Phototropism in gametophytic shoots of the moss Physcomitrella patens

Abstract

Shoot phototropism enables plants to position their photosynthetic organs in favorable light conditions and thus benefits growth and metabolism in land plants. To understand the evolution of this response, we established an experimental system to study phototropism in gametophores of the moss Physcomitrella patens. The phototropic response of gametophores occurs slowly; a clear response takes place more than 24 hours after the onset of unilateral light irradiation, likely due to the slow growth rate of gametophores. We also found that red and far-red light can induce phototropism, with blue light being less effective. These results suggest that plants used a broad range of light wavelengths as phototropic signals during the early evolution of land plants.

Introduction

Light, one of the most important factors in a plant's environment, provides energy for photosynthesis and also acts as a signal for photomorphogenesis, an adaptive response that allows plants to respond to their environment. For example, phototropism, plant growth toward (positive) or away from (negative) a light source enables plants to position their photosynthetic organs in advantageous light conditions.¹ Investigation of phototropism has revealed many fundamental findings, including the involvement of the plant hormone auxin, and identification of photoreceptors such as phototropin, as well as many other light signaling components.¹ Studies of phototropic responses in different species and in different tissues or organs suggest both conservation and diversification in the control of phototropism.^1-3

Although phototropism has been extensively studied for more than a century, many unknowns remain, especially regarding the evolution of shoot phototropism.⁴ Until now, most of our knowledge about phototropism of shoots, including stems, hypocotyls, and leaves, has come from studies of angiosperms. Generally, in angiosperms, blue light can induce a phototropic response in shoots.¹ In ferns, both red and blue light can induce a phototropic response in young leaves,⁵ as ferns have the photoreceptor neochrome, a chimera of a phytochrome red/far-red light receptor and a phototropin blue light receptor.³ Ferns likely acquired this receptor through horizontal gene transfer from hornworts.⁶

Non-vascular land plants, like the ancestors of mosses, are believed to have been among the first plants to occupy the land.⁷ Because of their ease of culturing and simple structure, moss protonemata have been extensively used for the study of phototropism.² For example, in the moss Physcomitrella patens, protonemata respond differently to light quality and quantity. Far-red light induces a positive phototropic response, which increases as the light fluence increases.² Red light induces a weak negative phototropic response^8,9 at fluence rates from 0.015 to 0.15 µmol m⁻² s⁻¹, a positive phototropic response⁸ from 0.5 to 5 µmol m⁻² s⁻¹ and an avoidance response⁸ above 5 µmol m⁻² s⁻¹. By contrast, blue light does not induce any tropic growth.⁸

At present, we cannot predict whether protonemata and gametophytic shoots show similar tropic responses, because the 2 organs have fundamental differences in structure and growth. The protonema is a linear file of single cells and responds to unilateral light exposure by tip growth of the apical cell.⁸ By contrast, the tropic bending of a multicellular gametophore is likely to be caused by differential growth between the irradiated side and the shaded side. On the other hand, at least 3 genes (designated ptrA, B and C) of P. patens have been reported, which are required for phototropism of both caulonemal apical cells and gametophores, indicating that phototropism of protonemata and gametophores share some components.¹⁰ We also cannot infer the mechanisms of phototropism of gametophores from our knowledge of vascular plants, as their development has fundamental differences. For example, gametophytic shoots develop in the haploid stage and the shoots of vascular plants develop in the diploid stage. Moreover, polar auxin transport, which is implicated in phototropism in angiosperms,¹ was not detected by a feeding experiment of radio-labeled auxin in gametophytic shoots of mosses, including P. patens.¹¹

Although examination of gametophores will likely provide insights into the evolution of phototropism, gametophores remain a challenging experimental system. Generally, it is difficult to accurately examine phototropism in white-light-grown gametophores, because they are short and covered with many leafy structures.¹² Thus, producing etiolated gametophytic shoots with rudimentary leaves could enable accurate analysis of the phototropic response in gametophores. Here, we developed an experimental system to efficiently induce etiolated and elongated gametophores of P. patens by keeping them in moist conditions in the dark. Using the etiolated and elongated gametophores, we quantitatively examined the phototropic response to red, far-red, and blue light and found that all 3 light conditions effectively induce a phototropic response in the gametophytic shoots. Also, the phototropic bending rate is much lower than that of angiosperms; gametophores show a maximum bending rate of 2.5 degrees hr⁻¹ while hypocotyls and coleoptiles of angiosperms exhibit about 10 degrees hr⁻¹ in effective light.¹

Results

An experimental system to study phototropism in gametophytic shoots of the moss P. patens

