Functional Characterization Of PhCESA3 In Petunia

Petunia(Petunia hybrida)is a perennial ornamental flower,often for a biennial cultivation.Petunia,with rich color and long flowering period,is an excellent material for flower beds and flower borders.Petunia plays an important role in landscaping.Moreover,petunia is often as the model for studying the ornamental plant growth and development.The plant type,plant height,flower type,florescence and fertility are important characteristics of the ornamental plant.Over the years,the breeders of ornamental plants tried to change the plant type,plant height,flower type,florescence,fertility and other characteristics of the ornamental plant to enrich the ornamental plant species.The breeding methods including introduction and domestication,cross breeding,mutation breeding,haploid breeding,polyploid breeding,cell engineering breeding,gene engineering breeding and so on.Cellulose is one of the main components of the cell wall,and it is the largest biomass energy source on earth.Cellulose biosynthesis is very complex,involving coordinated manipulation of multiple genes.Cellulose is mainly synthesized by cellulose synthase.Cellulose synthase complex is a large multisubunit complex and contains a number of cellulose synthase catalytic subunits.Cellulose synthase catalytic subunits(CESAs)play important roles in plant growth,development and disease resistance.Loss-of-function mutation or antisense transgenic plant in CESA3 resulted in the alteration of plant growth in Arabidopsis(Arabidopsis thaliana),including plant height,leaf and petal size and fertility,which are the important ornamental characteristics for ornamental plants,thus indicated an essential role of CESA3 in plant growth.However,little is known about the role of CESA3 in species other than Arabidopsis.In this work,we studied the function of PhCESA3 in petunia,a homolog of the Arabidopsis CESA3 gene.We analyzed the protein sequence and structure of PhCESA3 by using bioinformatics softwares.And we identified the function of PhCESA3 in petunia with the technology of VIGS.The results are as follows:(1)PhCESA3 was predicted to encode proteins of 1083 amino acids,PhCESA3 shares 86.5% and 94.8% amino acid sequence identity with AtCESA3 and SlCESA3,respectively.(2)PhCESA3 transcripts were present in all plant organs,including root,stem,leaf,and corolla,with varying patterns of expression.Moreover,in the different development stages of flower buds,the expression of PhCESA3 was different.From budding to flowering,the expression of PhCESA3 showed decreased first and then increased and then declined.(3)We successfully cloned 274 bp fragment of PhCESA3 3’UTR and constructed VIGS vector pTRV2-PhCESA3.And we infected petunia plants by using VIGS technique.(4)PhCESA3 silencing resulted in decrease of leaf cellulose content.The cellulose content of the leaves in PhCESA3 silenced plants decreased 31.6% compared to the control.(5)VIGS-mediated PhCESA3 silencing resulted in dwarf plants,short stems,pedicels,filaments,styles,petal tubes and epidermal hairs compared with those in control.The stems,pedicels,filaments,styles and epidermal hairs in PhCESA3 silenced plants were swollen compared with those in control.PhCESA3 silencing also resulted in smaller and thicker leaves and petals than those in control.Furthermore,PhCESA3 silencing led to abnormal stigmas,ovaries,fruits and seeds.(6)PhCESA3 silencing caused short and swollen cells.Compared with those in control,PhCESA3 silencing reduced cell length of the epidermal cells of stems,pedicels,filaments,styles,but increased the cell width.PhCESA3 silencing also resulted in thick stem and pedicel cortex,but the number of the cell layers in cortex didn’t increase,compared with those in control.The chloroplasts in the pedical cortex cells had smaller and less starch grains than those in control,some even did not have starch grains after the plants infected with pTRV2-PhCESA3.The adaxial and abaxial epidermal cells of leaves and petal limbs showed the reduced cell size in PhCESA3 silenced plants,but the internal cells of leaves and petals were swollen,compared with those in control.Besides,the epidermal cell walls of stems,petals and filaments in the PhCESA3 silenced plants were thinner than those in the wild type.Moreover,PhCESA3 silencing resulted in asymmetric structure of styles while those in control were symmetric.(7)PhCESA3 silencing led to abnormal pollen grains.The fertility was reduced in PhCESA3 silenced plants,with seed-setting rate decreased,compared with the wild type.