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Arabidopsis NPL1: A Phototropin Homolog Controlling the Chloroplast High-Light Avoidance Response

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Science  16 Mar 2001:
Vol. 291, Issue 5511, pp. 2138-2141
DOI: 10.1126/science.291.5511.2138

Abstract

Chloroplasts relocate their positions in a cell in response to the intensity of incident light, moving to the side wall of the cell to avoid strong light, but gathering at the front face under weak light to maximize light interception. Here, Arabidopsis thalianamutants defective in the avoidance response were isolated, and the mutated gene was identified as NPL1 (NPH-like 1), a homolog of NPH1 (nonphototropic hypocotyl 1), a blue light receptor used in phototropism. Hence, NPL1 is likely a blue light receptor regulating the avoidance response under strong light.

Plants need light not only for photosynthesis but also for precise regulation of their development. To compete successfully and effectively under varying environmental conditions, plants sense the surrounding light conditions, including wavelength, intensity, direction, duration, and in some instances even the plane of polarization. This information is used to regulate their development or prepare for forthcoming seasonal changes. Phytochromes and cryptochromes are the photoreceptors thought to mediate the multitude of responses involved (1).

To maximize photosynthesis, plants orient their growth and leaf angle to maximize light interception (a process known as phototropism), open their stomata in the presence of light to facilitate gas exchange, and relocate their chloroplasts within the cell. Plants use blue light in these responses, both to sense light direction and light intensity. However, a blue light receptor for increasing the efficiency of photosynthesis through the above three phenomena is known only for phototropism, where phototropin (NPH1) functions under weak light conditions (2, 3). Phototropin mediates phototropism of hypocotyls and roots, but to date has not been shown to mediate any other blue light responses. Although light-induced chloroplast movement is a phenomena well known since the 19th century (4), a blue light receptor that regulates this movement has not been identified. Phytochrome is known to be a photoreceptor for red light–induced chloroplast movement in the green alga Mougeotia scalaris (5) and the fernAdiantum capillus- veneris (6), but these are rare cases.

To identify the blue light receptor that regulates chloroplast movement in A. thaliana, a simple method for detecting chloroplast avoidance movements was developed (Fig. 1A) (7), and used to screen about 100,000 ethylmethane sulfonate (EMS)– mutagenized F2 seeds and 15,000 T-DNA–tagged lines. More than 10 mutants, named cav (defective in chloroplast avoidance movements) were obtained. Four of the mutants, cav1-1 tocav1-4, were found to belong to a single complementation group. All mutants were single, nuclear, and recessive.

Figure 1

(A) Leaves of wild-type (columbia) and cav1-1 (columbia) before and after partial irradiation with strong white light. Leaves covered with a non–light-transmitting black plate with open slits were irradiated through the slits with strong cool white light for 1.5 hours (7). The irradiated area of the wild-type (WT) leaf changed from dark green to pale green and was clearly distinguishable from nonirradiated area. In cav1-1, the area irradiated looked almost the same as the nonirradiated area. (B) The chloroplast avoidance movement in wild-type (columbia), cav1-2, and cav1-5 leaves. Chloroplast movement was observed and recorded under red light by methods described in (12). The cells were partially irradiated with a microbeam (20 μm in diameter shown as “white holes” in the photographs taken at 20 min) of weak blue light (2 μmol m−2 s−1) from 20 to 60 min and with strong blue light (40 μmol m−2 s−1) at the same position from 60 min to the end of experiments. (C) The movement of each chloroplast was traced during the experiment (upper panel) and the distance between the beam center and each chloroplast recorded (lower panel). These data were obtained using the same cells as shown in (B). Chloroplasts in both wild-type and cav1-5 accumulated in the spot of weak blue light (wB: 2 μmol m−2s−1). When the beam was changed to strong blue light (sB: 40 μmol m−2 s−1), chloroplasts in a wild-type cell moved away from the irradiated area but chloroplasts in the cav1-5 did not. (D) A part of a cell of dark-adapted cav1-1 was irradiated with a microbeam of strong blue light (40 μmol m−2 s−1, 20 μm in diameter) from 20 to 60 min. Chloroplasts showed accumulation movement without stopping at the edge of the microbeam and entered into the irradiated area. Other conditions are the same as in (C).