To study phototropism of gametophores, we established an experimental procedure to obtain etiolated, elongated gametophores. The procedure included 2 consecutive light steps: ~50 µmol m⁻² s⁻¹ for 5 days, and then ~10 µmol m⁻² s⁻¹ for 7 days, followed by culture in the dark for ~4 weeks. In the second light step, gametophores that were cultured on vertically oriented agar plates, tended to grow within the agar medium, which reduced the chance of dehydration of the shoots. In the subsequent dark culturing step, gametophores became etiolated. Some of them grew out of the agar medium, and then continued to elongate along the surface of the medium. These growth conditions, which kept the gametophores moist, proved critical for the continuous elongation of the etiolated shoots in the dark. However, up to 70% of the gametophores (from 81 etiolated and elongated gametophores) continued growing in the agar medium, and thus were not suitable for phototropic measurements (Fig. 1C). Further observations revealed that only gametophores with an enough growth rate exhibited a clear phototropic response (Fig. 1A and B). Therefore, we chose the gametophores that grew vertically upwards out of the agar medium with an elongation rate of more than 0.0083 mm hr⁻¹ in the 48-hr period of phototropic measurements. As a result, we found that 17% of gametophores (14 out of 81 etiolated and elongated gametophores) were suitable for phototropic measurements.

Figure 1.

Phototropic response of gametophytic shoots. The etiolated and elongated shoots were continuously irradiated with unilateral red light. (A and B), Representative images of a shoot showing continuous growth (A1 to A4) and of one showing inhibited growth (B1 to B4) before (0 hr, A1 and B1) and after irradiation for 12 (A2 and B2), 24 (A3 and B3) and 48 hr (A4 and B4) at 10 µmol m⁻² s⁻¹. The red arrows show the direction of unilateral light. The red dotted lines mark the same position at different time points. Note that photomorphorgenetic processes including shoot bending, stem growth and leaf enlargement occurred within 0.5 mm of the tip. The curvature was determined at the point of growth axis 0.2 mm basal to the tip (white broken line) against the gravity vector (black broken line). A shoot growing vertically upwards was measured as 0 degrees and a shoot growing horizontally to the light source was measured as +90 degrees. Note that the etiolated and elongated gametophytic shoot with continuous growth (A1 to A4) showed a smooth phototropic response, while the shoot with inhibited growth (B1 to B4) showed a delayed phototropic response; thus, the latter shoot was not examined in this study. The growth-inhibited shoot, as observed in B1 to B4, was likely to be affected by stress, which induced many protonemata on the shoot (black brackets). Bar: 0.5 mm. (C), A plant after continuous red light irradiation at 0.5 µmol m⁻² s⁻¹ for 72 hr, at a lower magnification, in which the phototropic response of gametophores was determined. Note that in this plant, 5 etiolated and elongated shoots were obtained; of the 5 shoots, 2 grew within the medium (black arrowheads), and 3 grew out of the medium, including 2 continuously growing gametophores (red circles) and a growth-inhibited gametophore (orange circle). Bar: 5 mm. All the pictures were taken under dim green light.

Phototropic bending and elongation take place within an apical 0.5-mm-long region

First, we used time-lapse measurements to check which region of the gametophores was responsible for phototropic bending and elongation. We found that both bending and elongation occurred within the apical 0.5-mm-long region under red, far-red, or blue light (Fig. 1A and B). Moreover, increase in leaf size, another photomorphogenic response, was also restricted to this region (Fig. 1A and B).

Red, far-red, and blue light effectively induce phototropism in gametophytic shoots

To examine the response of phototropism to different wavelengths and intensities of light, gametophores were continuously irradiated with unilateral red, far-red, or blue light at different light fluence rates and the fluence rate-response curve was determined for each color. This showed that red and far-red light effectively induced phototropic bending in gametophytic shoots, while blue light induced smaller and less certain responses, of which P values were between 0.05 and 0.1 (Fig. 2A). We also examined growth increment of gametophores during 48-hr of phototropic measurement. Generally, we observed similar growth rates under each light condition except in blue light, which slightly inhibited elongation of gametophores (Fig. 2B). Consistent with the above fluence rate-response curve, red light induced the fastest response, with maximum bending rates of about 2.5 degrees hr⁻¹ (Fig. 2C). Blue light showed the slowest response, which was first detectable 48 hr after onset of irradiation (Fig. 2C). Red and blue light also induced the increase in leaf size more effectively than far-red light (not shown).

Figure 2.

Phototropic bending and growth of etiolated and elongated gametophytic shoots during irradiation with unilateral red, far-red, or blue light. The fluence rate-response curves of curvature (A) and growth increment (B) were determined 48 hr after onset of unilateral light irradiation. (C), The time-course of phototropic curvature at 10 µmol m⁻² s⁻¹. The data shown at each point are means ± SD from 3 to 6 gametophytic shoots. The asterisk shows a statistically significant difference between light irradiated and dark-cultured shoots (A and B) and between before and after irradiation (C) (*, 0.05 < P < 0.1; **, P < 0.05 by Student's t-test).

