Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation

Abstract

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1 npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1 npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

Figure 1

Biochemical and photochemical properties of Arabidopsis npl1. (A) Western blot analysis of nph1 and npl1 expressed in insect cells. Soluble protein fractions prepared from insect cells expressing either nph1 or npl1 were probed with anti-His antibody. Positions of molecular mass markers are indicated on the left in kilodaltons. (B) Autoradiograph showing the in vitro light-dependent phosphorylation of soluble protein fractions prepared from insect cells expressing either nph1 or npl1 (indicated by arrows). All manipulations were carried out under dim red light. Samples were given a mock irradiation, D, or irradiated with white light, L, at a total fluence of 30,000 μmol⋅m⁻². Arrows on the left and right indicate the approximate molecular masses of recombinant nph1 and npl1 (125 kDa and 110 kDa, respectively). (C) Absorption spectra (Insets) and light-minus-dark difference spectra (main panels) of oat nph1 and Arabidopsis npl1 expressed and purified from E. coli. The difference spectra show dark recovery to the ground state after a light flash and were taken at 1-s intervals after the light flash, except for nph1 LOV2, for which the time interval was 5 s.

Figure 2

Hypocotyl phototropism in etiolated wild-type, nph1, npl1, and nph1npl1 mutant seedlings of Arabidopsis. Hypocotyl curvatures of 3.5-day-old seedlings were measured as indicated by χ° in the Inset. Curvatures were measured after a 12-hr exposure to unilateral blue light at the fluence rates indicated. Hypocotyl curvatures of 9 to 16 seedlings were measured in each case and average curvatures were calculated. Values shown represent the average of the three independent experiments. Error bars represent the mean ± the standard deviation.

Figure 3

Light-activated chloroplast relocation in wild-type plants and the nph1npl1 double mutant. (A) Slit assay for chloroplast relocation. Leaves from wild-type (Columbia), npl1-101, and the nph1npl1 double mutant were partially irradiated with high-intensity blue light for 1 hr. Photographs taken in dark (upper section) and bright (lower section) fields of vision are shown in each case. (B) A series of images monitoring chloroplast relocation in single mesophyll cells from wild-type (Columbia) plants and the nph1npl1 double mutant. Chloroplast accumulation movement was induced by continuous microbeam irradiation with low-intensity blue light (LB, 2 μmol⋅m⁻²⋅s⁻¹) from the 20th to 60th minute after the onset of the experiment (D, without blue light irradiation). Chloroplast avoidance movement was induced with high-intensity blue light (HB, μmol⋅m⁻²⋅s⁻¹) from the 60th to the 90th min. The microbeam (20 μm in diameter) used can be seen as a light circle in each image taken after 20 min. (C) Movement tracks of individual chloroplasts. The movement of each chloroplast numbered in B was traced during the experiment and is shown in Upper. Circles in the cells represent the irradiated areas. Distances (μm) between the beam center and each chloroplast were also recorded and are shown in Lower. The 10-μm distance represents the range of the irradiated area (the radius of the blue light microbeam).

Figure 4

Proposed photosensitivities required for nph1 and npl1 function. (A) The range of fluence rates effective for nph1 and npl1 action in hypocotyl phototropism. (B) The range of fluence rates expected to be effective for nph1 and npl1 function in chloroplast relocation. Wild-type plants typically show a chloroplast accumulation response to blue light from 0.4 to between 16 and 32 μmol⋅m⁻²⋅s⁻¹ (blue arrow) and an avoidance response to blue light of 32 μmol⋅m⁻²⋅s⁻¹ or higher (red arrow). The fluence rate boundary between the accumulation and the avoidance responses is estimated to fall between 16 and 32 μmol⋅m⁻²⋅s⁻¹ (19). On the basis of our findings with the nph1npl1 double mutant in addition to earlier studies (17, 19), nph1 appears to mediate chloroplast accumulation movement to light at fluence rates from 0.4 to 40 μmol⋅m⁻²⋅s⁻¹, or higher (blue arrow). npl1 regulates the light-activated chloroplast accumulation at fluence rates from 2 μmol⋅m⁻²⋅s⁻¹ to between 16 and 32 μmol⋅m⁻²⋅s⁻¹ (blue arrow). The light-induced chloroplast avoidance movement is mediated by npl1 at a fluence rate of 32 μmol⋅m⁻²⋅s⁻¹ or higher (red arrow).