The Function Of PhACL And PhmCS In Petunia

Flower color is an important characteristic of ornamental plants.Anthocyanins play an important role in the color formation of flower.The synthetic route from acetyl-CoA to anthocyanin has been well studied.However,the source of acetyl-CoA for anthocyanin synthesis is still unclear.Acetyl CoA in cytoplasm is mainly generated by citrate catalyzed by ATP-citrate lyase(ACL).Citrate could be synthesized by citrate synthase(CS)catalyzing oxaloacetic acid(OAA)and acetyl-CoA.Studies on ACL andCS mostly focus on plant growth and development,but whether ACL andCS participate in anthocyanin synthesis is still unknown.Petunia hybrida is an important model system.In this study,our results showed that PhACL and PhCS participated in anthocyanin synthesis.These results are as follows:(1)Four genes encoding two subunits of PhACL,PhACLA1,PhACLA2,PhACLB1 and PhACLB2 were cloned in petunia.The interactions between subunits were identified by yeast two-hybrid assays: both PhACLA1 and PhACLA2 interacted with PhACLB1 and PhACLB2,PhACLB1 interacted with PhACLB2,and PhACLB2 interacted with PhACLB2.The protein complex modle of PhACL was speculated.(2)Silencing of PhACLA1,PhACLA2,PhACLB1 or PhACLB2 alone did not result in visible phenotype changes compared with control,but PhACLA1-A2 or PhACLB1-B2-silenced plants exhibited similar phenotype changes: dwarfing plant,shortening branches and stems,small and dark leaves,and light color and small petals.(3)Between PhACLA1-A2-and PhACLB1-B2-silenced plants had similar phenotype,and our following observation and experiment were focused on PhACLB1-B2-silenced plants.PhACLB1-B2-silenced plants exhibited abnormal reproductive growth: malformed anthers,less pollens,abnormal seeds and pale color of seed coat.Silencing of PhACLB1-B2 decreased the activity of PhACL and increased the content of citrate.PhACLB1-B2-silenced plants displayed features of senescence,including shorter vase-life,more ethylene evolution of corollas,and increasing several senescence-related genes expressions.PhACLB1-B2-silenced plants might be in a state of oxidative stress: the increasing of reactive oxygen species(ROS)activity and the proline content.(4)Three PhCSs,PhmCS,PhCSY1 and PhCSY2,were all cloned in petunia.The subcellular localization technique was used to determine the location of three PhCSs in petunia leaf protoplasts.PhmCS was located in mitochondria,while PhCSY1 and PhCSY2 were located in peroxisome.(5)The plants of PhCSY1,PhCSY2,and PhCSY1-Y2 silencing had no visible phenotype compared with control,while PhmCS-silenced plants exhibited abnormal phenotype: yellow young leaves(then recovering during growth),larger aspect ratio of leaves,malformed petals,pale and blue flower,decreasing citrate content of corolla and leaves,increasing p H of corolla and leaves,and increasing content of amino acid.(6)In both PhACLB1-B2-and PhmCS-silenced plants,the contents of acetyl-CoA and anthocyanin in petunia corolla were decreased significantly,metabolites in corolla were altered greatly,and the content of malonyl CoA in petunia corolla also was decreased in PhmCS-silenced plants.