Arabidopsis ABCG14 forms a homodimeric transporter for multiple cytokinins and mediates long-distance transport of isopentenyladenine-type cytokinins

Abstract

Cytokinins (CKs), primarily trans-zeatin (tZ) and isopentenyladenine (iP) types, play critical roles in plant growth, development, and various stress responses. Long-distance transport of tZ-type CKs meidated by Arabidopsis ATP-binding cassette transporter subfamily G14 (AtABCG14) has been well studied; however, less is known about the biochemical properties of AtABCG14 and its transporter activity toward iP-type CKs. Here we reveal the biochemical properties of AtABCG14 and provide evidence that it is also required for long-distance transport of iP-type CKs. AtABCG14 formed homodimers in human (Homo sapiens) HEK293T, tobacco (Nicotiana tabacum), and Arabidopsis cells. Transporter activity assays of AtABCG14 in Arabidopsis, tobacco, and yeast (Saccharomyces cerevisiae) showed that AtABCG14 may directly transport multiple CKs, including iP- and tZ-type species. AtABCG14 expression was induced by iP in a tZ-type CK-deficient double mutant (cypDM) of CYP735A1 and CYP735A2. The atabcg14 cypDM triple mutant exhibited stronger CK-deficiency phenotypes than cypDM. Hormone profiling, reciprocal grafting, and ²H₆-iP isotope tracer experiments showed that root-to-shoot and shoot-to-root long-distance transport of iP-type CKs were suppressed in atabcg14 cypDM and atabcg14. These results suggest that AtABCG14 participates in three steps of the circular long-distance transport of iP-type CKs: xylem loading in the root for shootward transport, phloem unloading in the shoot for shoot distribution, and phloem unloading in the root for root distribution. We found that AtABCG14 displays transporter activity toward multiple CK species and revealed its versatile roles in circular long-distance transport of iP-type CKs. These findings provide new insights into the transport mechanisms of CKs and other plant hormones.

Keywords: circular transport; half-size ABC transporter; homodimer; isopentenyladenine-type cytokinins; long-distance transport.

Figure 1

AtABCG14 forms homodimers in vivo. (A) Reciprocal co-immunoprecipitation (coIP) assay of Myc-AtABCG14 and FLAG-AtABCG14 in human HEK293T cells. Total protein was extracted from human HEK293T cells transfected with Myc-AtABCG14 and FLAG-AtABCG14. (B and C) CoIP assay of GFP-AtABCG14 and Myc-AtABCG14 expression in tobacco leaves (B) and Arabidopsis(C). Total protein was extracted from tobacco leaves (B) and 25-DAG transgenic plants (C) co-expressing GFP-AtABCG14 and Myc-AtABCG14.

Figure 2

AtABCG14 displayed efflux transport activities toward multiple CK species in BY-2 cells and Arabidopsis cells. (A) Quantification of tZR, iP, iPR, cZ, and cZR in BY-2 cells transformed with the 35S::GFP (RCS2) and 35S::GFP-AtABCG14 vectors. BY-2 cells were collected 5 days after transfer to fresh culture medium. Data are means ± SD (n = 4). (B) Quantification of tZR, iP, iPR, cZ, and cZR in BY-2 cells transformed with 35S::GFP (RCS2) and 35S::GFP-AtABCG14. Cells were collected 5 days after subculture. Data are means ± SD (n = 4). (C) Quantification of iP, iPR, cZ, and cZR in Arabidopsis cells transformed with 35S::GFP-AtABCG14 and WT cells 5 days after subculture. Data are means ± SD (n = 3). (D–G) Quantification of the content of iP (D), iPR (E), cZ (F), and cZR (G) in 35S::GFP-AtABCG14 transformed Arabidopsis cells and WT cellscollected 5 days after subculture. Data are means ± SD (n = 4). ∗P < 0.05, ∗∗P < 0.001, Student’s t-test. tZR, trans-zeatin ribotide; iP, isopentenyladenine; iPR, iP ribotide; cZ, cis-zeatin; cZR, cZ ribotide; G14, AtABCG14; N.D., not detected; FW, fresh weight.

Figure 3

AtABCG14 displayed different efflux transport activities toward multiple CK species in YPH499 yeast cells. (A) Immunoblotting of the heterologous expression of GFP-AtABCG14 in YPH499 yeast cells harboring GFP-AtABCG14 using GFP antibody. YPH499 yeast cells transformed with an empty vector were used as a control. (B and C) Quantification of the isotope-labeled CKs ²H₅-tZ, ²H₅-tZR, ²H₆-iP, ²H₆-iPR, ¹⁵N₄-cZ, or ²H₃-DHZ in YPH499 yeast cells harboring GFP-AtABCG14 or an empty vector after feeding with the respective isotope-labeled CKs (B) or the respective isotope-labeled CKs and vanadate (C) for 20 min. Data are means ± SD (n = 4). (D) Exported ²H₅-tZ content from YPH499 yeast cells harboring GFP-AtABCG14 or an empty vector after feeding with ²H₅-tZ. Data are means ± SE (n = 4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Student’s t-test.

