Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana

Abstract

The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

Keywords: ABC transporter; Cytokinin; Endoplasmic reticulum; HY5; Seedling growth.

Fig. 1

ABCI19, ABCI20, and ABCI21 are closely related NBD-type ABC proteins expressed at young seedling stages. (a) Phylogenetic tree of 9 NBD-type ABCI proteins in A. thaliana. Protein sequences were aligned using ClustalW program (http://www.genome.jp/tools/clustalw/), and then phylogenetic analysis was undertaken to produce a midpoint rooted tree. ABCI19, ABCI20 and ABCI21 are marked in red. (b) Relative expression levels of ABCI19, ABCI20 and ABCI21 measured by qRT-PCR at seedling stage after 3–14 days of seed sowing. Data were normalized using the expression level of ACTIN1. Mean values (±SD) of two independent experiments (n=3) are given. (c-h) The GUS signals of proABCI20:GUS transgenic plants observed in 1-day-old (c), 3-day-old (d), 7-day-old (e and f), and 2-week-old (g) seedlings and in flowers (h).

Fig. 2

Generation of abci19 abci20 abci21 knockout mutants. The locations of gene fragment deletions, point mutation, or T-DNA insertion in the abci19 abci20 abci21 mutants are presented. (a) The premature stop codons and amino acid substitution in abci20–1 and abci20-2 produced by CRISPR/Cas9. PCR-amplified genomic DNA of the mutants were resistant, whereas the wild type PCR product was digested successfully by the restriction enzymes Bsl I and Xmn I. (b) T-DNA insertional position in abci21 (SALK_064144). RT-PCR analysis confirmed the absence of ABCI21 transcript in abci21 mutant. (c) Two sets of guide RNA pairs targeting ABCI19 gene produced two different fragment deletions in ABCI19 in the background of abci20–1 abci21. PCR using GT1/2 primers confirmed ABCI19 gene deletion.

Fig. 3

Cytokinin-hypersensitive seedling growth of abci19 abci20 abci21 mutants. (a) Plants were vertically grown on ½ MS-agar plates in the absence or presence of trans-zeatin. Pictures were taken at 12 days after seed sowing. Scale bar = 1 cm. (b, c) Plant growth under conditions given in (a) quantified by measuring the total shoot fresh weight per plate and the primary root length. Mean values (±SD) of two independent experiments (3 biological replicates each) are presented (*; p < 0.03, Student’s t test). (d) The numbers of cortical cells in RAM under trans-zeatin treatment condition. The wild type and mutants were grown on 1/2 MS-agar media containing 10, 20, or 50 nM trans-zeatin for 8 days. Mean values (±SD) are presented (*; p < 0.03, Student’s t test, N=2, n=6).

Fig. 4

Enhanced cytokinin signaling in abci20 abci21. (a) Representative root tip images of multiple TCS:GFP-expressing wild type and abci20–1/21 lines grown in the absence or presence of 20 nM tZ. Scale bar = 20 μm. (b) TCS:GFP fluorescence intensity measured in the root tips of the wild type or abci20–1/21 transgenic lines expressing TCS:GFP. Seeds were germinated and grown on ½ MS-agar media supplemented with or without 20 nM trans-zeatin. Images were taken at 8 days after seed sowing. The fold increase of TCS:GFP level in tZ-treated root tips over in non-treated control root tips was calculated. The combined results (mean ± SD) of two independent experiments (total 7 biological replicates) are given. Different alphabets indicate that the means are significantly different between genotypes by Tukey’s Honest Significant Difference test at p ≤ 0.01.

Fig. 5

Localization of ABCI20 and ABCI21 at endoplasmic reticulum. (a) Representative root tip images of ABCI20-GFP transgenic A. thaliana seedlings. The plasma membrane was labeled by brief staining with FM4–64. The boxed region of the root tip is magnified in the bottom panels. Scale bars = 10 μm (top panel) and 2 μm (bottom panel). (b) Membrane fractionated by sucrose density gradient and probed with antibodies to marker proteins of different membranes. (c) ABCI20-GFP was released to soluble fraction (S) from the membrane fractions (pellet, P) when subjected to alkaline (100 mM Na₂CO₃) buffers but not to high salt (100 mM KCl). (d) Representative images of tobacco leaf epidermis cells that transiently express RFP-ABCI21. Scale bars = 10 μm.

Fig. 6

ABCI20 is a component of a large protein complex. Whole protein extracts from the wild type and abci20–1/21 double knockout Arabidopsis seedlings were analyzed by BN-PAGE and Western blot with anti-ABC20 antibody. Anti-ABCI20 antibody detected a protein complex around 300–400 kDa (red arrowhead).

Fig. 7

HY5-dependent induction of ABCI20 and ABCI21 upon light exposure. (a) Promoters of ABCI19, ABCI20 and ABCI21 contain HY5-responsive elements (ACGT containing elements and/or G-box). (b) Relative transcript levels (mean values ± SD) of ABCI19, ABCI20 and ABCI21 in hy5–1 and the wild type (Ler) under various light/dark conditions. Gene expression levels were measured by qRT-PCR and normalized by the β–Tubulin 8 expression level. Asterisks indicate significant differences between Ler and hy5–1 (p < 0.05, Student’s t test), and hashtags indicate significant differences between light-exposed samples and dark control (p < 0.05, Student’s t test; N=2, n=3).