Chromosome mapping of the CAV1 gene placed it between the ILL2 and PLC1a markers at 113 centimorgans on chromosome V (Fig. 2A) (8). This region includes the recently described NPL1 gene, which encodes a protein similar in sequence to phototropin (9), a blue light receptor for phototropic responses (2, 3). Thus, the NPL1 protein is a strong candidate to be encoded by CAV1. After screening a library of T-DNA–tagged lines, we obtained one line with an insertion in NPL1 (10), which lacked the avoidance movement of chloroplasts. We designated this mutantcav1-5, because it was allelic to the previously isolatedcav1 mutants. The mutation sites of all the cav1mutants were located within the NPL1 gene (Fig. 2B), confirming that CAV1 and NPL1 are the same. Incav1-2, the conserved threonine residue at 727 in subdomain VIII for substrate recognition of serine/threonine kinases region (11) was mutated to isoleucine, indicating that a signal for the chloroplast avoidance movement might be generated by phosphorylation. Accumulation of NPL1 mRNA was not detected in cav1-5 by reverse transcription polymerase chain reaction (RT-PCR), suggesting that cav1-5 is a null mutant of NPL1.

Figure 2

cav1 cloning and gene structure. (A) cav1 map on chromosome V. Thecav1-1 mutation was first found to be located between two markers AthPHYC and LHYa by cleaved amplified polymorphic sequences (CAPS) (8), and then between ILL2 and PLC1a where theNPL1 gene is found. Recombination rates for each marker when crossed between cav1-1 (columbia) and WT (Lansberg) are shown underneath and the rate of NPL1appearance is also shown in parenthesis. (B) Structure of the NPL1 gene and positions of cav1 mutants. The locations of start (AGT) and stop (TAG) codons are indicated. Twenty-three exon (boxes) and 22 intron (lines) positions were determined by comparing the genomic sequence with the cDNA sequence. Mutations of cav1-1, cav1-2,cav1-3, cav1-4, and cav1-5 are as follows: base 3121 from the start codon, the last nucleotide in intron 11 was changed from G to A in cav1-1; base 4425 was changed from C to G, changing threonine (T) to isoleucine (I) incav1-2; a deletion of G at base 2408 caused a frame shift incav1-3; and deletion of 79 base pairs (bp) from 2192-2270 and addition of 4 bp in cav1-4; and insertion of T-DNA into exon 9 in cav1-5. (C) Structural features of some plant proteins that have two LOV domains. Shown are A. thaliana NPL1 (atNPL1) (9), phototropin (atNPH1) (2), and Adiantum capillus-veneris phytochrome 3 (acPHY3) (21). LOV, kinase, and phytochrome domains are shown as meshed, black, and slashed boxes, respectively.

The movement of an individual chloroplast in a mesophyll cell at the top layer of the palisade tissue was also examined, using a recording system to monitor chloroplast movement every 1 min under a microbeam irradiator (12). A part of the cell surface was irradiated with a small beam of weak or strong blue light (20 μm in diameter) (Fig. 1, B and C) (13). Chloroplasts in cav1mutant cells moved toward the area irradiated with weak blue light (2 μmol m−2 s−1) as did chloroplasts in wild-type cells. Unexpectedly, however, chloroplasts in these mutant cells also accumulated in the area irradiated with strong blue light (more than 40 μmol m−2 s−1) (Fig. 1D); chloroplasts in a wild-type cell did not enter into the area irradiated with strong light (12). These results show that a blue light receptor for the accumulation movement is present in the irradiated area of the mutant cells and that the strong blue light irradiation itself can generate a signal for accumulation response, at least when the avoidance response is inactivated. In a wild-type cell, a signal for the accumulation response might also be generated under strong blue light. However, because no chloroplast was found to enter into the microbeam-irradiated area, the signal for avoidance is probably stronger than that for accumulation within the microbeam-irradiated area.