Discussion

To allow us to quantitatively examine phototropism, we produced etiolated and elongated gametophores of P. patens. In our study, we found that keeping the gametophores moist was critical for obtaining etiolated, elongated shoots in the dark. By improving the culture method, we succeeded in obtaining enough etiolated and elongated samples to allow us to conduct experiments under multiple conditions and with robust statistical analysis. Consistent with our observations, gametophores cultured on horizontal agar plates, where they stood upright in the air, sometimes did not show significant growth.¹³ Our method of culturing etiolated and elongated gametophores (Fig. 1C) will be useful for researchers interested in the physiology and development of gametophytic shoots. However, with the current method, about a half of the etiolated and elongated gametophores that grew out of the medium in the dark showed growth inhibition (Fig. 1B), suggesting that growth conditions varied, even in a single plate. Thus, further effort will be necessary to improve the culture conditions in the dark. In this regard, a choice of medium also needs to be considered, since BCDATG agar medium that we used contains ammonium tartrate, which tends to reduce gametophore production¹⁰. Alternatively, gametophores with small leaves and an elongated stem, which are obtained under continuous far-red light,¹³ could be used for phototropic studies.

With respect to light quality and induction of phototropism in protonemata, bryophytes and pteridophytes, including mosses, liverworts and ferns show a great diversity, with red, far-red, or blue light effective in certain plant species. In the liverwort Marchantia polymorpha, blue light effectively induces phototropism of protonemata, but red light does not.¹⁴ In the mosses, P. patens^2,15 and Physcomitrium turbinatum,¹⁶ both red and far-red light induce phototropism, but blue light induces no phototropism in P. patens⁸ and in P. turbinatum.¹⁶ In the moss Ceratodon purpureus, red light induces positive phototropism and far-red light does not; by contrast, blue light markedly randomizes the growth angle of the protonemata.^17,18 In the fern Adiantum capillus-veneris,^19,20 red and blue light induce phototropism, but far-red light does not. In another fern Schizaea pusilla either red, far-red, or blue light induces negative phototropism.²¹ This diversity of responses indicates divergence in downstream signaling in different species.

To our knowledge, only a few examples of light-mediated tropic responses in gametophores in bryophytes and pteridophytes have been reported, one in the thalli of M. polymorpha²² and the others in the gametophytic shoots of P. patens.^12,23 In the case of M. polymorpha, far-red light induces upward growth in the tip of gametophytic thalli, which grow horizontally on the surface of the medium under white light. This response can be reversed by red light, indicating that phytochrome is involved.²² However, whether such upward growth of thalli tips is a phototropic response remains unknown, because the thalli were not irradiated unilaterally.²² Here, we have demonstrated that, as in protonemata,² far-red light induces positive phototropism in P. patens gametophores (Fig. 2A). We also demonstrated that the phototropic effectiveness of red and blue light differs in gametophores and protonemata. In protonemata, red light induces an avoidance response at light fluence rates above 5 µmol m⁻² s⁻¹,⁸ but in gametophores, red light only induces positive responses in the higher range of fluence rates examined (Fig. 2A). Furthermore, blue light does not induce tropism in protonemata,⁸ but randomizes the direction of growth in dark-adapted protonemata (unpublished data). By contrast, in gametophores, blue light appears to induce a weak positive phototropism when they are irradiated at higher fluence rates and for as long as 48 hr (Fig. 2A). Such differences in phototropic response between gametophores and protonemata suggest that the light-signaling components involved might be distinct between the 2 organs.

Considering that phytochromes function as the photoreceptors for phototropism toward continuous red light^8,9 and far-red light^2,15 in protonemata of P. patens, phytochromes likely also function in phototropism of gametophores in response to red and far-red light. Because the fluence rate-response curves of gametophores differ between red/far-red light and blue light responses, phytochrome likely does not function as the photoreceptor of blue light-induced phototropism. The P. patens genome does not contain genes encoding neochrome^3,6 or Zeitlupe family proteins,²⁴ which could act as blue-light photoreceptors. In Arabidopsis thaliana hypocotyls, phototropism and randomization of growth occur through phototropin and cryptochrome, respectively,²⁵ suggesting that phototropin may act in blue light-induced phototropism of gametophores in P. patens. At present, phototropin²⁶ and cryptochrome²⁷ mutants, as well as phytochrome⁸ and phytochrome chromophore⁹ deficient mutants are available in P. patens; thus one promising avenue for future research will be to conclusively identify the photoreceptor for phototropism for each color in gametophores. Besides photoreceptors, characterization of other signaling components including ptrA, ptrB and ptrC¹⁰ as well as PIN²³ proteins will be necessary to understand the mechanisms of phototropism in gametophores.

