The Mechanism Of GhBBXs And GhGSTFs Regulating Anthocyanin Synthesis And Transport In Cotton

Anthocyanin accumulation can protect cotton plants against pathogen and pest attacks and increase the tolerance to abiotic stresses,including UV light,drought and saline-alkali.Besides,anthocyanin accumulation in cotton leaves and flowers are also important agronomic traits that have been used in modern agronomic and genetic research.Therefore,it has important practical significance to study the mechanism underlying the accumulation of anthocyanins in cotton plants in response to internal and external factors.The red-leaf phenotype of T586 is caused by the repeated insertion of promoter of GhPAP1D(proB).In this study,T586 was used as the main material,the mechanism of GhBBXs regulating the expression of GhPAP1 D and the involvement of GhGSTFs in the intracellular transport of anthocyanins were studied.The main research contents are as follows:1.GhBBXs regulates the synthesis of anthocyanins in red-leaf cotton T586.GUS staining assays showed that the transcription activity of GhPAP1 D was significantly enhanced by the insertion of repeated fragments in the promoter region.A G-box cis-element,which is responsive to light,was found in the repeated fragment.Yeast one-hybrid assays indicated that GhHY5,an important transcription factor for photomorphogenesis,could bind to the G-box cis-element.However,dual luciferase assay showed that GhHY5 could not significantly improve proB activity.Forthermore,the IV subgroup genes of BBX family,which are important for photomorphogenesis,regulated the expression of GhPAP1 D.When T586 seedlings were treated by UV-B light,GhPAP1 D,GhHY5 a and GhHY5 b were significantly up-regulated in cotyledon,and among IV subgroup of BBX family,GhBBX21 a and GhBBX21 b were slightly regulated while GhBBX25 a and GhBBX25 b were sharply down-regulated.Dual-luciferase assays demonstrated that GhBBX21 a and GhBBX21 b could significantly activate the expression of downstream genes of proB,either alone or together with GhHY5 b.GhBBX25 b inhibited the expression of downstream genes of proB,while the presence of GhHY5 b weakened this inhibition.BiFC assays indicated that GhBBx21 b and GhBBx25 b can physically bound with GhHY5 b.The above experiments showed that the IV subfamily genes of BBX family,together with GhHY5 b,regulated the expression of GhPAP1 D and promoted the synthesis of anthocyanins under UV-B light.2.GhGSTFs are involved in the intracellular transport of anthocyanins in T586.Two glutathione S-transferases,GhGSTF1 and GhGSTF2,were highly expressed in T586 leaves by transcriptome data and q RT-PCR assay.Phylogenetic analysis presented that they were clustered as anthocyanin transporters belonging to the Phi(F)subfamily.CDS sequences of their alleles were analyzed in TM-1,T586,3-79 and Xinhai-21.The sequence of GhGSTF1 is consistent in the four materials.GhGSTF2 is consistent in TM-1 and T586.GbGSTF2 a,the allele gene of GhGSTF2 in 3-79,had sequence deletion in the N-terminal domain,resulting in a shorter protein sequence.GbGSTF2 b,the allele gene of GhGSTF2 in Xinhai-21,had sequence deletion in the C-terminal domain,resulting in premature termination.The complementary experiments conducted in Arabidopsis mutant tt19-7 confirmed that GhGSTF1 and GhGSTF2 had the function of anthocyanin transport,while GbGSTF2 a and GbGSTF2 b lost this function.The GST enzyme activity assays were performed in vitro by using the recombinant proteins and the results showed that GhGSTF1 and GhGSTF2 had GST enzyme activity,while GbGSTF2 a and GbGSTF2 b did not.In vitro binding experiments showed that the binding ability of GhGSTF1,GhGSTF2,GbGSTF2 a and GbGSTF2 b to Cya was remarkablely higher than that of C3 G,and the binding ability of GhGSTF2 to C3 G was notably higher than that of another three recombinant proteins.Different binding abilities of recombinant proteins to Cya and C3 G affects the types and content of anthocyanins in cotton.In conclusion,this research was the first to investigate the cotton BBX family IV subfamily genes involving in anthocyanin accumulation under light.The mechanism of GhGSTF1 and GhGSTF2 involved in the intracellular transport of anthocyanins was also studied.This experiment provides a theoretical basis for exploring the synthesis and transport mechanism of anthocyanins in cotton plants,and lays a foundation for investigating the synthesis and accumulation of secondary metabolites in cotton plants in response to internal and external factors.