Mechanism Of AtABCG14 Transport Of Isopentene Adenine Type Cytokinin In Arabidopsis Thaliana

Cytokinin(CK)is an irreplaceable plant hormone during plant growth and development.It plays an important role in plant resistance to biological and abiotic stresses.CK synthesis and response sites often occur in different tissues,so it is very important to study the transport mechanism of CK from source to target cell.ABCG14,located in the plasma membrane,is a long-distance transporter and is responsible for the transport of trans-Zeatin(t Z)synthesized from roots to shoot.It has been suggested that iP-type CKs(N⁶-(?²-isopentenyl)adenine)are transported from shoot to root through phloem,but the mechanism of iP-type CKs transport is still unclear.In this study,abcg14 cypDM triple mutant was obtained by genetic method,and the transport mechanism of ABCG14 on iP-type CKs was studied by physiological and biochemical experiments.(1)ABCG14 is induced by the iP-type CKs and has the activity of transporting the iP-type CKs.The expression level of ABCG14 in cypDM(cyp735a1,cyp735a2)was detected by qRT-PCR,and the results showed that the expression level of ABCG14 was significantly higher than that of wild type(WT).The expression of ABCG14 was also significantly increased when iP was exogenously applied to cypDM,suggesting that iP-type CKs induced the expression of ABCG14.Western Blotting showed that ABCG14 could form a homodimer to function.The analysis of yeast transport activity showed that ABCG14 had efflox transport activity on tZ,cZ,DHZ,iPR.(2)ABCG14 is involved in the transport of iP-type CKs from root to shoot and is responsible for the distribution of iP-type CKs in shoot.Triple mutant abcg14 cypDM was obtained by crossing cypDM with abcg14 mutant.The mutant showed short roots,small rosettes,few inflorescences,nanous plants and delayed development.Hormone analysis of endogenous hormones,xylem wound fluid and isotope absorption experiments showed that iP-type CKs were accumulated in root and decreased in shoot.The results indicated that ABCG14 was responsible for the transport of iP-type CKs to the shoot.After scion of abcg14 cypDM was grafted on the root stock of cypDM,the root could transport iP-type CKs to abcg14 cypDM,but abcg14 cypDM did not recover to cypDM phenotype,indicating that ABCG14 was involved in the unloading and distribution of iP-type CKs in shoot.The indicative ²H₆-iP was incubated on the 3^rd and 4^th rosette of abcg14 cypDM.Compared with cypDM,the content of ²H₆-iP in the young rosettes was significantly reduced,which further suggested that ABCG14 was involved in the distribution of iP-type CKs in shoot.(3)ABCG14 is involved in the transport of iP-type CKs from shoot to root.After²H₆-iP was incubated on the rosettes of abcg14 cypDM,it was found that the content of ²H₆-iP in the root was significantly reduced compared with that of cypDM.Hormone analysis of the phloem wound flow in the rosettes of abcg14 cypDM showed that iP-type CKs were accumulated in the phloem.These results indicate that ABCG14 is involved in the root unloading of iP-type CKs during the long-distance transport of iP-type CKs from shoot to root.In conclusion,ABCG14 is involved in the transport of iP-type CKs in Arabidopsis.iP-type CKs flow from the shoot to the root via phloem,and ABCG14unloads iP-type CKs from the phloem in root to the intercellular substance for normal growth and development of root tissues.At the same time,ABCG14 excrete iP-type CKs produced by the root into the intercellular.iP-type CKs are loaded into xylem and transported to the apex with transpiration,and unloaded to the apoplast by ABCG14.The CK on shoot cause CK signal response on the plasma membrane,or enter the cell through various transporters for metabolism.These results indicate that ABCG14 plays an important role in the cyclic transport of iP-type CKs in Arabidopsis thaliana,which is of great significance to elucidate the mechanism of CK transport.