The cytokinin efflux transporter ABCC4 participates in Arabidopsis root system development

Abstract

The directional and sequential flow of cytokinin in plants is organized by a complex network of transporters. Genes involved in several aspects of cytokinin transport have been characterized; however, much of the elaborate system remains elusive. In this study, we used a transient expression system in tobacco (Nicotiana benthamiana) leaves to screen Arabidopsis (Arabidopsis thaliana) transporter genes and isolated ATP-BINDING CASSETTE TRANSPORTER C4 (ABCC4). Validation through drug-induced expression in Arabidopsis and heterologous expression in budding yeast revealed that ABCC4 effluxes the active form of cytokinins. During the seedling stage, ABCC4 was highly expressed in roots, and its expression was upregulated in response to cytokinin application. Loss-of-function mutants of ABCC4 displayed enhanced primary root elongation, similar to mutants impaired in cytokinin biosynthesis or signaling, that was suppressed by exogenous trans-zeatin treatment. In contrast, overexpression of the gene led to suppression of root elongation. These results suggest that ABCC4 plays a role in the efflux of active cytokinin, thereby contributing to root growth regulation. Additionally, cytokinin-dependent enlargement of stomatal aperture was impaired in the loss-of-function and overexpression lines. Our findings contribute to unraveling the many complexities of cytokinin flow and enhance our understanding of the regulatory mechanisms underlying root system development and stomatal opening in plants.

Figure 1.

Quantification of exported cytokinins from ABCC4-overexpressing tobacco leaf cells. A) Tobacco leaf disks expressing ABCC4 were incubated in an incubation buffer for 12 h, followed by measurements of cytokinin levels in the buffer. Data are means ± SD (n = 3). Asterisks represent the Student's t-test significance compared with VC (**P < 0.01). B) Time course analysis of the exported cytokinins. The leaf disks were incubated for the indicated times, and the cytokinin levels in the buffer were quantified. Data are means ± SD (n = 4). Asterisks represent the Student's t-test significance compared with VC (*P < 0.05, **P < 0.01, ***P < 0.001). C) The effect of ABC transporter inhibitors on the levels of exported cytokinins from ABCC4-overexpressing tobacco leaf cells. Tobacco leaf disks expressing ABCC4 were incubated in incubation buffer without any inhibitors (Mock), with 1 mm orthovanadate (Vana), or with 0.1 mM glibenclamide (GC) for 12 h, after which cytokinins in the buffer were quantified. Data are means ± SD (n = 3). Asterisks in this figure represent the Student's t-test significance compared with VC (*P < 0.05, **P < 0.01, ***P < 0.001). VC, empty vector control; Conc., concentration; gDW⁻¹, grams per dry weight; iP, N⁶-(Δ²-isopentenyl)-adenine; tZ, trans-zeatin; cZ, cis-zeatin.

Figure 2.

Quantification of exported cytokinins from ABCC4-overexpressing Arabidopsis seedlings. Seedlings of two independent transgenic Arabidopsis lines (Line 1 and Line 2) expressing ABCC4 under control of the β-estradiol-inducible promoter were treated with (+) or without (−) 10 μM of β-estradiol for 24 h, followed by measurements of cytokinins in the culture medium. Data are means ± SD (n = 4). Asterisks in this figure represent the Student's t-test significance compared with the mock (−) treatment (*P < 0.05, **P < 0.01, ***P < 0.001). Conc., concentration; gFW⁻¹, grams per fresh weight; iP, N⁶-(Δ²-isopentenyl)-adenine; iPR, iP riboside; iPRPs, iP ribotides; tZ, trans-zeatin; tZR, tZ riboside; tZRPs, tZ ribotides; cZ, cis-zeatin; cZR cZ riboside; cZR, cZ ribotides.

Figure 3.

Heterologous expression of ABCC4 in yeast cells. A) Immunoblot detection of ABCC4 in yeast cells. Yeast (strain YPH499) harboring the pYES-empty vector (VC) or pYES-ABCC4 (ABCC4) was cultured, and total protein was extracted. Total proteins (60 μg) were subjected to immunoblot analysis using an anti-ABCC4 antibody. Sizes of the molecular mass markers (M) are indicated on the left. B) Cytokinin transport assay in yeast. VC and ABCC4 yeast strains treated with stable isotope-labeled 50 nm tZ (+10) or tZR (+15) were incubated in isotope free-buffer for 0 and 10 min, followed by quantification of the labeled compounds in the cells. The relative concentration was calculated by defining the concentration at 0 min as 100%. Data are means ± SD (n = 4). Asterisks in this figure represent Student's t-test significance compared with VC (**P < 0.01). conc., concentration; tZ, trans-zeatin; tZR, tZ riboside.

Figure 4.

