Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum

Abstract

Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants.

Fig. 1.

The Arabidopsis cytokinin receptor AHK3 localizes to the ER in transiently transformed tobacco leaf cells and Arabidopsis seedlings. (A–D) and (F) Confocal images of transiently transformed tobacco epidermal leaf cells co-expressing the indicated AHK3 fusion protein under the control of the 35S promoter or the UBQ10 promoter with the ER marker ER-rk CD3-959. (E) Confocal images of transiently transformed tobacco epidermal leaf cells expressing an AHK3–mCherry fusion protein under the control of the estradiol-inducible promoter (XVE). Images were recorded 2 h (I), 4 h (II), and 24 h (III) after application of 20 μM β-estradiol. Images (I) and (II) were recorded at the highest sensitivity settings of the microscope at which the mCherry fluorescence was just detectable. (G) Confocal images of transiently transformed Arabidopsis cotyledon cells co-expressing the indicated AHK3 fusion protein with the ER marker ER-rk CD3-959. Bars represent 10 μm.

Fig. 2.

AHK3–GFP and GFP–AHK3 fusion proteins co-localize with ERS1–RFP. (A–D) Confocal images of transiently transformed tobacco epidermal leaf cells co-expressing the indicated AHK3 fusion protein under the control of the 35S or the UBQ10 promoter and an RFP fusion of the ethylene receptor ERS1 (ERS1–RFP). Bars represent 10 μm.

Fig. 3.

AHK3–GFP and GFP–AHK3 fusion proteins do not co-localize with the plasma membrane-localized fusion protein NHL3–RFP. (A–E) Confocal images of transiently transformed tobacco epidermal leaf cells co-expressing the indicated AHK3 fusion protein under the control of the 35S promoter or the UBQ10 promoter and the plasma membrane-localized fusion protein NHL3–RFP. (F) Confocal images of transiently transformed Arabidopsis cotyledon cells co-expressing the indicated AHK3 fusion protein and the plasmalemma-localized fusion protein NHL3–RFP. Bars represent 10 μm.

Fig. 4.

AHK3–mCherry does not co-localize with a GFP fusion of the plasma membrane-bound Arabidopsis histidine kinase 1 (AHK1–GFP). Confocal images of different magnification of transiently transformed tobacco epidermal leaf cells co-expressing AHK3–mCherry under the control of the estradiol-inducible promoter (XVE) and AHK1–GFP under control of the 35S promoter. The image series of the second row represents a magnified detail of the images of the first row. The image series of the third row derives from an independent cell. Images were recorded 4 h after application of 20 μM β-estradiol. Bars represent 10 μm.

Fig. 5.

The cytokinin receptor AHK4 co-localizes with AHK3 in the ER and interacts with AHK3 in vivo (yeast). (A) Confocal images of transiently transformed tobacco epidermal leaf cells co-expressing RFP–AHK4 and GFP–AHK3 under control of the 35S promoter. Bars represent 10 μm. (B) Yeast mbSUS protein–protein interaction analysis. The AHK4-Cub-PLV construct was transformed in yeast strain THY.AP4 (MATa), and the Nub constructs of AHK3 and ERS1 were transformed in yeast strain THY.AP5 (MATα). After mating, activation of the reporter gene was determined by growth of the transformants in a dilution series (OD_600nm from 1 to 0.01) on SC medium (SC). The presence of the plasmid was assayed by growth on SC medium supplemented with adenine and histidine (SC+Ade., His.). Co-transformations of the AHK4–Cub-PLV fusion with NubG served as negative control and co-transformation with NubWT served as positive control.

Fig. 6.

The GFP fusion proteins of AHK3 show EndoH sensitivity and are glycosylated in vivo. (A) Amino acid sequence of AHK3. The transmembrane domains are shown in blue, the histidine kinase domain in red, and the pseudo receiver domain and the receiver domain in purple. N-X-S/T sequons are framed. The predicted signal peptide is italicized, and the putative ER export signals are underlined. The green triangle marks the site where GFP was inserted into AHK3intGFP. (B) Representations of AHK3, AHK1, and the ethylene receptor ERS1. Putative glycosylation sites are indicated with asterisks. (C) The electrophoretic mobility of AHK3 is endoglycosidase H (EndoH) sensitive. Equal volumes of protein extracts from transiently transformed tobacco leaves expressing the indicated fusion proteins were treated with EndoH (+) or mock treated (–), followed by western blot analysis and immunodetection using an anti-GFP antibody. The fusion proteins are indicated by arrowheads.

Fig. 7.

The GFP fusions of AHK3 are able to complement the mutant phenotype of the ahk2ahk3 receptor mutant and locate to the ER in the complemented lines. (A) Adult plants of the wild type (Col 0), the ahk2ahk3 mutant, and plants expressing AHK3–GFP or GFP–AHK3 from the UBQ10 promoter in the ahk2ahk3 background (AHK3–GFP and GFP–AHK3). (B) Genotypic analysis of AHK3–GFP and GFP–AHK3 lines by PCR using the indicated primer pairs. (C) Inhibition of root elongation by increasing amounts of exogenously applied cytokinin (kinetin). Seedlings of the wild type (Col 0, white columns), the ahk2ahk3 mutant (black columns), and the complemented, heterozygous AHK3–GFP and GFP–AHK3 lines (dark and light grey columns) were grown on 0.5× MS agar plates supplemented with the indicated concentrations of kinetin. Mean values (n=18; standard errors) relative to the root length of non-treated controls and P-values for statistical significance are given. (D) AHK3–GFP fluorescence is detectable in the ER of the AHK3-GFP-complemented Arabidopsis line. CLSM images of epidermis and stomatal cells from cotyledons of etiolated seedlings are shown. N indicates the nucleus. Bars represent 10 μm. (E) EndoH sensitivity of the AHK3–GFP fusion protein in the AHK3-GFP-complemented line. Equal amounts of protein extract from seedlings of the indicated lines were treated or not with endoglycosidase H (EndoH), followed by western blot analysis and immunodetection with an anti-GFP antibody. The fusion proteins are indicated by arrowheads.