Gene Identification And Functional Analysis Of Hexokinases Involving In The Latex Regeneration Of Hevea Brasiliensis

In higher plants,hexose kinases can phosphorylate hexoses,such as glucose,fructose to initiate glycolysis metabolism.Hexokinases(HXKs)is a crucial enzyme that phosphorylates glucose,and fructokinases(FRKs)are mainly functioning in the phosphorylation of fructose.In the rubber tree laticifers,the phosphorylation of glucose initiates glycolysis and pentose phosphate pathways,which provide energy and other compounds required for latex regeneration.However,the mechanism of how HXKs participate in the latex regeneration in laticifers is currently unknown.In this study,two large hexose kinases family,i.e.hexokinase and fructokinase were studied and further investigate the mechanisms of these functional enzymes involving in rubber biosynthesis.The major results as follows:(1)The cDNAs of four HXK(HbHXK1-4)and five FRK(HbFRK1-5)were isolated from the rubber tree(Hevea brasiliensis)and sequenced.Nucleotide-derived amino acid sequences revealed the presence of conserved amino acids that are required in the catalyzation of glucose and fructose.Among these HXK genes,HbHXK2 and 4 were found to be the predominant isoforms in latex(cytoplasm of laticifers)when compared with others.Of the five FRK genes,HbFRK1,2 and 3were identified as predominant members.(2)Phyhlogenetic tree analysis revealed that HbHXK2 was classified into Type-A which amino acid targeted to plastid,and HbHXK1,3 and 4 were clustered to Type-B which amino acid targeted to mitrochondria.And,HbFRK1,HbFRK2,HbFRK3 were classified into clade II,clade V,clade I,respectively.Furthermore,subcellular localization analysis revealed that HbHXK2 was presumably targeted to the chloroplast and HbHXK4 to the mitochondria.Both HbFRK2 and HbFRK3 were highly possible to be expressed in cytosol.(3)In enzymatic function,both HbHXK2 and 4 were confirmed to have catalytic activities towards glucose and fructose with a significantly higher affinity for glucose.HbFRK2 also showed the highest affinity for fructose compared with HbHXK2 and HbHXK4.(4)In two yield-promoting experiments,both tapping and ethphon can induce the increase of rubber tree yield,and accompanied with the changes of sucrose and glucose.HbHXK4 was strongly induced and lined with HXK catalytic activity and latex yield by tapping and ethephon treatments,whereas,HbHXK2 had the opposite expression patterns.Likewise,using either method to induce an increase in latex yield,the abundance of HbFRK2 and HbFRK3 in transcripts,was increased especially HbFRK2.