Expression patterns of ABCC4 in Arabidopsis. A) Expression patterns of ABCC4 in plant organs. Total RNAs were extracted from shoots and roots of 10-d-old Arabidopsis seedlings (left panel) and from the indicated organs of 45-d-old plants (right panel) and subjected to RT-qPCR analysis. Expression levels of ABCC4 were normalized to that of TIP41, a housekeeping gene. Data are means ± SD (n = 4). B, C) Effect of cytokinin and auxin treatments on the expression of ABCC4 in shoots B) and roots C) Arabidopsis seedlings grown for 10 d on 1/2 agar plates were sprayed with 0.01% dimethyl sulfoxide (Mock),1 μM N⁶-(Δ²-isopentenyl)-adenine (iP), 1 μM trans-zeatin (tZ), or 0.4 μM indole-3-acetic acid (IAA). The shoots and roots were separately harvested after 30 min and 2 h. Total RNAs were extracted and subjected to RT-qPCR analysis. Expression levels of ABCC4 were normalized to that of ACT8. Data are means ± SD (n = 5). Asterisks in this figure represent Student's t-test significance compared with Mock (*P < 0.05, **P < 0.01, ***P < 0.001). exp., expression.

Figure 5.

Root growth phenotypes of abcc4 mutants and ABCC4-overexpression lines. A) A representative image of WT (Col-0), a T-DNA insertion mutant (abcc4-1), a genome-edited mutant (abcc4-2), and two ABCC4-overexpression lines (OX-1 and OX-2). The plants were grown on 1/2MS agar plates for 12 d. Scale bar, 1 cm. B) Primary root length of WT, abcc4-1, abcc4-2, OX-1, and OX-2 seedlings grown on 1/2MS agar plates for 14 d. Data are means ± SD (n_WT = 37, n_abcc4-1 = 34, n_abcc4-2 = 36, n_OX-1 = 33, n_OX-2 = 36). C) Representative images of the root meristem of WT, abcc4-1, abcc4-2, OX-1 and OX-2 seedlings grown for 6 d. Red and black arrowheads indicate the quiescent center and the boundary of first elongated cortex cell, respectively. D) Root meristem cell number of WT, abcc4-1, abcc4-2, OX-1 and OX-2 seedlings grown for 6 d. Data are means ± SD (n_WT = 6, n_abcc4-1 = 7, n_abcc4-2 = 9, n_OX-1 = 5, n_OX-2 = 5). E) The effect of cytokinin treatment on primary root length of WT, abcc4-1, and abcc4-2. Seedlings were grown on 1/2MS agar medium with 0.01% DMSO (Mock) or the indicated concentration of trans-zeatin (tZ) for 11 d. Data are means ± SD (n_mock-WT = 32, n_mock-abcc4-1 = 29, n_mock-abcc4-2 = 32, n_tZ10nM-WT = 32, n_{tZ10nM-abcc4-1} = 26, n_{tZ10nM-abcc4-2} = 25, n_tZ100nM-WT = 21, n_{tZ100nM-abcc4-1} = 18, n_{tZ100nM-abcc4-2} = 25, n_{tZ1 µM-WT} = 36, n_{tZ1 µM-abcc4-1} = 34, n_{tZ1 µM-abcc4-2} = 35). Asterisks in this figure represent Student's t-test significance compared with WT (**P < 0.01, ***P < 0.001).

Figure 6.

Comparison of TCSn:GFP fluorescent pattern in the root of WT, abcc4 mutant and ABCC4-overexpression lines. A) Representative fluorescent images of WT (Col-0), abcc4-1, and ABCC4-overexpression line (OX-1) grown for 10 d. Scale bar, 100 µm. Arrowhead line in orange shows the measured GFP fluorescence. B) The range of GFP fluorescence observed in the epidermal tissue from the root tip was measured by ImageJ. Data are means ± SD (n_WT = 10, n_abcc4-1 = 10, n_OX-1 = 14). Asterisks represent Student's t-test significance compared with WT (***P < 0.001).

Figure 7.

Stomatal aperture in WT, abcc4 mutant and ABCC4-overexpression lines. A) Measurement of stomatal aperture in WT (Col-0), abcc4-1, OX-1 and OX-2 in the dark or light. Detached leaves were placed in dark (Dark) or lighted (Light) conditions for 2 h. Data are means ± SD (n = 30). Asterisks represent Student's t-test significance compared with WT (***P < 0.001). B) Effect of trans-zeatin (tZ) application on the stomatal aperture in the dark. Detached leaves of WT, abcc4-1, OX-1 and OX-2 were placed in dark for 3 h followed by 0.01% DMSO (Mock) or 1 μM tZ (tZ) treatment. Data are means ± SD (n = 30). Asterisks represent Student's t-test significance compared with Mock (***P < 0.001).