Figure 4

Morphological phenotype of the atabcg14 cypDM triple mutant line at different developmental stages. (A) Phenotypes of the WT, atabcg14, cypDM, and atabcg14 cypDM 8 days after germination (DAG). Scale bar, 2 cm. (B and C) Quantification of the root lengths (B) and chlorophyll contents (C) of the plants in (A). (D) Phenotypes of the WT, atabcg14, cypDM, and atabcg14 cypDM at 22 DAG. Scale bar, 2 cm. (E) Quantification of rosette diameters of the plants in (D). (G) Phenotypes of the WT, atabcg14, cypDM, and atabcg14 cypDM at 35 DAG. Scale bar, 2 cm. (F, H, and I) Quantification of plant height (F), number of primary branches per plant (H), and number of siliques per plant (I) of the plants in (G). Data are means ± SD. n = 30 in (B), n = 4 in (C), n = 15 in (E), n = 17 in (F), n = 19 in (H), n = 15 in (I). Groups marked with different letters are significantly different (P < 0.05, ANOVA). g14cypDM represents atabcg14 cypDM.

Figure 5

Shootward long-distance transport of iP-type CK species is mediated by AtABCG14. (A and B) Quantification of iP-type CKs in the shoot (A) and root (B) of cypDM and atabcg14 cypDM. The seedlings were grown on half-strength Murashige and Skoog agar medium for 8 days before harvest of shoots and roots. (C) Translocation of exogenous iP and iPR in atabcg14 cypDM. Roots of 8-DAG cypDM and atabcg14 cypDM seedlings were incubated with 50 nM ²H₆-iP for 1 h. The shoots were harvested for CK quantification. (D and E) CK concentrations of xylem sap in cypDM, atabcg14 cypDM(D), WT, and atabcg14(E). Xylem sap was collected from 25-DAG plants. (F–I) Quantification of CK content in the shoots (F), roots (G), apoplast (H), and phloem sap (I) of cypDM/cypDM, atabcg14 cypDM/atabcg14 cypDM, atabcg14 cypDM/cypDM, and cypDM/atabcg14 cypDM plants at 25 DAG. Data are means ± SD. n = 3 in (A)–(E) and (I), n = 4 in (F)–(H). Groups marked with different letters are significantly different (P < 0.05, ANOVA). ∗P < 0.05, ∗∗P < 0.01 (Student’s t-test).

Figure 6

Rootward long-distance transport of iP-type CKs is mediated by AtABCG14. (A) Morphological phenotypes of 25-DAG cypDM and atabcg14 cypDM. Scale bars, 1.5 cm. A solution of 5 ppm ²H_6^-iP was dripped onto the third and fourth leaves of plants (7 μl/cm² leaf area) and incubated for 6 h. The roots (B), younger (fifth to tenth) leaves (C), and third and fourth leaves (D) were then harvested for CK quantification. (E and F) Quantification of iP and iPR content in phloem sap of cypDM, atabcg14 cypDM(E), WT, and atabcg14(F). Phloem sap from 25-DAG plants was collected for CK quantification. (G) A solution of 5 ppm ²H₆^-iP was dripped onto the third and fourth leaves of 25-DAG plants and incubated for 6 h. The roots were harvested for CK quantification. (H) WT plants were incubated under split-root conditions with or without ²H₆-labeled iP. (I) Quantification of ²H₆-labeled iP and iPR contents in the shoot and untreated root (which did not receive ²H₆-labeled iP) after feeding with ²H₆-iP for 4 h in (H). Data are means ± SD. n = 3 in (B)–(E), n = 4 in (F), (G), and (I). ∗P < 0.5, ∗∗P < 0.01 (Student’s t-test). Groups marked with different letters are significantly different (P < 0.05, ANOVA).

Figure 7

Diagram of AtABCG14-mediated circular long-distance transport of iP-type CKs. In the root, iPR/iP produced in pericycle cells (PCs) or retrieved from the apoplast by an unknown transporter (?) are loaded into the root xylem by G14 (AtABCG14) on the PC PM. Then iPR/iP are transported via the xylem in the leaf transpiration stream. In the shoot, iPR/iP released from the xylem or synthesized in the shoot are loaded into the phloem companion cells (PCCs) in the stems or leaf veins by an unknown transporter (?). iPR/iP in the phloem are then transported upstream to the leaf minor veins and unloaded into the apoplast by G14 on the PCC PM for shoot distribution. iPR/iP in the phloem are transported downstream to the root. In the root, iPR/iP are unloaded from the phloem to the apoplast by G14 on the PCC PM. Finally, the released iPR/iP can be used for another round of circular long-distance transport.