The cav1 plants are defective in chloroplast avoidance movement but, to date, are normal in other blue light–induced physiological responses examined, such as phototropism, the chloroplast accumulation movement, inhibition of hypocotyl elongation, and the timing of flowering (14). The level ofNPL1 mRNA accumulated in wild-type plants was higher in leaves than in stems and flowers, and was very low in roots (Fig. 3A). These results are perhaps not surprising given that chloroplast movement occurs mostly in leaves. When etiolated seedlings of wild-type plants were irradiated with red, blue (10 μmol m−2 s−1, respectively), or white light (30 μmol m−2s−1) for 4 hours, the level of accumulation ofNPL1 mRNA increased compared to a dark control (Fig. 3B). The blue light on the level of NPL1 mRNA was tested in more detail by varying the fluence rate of blue light. It was found that the accumulation level more than doubled at the higher fluences (Fig. 3C) (15). Similarly, the mRNA level of the OsNPH1b gene, a NPL1 homolog in rice, increased in etiolated leaves after light irradiation (16).

Figure 3

NPL1 gene expression inArabidopsis wild-type (Landsberg erecta). RNA gel blot hybridization was performed with the 32P-radiolabeledNPL1 cDNA and 18S ribosomal DNA as probes against 5 μg of total RNA. The graphs show the relative abundance ofNPL1 mRNA measured by quantitative RT-PCR. The value was calculated relative to that of the leaf tissue sample in (A) or the sample mock irradiated for 4 hours in (B and C) versus the 18S ribosomal RNA signal. (A) NPL1gene expression in different tissues of adult plants grown on soil under continuous white light at 21°C for 4 to 5 weeks. Tissues were at an early flowering stage. (B) NPL1 gene expression in 4-day-old etiolated seedlings after irradiation for 4 hours under various light conditions. (C) NPL1gene expression in 4-day-old etiolated seedlings after 4 hours exposure to blue light at the various intensity as described (22).

The NPL1 protein and phototropin are highly similar in their amino acid sequences (Fig. 2C) (9). The NPL1 protein has two highly conserved LOV (light, oxygen, and voltage) domains, to which FMN (flavin mononucleotide), a chromophore for blue light perception, was found to bind in phototropin from oat and phytochrome 3 from the fern A. capillus-veneris (17). Because NPL1 LOV domains are known to absorb blue light (18), NPL1 is likely to be the fourth blue light receptor found in A. thaliana. Physiological experiments using polarized blue light suggest that the photoreceptors for the avoidance response in A. thaliana are localized on or close to the plasma membrane with their dipole moment arranged parallel to the membrane (12), as is the case with lower plants (6). Together with the fact that phototropin is located on the plasma membrane (19), NPL1 may also function at the plasma membrane as a blue light photoreceptor.

The action spectra for chloroplast photorelocation movements under weak and strong light conditions are very similar, both having peaks at 450 and 480 nm in the water plant Lemna trisulca and the moss Funaria hygrometrica(20). It has been suggested that photoreceptors for both weak and strong light responses are the same. Because the accumulation movement occurs in the cav1 mutants under strong blue light and because NPL1 protein could be the photoreceptor for the avoidance movement, there must exist a different blue light receptor for the accumulation movement. We obtained more than 10 npl1 mutants for the avoidance movement but did not find any candidates for photoreceptor mutants for the accumulation response. A mutant defective in the photoreceptor for the accumulation movement might be lethal or the photoreceptors may be genetically redundant and necessary for this response under weak light conditions.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: wada-masamitsu{at}c.metro-u.ac.jp

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