive to dicots and monocots,respectively.In this study,it was found that PIN3 was retained and PIN10 was lost in all four palm trees.it was found that PIN3 was retained and PIN10 was lost in all four palm trees.We also found that the Ka/Ks ratio of all AcPIN and AcPILS homologous gene pairs was less than 1,indicating that these genes underwent purifying selection.2.The expression patterns of PIN and PILS family genes in different tissues and organs of Areca catechu(crown root,brace root,leaf,vein,female flower and male flower)were analyzed by RNA-seq and q RT-PCR.The PILS family genes(except AcPILS6a)were expressed in all six organs.In the PIN family,AcPIN5 a and AcPIN5 b are expressed only in leaves,and AcPIN8 is detected only in male flowers.We found that the expression level of AcPIN6 in the brace root of Areca catechu was the highest among all the genes of PIN and PILS family in different organs,and AcPIN6 was not expressed in the crown root of Areca catechu,indicating that AcPIN6 is a key gene affecting the growth of brace root of Areca catechu in PIN and PILS family.3.The expression of AcPIN6 in various growth stages and tissue regions of Areca catechu brace roots was examined using q RT-PCR,and it was found that the expression level of AcPIN6 was mainly high in the middle of the middle growth stage.At the later stage of growth,AcPIN6 was mainly expressed in brace taproots,but was less expressed in primary and secondary lateral roots.In addition,the expression of AcPIN6 was higher in pericycle in brace roots than in cortex,and the expression level was lower in vascular tissues.Divide pericycle recovery ability form lateral root primordium,lateral root primordium AcPIN6 amount of expression is significantly higher than surrounding tissue area,and lateral root primordium of only 65 ng/g FW,IAA content is lower than the surrounding tissue.This suggests that AcPIN6 plays an important role in brace root growth and formation,especially during lateral root formation.Compared with crown root,the primary structure of bracee root tip of Areca catechu lacks elongation zone.4.brace roots have been developed in many plants of the palm family.Through phylogenetic analysis of PIN6 in four species of the palm family,it was found that Areca catechu and Cocos nucifera PIN6 are closely related.By protein sequence alignment,it was found that PIN6 of the four species of palm was highly conserved during the evolutionary process,with a 93.52% amino acid sequence agreement and a TPRXS domain in the hydrophilic loop.The expression level of PIN6 in brace roots of Cocos nucifera,Elaeis guineensis and Phoenix dactylifera was significantly higher than that in crown roots,suggesting that some species of palm play similar roles in brace root growth and development.5.In order to verify the function of AcPIN6 in Areca catechu,Arabidopsis thaliana with overexpression of AcPIN6-e GFP and AcPIN6 was constructed.The results showed that AcPIN6-e GFP protein was localized to the cell membrane.Overexpression of AcPIN6 resulted phenotypes in transgenic Arabidopsis thaliana,such as shorter taproot length,longer root hairs,increased plant height,longer silique length,and increased silique yield per plant.Therefore,AcPIN6 can be considered as a cell membrane localization protein associated with auxin transport.In conclusion,this study took Areca catechu as the main research material,analyzed the PIN and PILS families of Areca catechu through bioinformatics,and found the key role of AcPIN6 in brace roots by analyzing the expression levels of genes in different tissue families of Areca catechu.The expression of AcPIN6 in different tissues of brace roots was further analyzed,and the content of IAA was determined to confirm the key role of AcPIN6 in the root primordium of brace roots.Finally,the relevant function was verified by constructing transgenic Arabidopsis thaliana,and it was found that AcPIN6 was localized to the cell membrane,and the transgenic Arabidopsis thaliana showed an auxin transport-related phenotype.The results of this study will provide a theoretical basis for better understanding of the evolution of PIN and PILS in monocotyledonous plants and for improving the understanding of the root system architecture of the large palm family.