Compared to hypocotyls and coleoptiles of angiosperms, which show faster phototropic bending rates of about 10 degrees hr⁻¹,¹ moss gametophores showed a slower phototropic response, with maximum bending rates of 2.5 degrees hr⁻¹ (Fig. 2C). Generally, bending rates may depend on the position of the bending site and/or the rates of elongation. The farther the bending region is apart from the apical tip where the growth angle is measured, the faster the growth angle tends to change. However, gametophores bend in the apical 0.5-mm region (Fig. 1A), which is not so different from the apical 1.1-mm region of the hypocotyl in A. thaliana.²⁸ By contrast, the growth rates of 0.01–0.03 mm hr⁻¹ in P. patens gametophores (Fig. 2B) were much slower than the growth rates of about 0.3 mm hr⁻¹ in A. thaliana hypocotyls.²⁹ Thus, the slow bending rate might be caused by the slow elongation rate in gametophores. If this is the case, it may be concluded that the 2 multicellular organs, gametophores and hypocotyls, have similar bending mechanisms.

How did ancient shoots respond to light changes? The shoot is inferred to have evolved in land plants.⁷ While plants mainly use red and blue light for photosynthesis, how early land plants gained the ability to find favorable light environments through shoot phototropism remains a fundamental question in evolution. In this study, we found that red, far-red, or blue light induces phototropism in gametophytic shoots of the moss P. patens, suggesting that plants used a broad range of light wavelengths as phototropic signals in the early evolution of the shoot system. In line with this, while blue light has well-known functions in inducing phototropic response in shoots of angiosperms through phototropin,¹ red and far-red light-mediated phototropism through phytochrome sometimes occurs in particular organs in specific conditions, such as in the mesocotyl of maize,³⁰ light-grown cucumber hypocotyl³¹ and A. thaliana hypocotyl in microgravity conditions.³² So we speculate that the modern angiosperms might have lost the use of red or far-red light as phototropic signals during evolution.

Materials and Methods

Plant materials and growth conditions

To obtain etiolated and elongated gametophytic shoots, protonemal tissue of Physcomitrella patens (Hedw.) Bruch & Schimp subsp. patens³³ were cultured in BCDATG agar medium³⁴ in a 9-cm Petri dish sealed with surgical tape at 22°C. In each plate, 6 to 8 plants were inoculated and grown under continuous white light (~50 µmol m⁻² s⁻¹) provided by 2 40-W white fluorescent tubes (FLR40S-EX-N/M/36; Mitsubishi-Osram, Yokohama, Japan) for 5 d. The plates were then oriented vertically and the resultant gametophytic shoots were grown under weaker white light (~10 µmol m⁻² s⁻¹) for 7 d at 22°C. Then, 3 quarters of the perimeter of the plates was sealed with black vinyl tape (No. 121; Nichiban, Tokyo, Japan), which kept water inside the medium from leaking but allowed exchange of air. The sealed plates were wrapped with sheets of black paper and aluminum foil and kept vertically in the dark for ~4 weeks at 25°C.

Time-lapse measurements of phototropic responses

The etiolated and elongated gametophytic shoots in vertically oriented Petri dishes were unilaterally irradiated with blue (λmax = 470 ± 30 nm; Stick-mB, Tokyo Rikakikai, Tokyo, Japan), red (λmax = 660 ± 20 nm; Stick-mR), or far-red (λmax = 735 ± 25 nm; Stick-mFR) light-emitting diodes through the side of the Petri dish that was covered only with surgical tape. Time-lapse imaging was conducted using a microscope (SZX12; Olympus, Japan) equipped with a digital camera (DMC-G2; Panasonic, Tokyo, Japan) under dim green light obtained with a green filter (No. 362; Mitsubishi, Tokyo). After taking pictures at different time points, the plates were put back into the original vertical position. For each measurement, at least 3 gametophores were examined. Curvatures were measured from images at the point of growth axis 0.2 mm basal to the tip against the direction of the gravity vector (Fig. 1A and B) by ImageJ software. The bending rates were calculated at the time point when a significant phototropic response began to be observed after onset of unilateral light irradiation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Takayuki Kohchi and Dr. Ryuichi Nishihama of Kyoto University, Dr. Masamitsu Wada of Kyushu University and Dr. Tatsuya Sakai of Niigata University for their helpful discussion. L. B. thanks for Hokkaido University Special Grant Program and Monbukagakusho Honors Scholarship for Privately Financed International Students (Japan Student Services Organization).

Funding

This work was supported, in part, by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS) to T. F.