The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning

Abstract

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

Figure 1.

PKS1/PKS2/PKS4 regulate leaf flattening and act in the phot2 pathway. A, Plants were grown for 25 d under 80 ± 8 μ mol m⁻² s⁻¹ white light (WL) with a 16-h-light photoperiod at 20°C (until the wild type [WT] reached growth stage 1.11; Boyes et al., 2001). The flattening index of leaf 5 was calculated by dividing the projection area of intact curled leaves (inset, left) with that of manually uncurled leaves (inset, right). The graph shows average values ± 95% confidence intervals for 17 or 18 plants. Images of leaf sections at bottom illustrate leaf curling. B, Positions of PKS1/2 and NPH3 based on the interpretation of epistasis data.

Figure 2.

PKS2 regulates leaf positioning. Leaf positioning was determined after light treatments by measuring the hypocotyl-petiole angle; 90 ° was subtracted to provide an indication of petiole position relative to horizontal (inset in A). Light blue histogram bars correspond to 50 μ mol m⁻² s⁻¹ red light plus 0.3 μ mol m⁻² s⁻¹ blue light; dark blue bars correspond to red light plus 5.0 μ mol m⁻² s⁻¹ blue light. A, Leaf positioning in pks1, pks2, and pks4 mutants and in the triple mutant. B, Leaf positioning in PKS2-overexpressing plants. Values indicate means ± 95% confidence intervals for 21 < n < 31 (A) and 34 < n < 57 plants (B). WT, Wild type.

Figure 3.

Genetic analysis of PKS2 and NPH3 roles within phot1 and phot2 pathways controlling leaf positioning. Plants were grown as described in Figure 2. A, Epistasis between nph3 and phot mutants. B, Epistasis between pks2, nph3, and phot mutants. Bars indicate means ± 95% confidence intervals for 32 < n < 52 plants (A) and 32 < n < 55 plants (B). C, Visual comparison of selected mutants grown under high blue light. Side views of plants illustrate the positioning of petioles and the flatness of laminae of the first pair of true leaves. Top views further show lamina epinasty and reduction in light capture. D, Positions of NPH3 and PKS2 in phot1 and phot2 pathways in both LBL and high blue light based on the interpretation of epistasis data. BL, Blue light; WT, wild type.

Figure 4.

PKS2, PKS1, and NPH3 are associated with phot1 and phot2 in vivo. Solubilized microsomal proteins were obtained from green tissues of 14-d-old plants grown under 100 μ mol m⁻² s⁻¹ white light and were subjected to anti-GFP immunoprecipitation using anti-GFP antibodies. The following genotypes were analyzed: wild type (lane 1), 35S:GFP-LTi6b (plasma membrane-associated protein; lane 2), PHOT2:PHOT2-GFP phot1-5 phot2-2 (lane 3), PHOT1:PHOT1-GFP phot1-5 (lane 4). Input represents solubilized microsomes used for the immunoprecipitated material (IP). DET3 served as a loading control.

Figure 5.

PKS1/PKS2/PKS4 are not required for blue light (BL)-induced chloroplast relocation or stomatal opening. A to D, Chloroplast movements in pks1pks2pks4 mutants. Plants were grown for 6 weeks under 100 to 120 μ mol m⁻² s⁻¹ white light at 24°C with a 12-h photoperiod. Leaves were dark adapted for 18 h and then exposed to a progressive increase of blue light fluence rate from 0.1 to 120 μ mol m⁻² s⁻¹. Plots show dose-response curves corresponding to the change (in percentage) of red light (RL) transmittance of leaves relative to the average transmittance measured in dark-treated leaves. Data points show averages ± sd of 9 < n < 13 plants. E, Isolated epidermal peels were obtained from rosette leaves of 4-week-old plants and irradiated for 3 h at 24°C under red light (60 μ mol m⁻² s⁻¹; R) or red light (50 μ mol m⁻² s⁻¹) plus blue light (10 μ mol m⁻² s⁻¹; R+B). The average aperture of 45 stomata was calculated per experiment. The graph shows averages ± sd of three separate experiments. D, Dark.

Figure 6.

Growth of wild-type and epinastic mutant plants under intermediate white light fluence rates. Plants were grown at 20.5°C ± 1°C under 150 ± 15 μ mol m⁻² s⁻¹ white light with a 16-h-light photoperiod and were shuffled around to even out the effects of varying microenvironments. Fresh weight (FW) of green tissues was measured at 14 (A), 19 (B), and 24 (C) d after incubation (dai). Graphs show average values ± 95% confidence intervals for 20 < n < 36 plants. Images at bottom show one representative plant for each genotype. Bars = 1 cm.

Figure 7.

Morphological and physiological parameters of wild-type (WT) and epinastic mutant leaves. A and B, Morphological parameters of leaf 5 of plants shown in Figure 6C. Light interception area of curled leaves and total leaf area were calculated as in Figure 1. C, Light-induced transpiration in whole leaves. Plants were grown for 8 to 10 weeks under 200 μ mol m⁻² s⁻¹ white light with an 8-h-light (22°C)/16-h-dark (16°C) cycle. After overnight dark adaptation, the adaxial side of mature leaves was exposed to 500 μ mol m⁻² s⁻¹ red light (black bars) for 60 min and then 25 μ mol m⁻² s⁻¹ blue light (white bars) was superimposed for 60 min. Transpiration on the leaf abaxial side was measured over time by infrared gas analysis technique. Graphs show average transpiration levels 10 min before and 0 to 35 min after switching on blue light for 5 < n < 9 plants (±se). D, Stomatal density of abaxial epidermis. Prints were obtained from similar leaves as in Figure 1. Average stomatal density was calculated by counting the number of stomata within a measured area comprising 60 to 120 epidermal pavement cells. Plots show means ± sd of five leaves. Different leaf regions were analyzed (margin to midvein, apex to base).

Figure 8.

PKS2 may control leaf flattening and positioning by acting on auxin transport regulation. A, Leaf positioning in PKS2-overexpressing plants under red light. Bars indicate means ± 95% confidence intervals for 34 < n < 57 plants. B, Expression pattern of PKS2 reported by GUS expression. Plants were grown for 2 weeks on agar under 100 μ mol m⁻² s⁻¹ continuous white light at 22°C and were incubated with 5-bromo-4-chloro-3-indolyl- β -glucuronic acid substrate for 24 h at 37°C for coloration. C, Auxin loading in mesophyll protoplasts of the wild type (WT) and pks1, pks2, pks1pks2, and aux1 mutants. Data are averages ± sd (n = 3). Asterisks mark significantly different means from the wild type (t test, P < 0.05). IAA, Indole-3-acetic acid.