Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Abstract

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.

Abbreviations

LOV

light, oxygen, or voltage

FMN

flavin mononucleotide

phy-module

light-sensing module of phytochrome

phot-module

light-sensing module of phototropin

Most plants bend toward the incident light in a response that is regulated by phototropins.¹ Phototropin is a blue light receptor composed of tandemly repeated LOV (light, oxygen, or voltage) domains (LOV1, LOV2) and a Ser/Thr protein kinase domain.² Each LOV domain contains a conserved cysteine residue that binds to a chromophore flavin mononucleotide (FMN). In response to blue light irradiation, FMN forms a cysteinyl-adduct in both LOV domains³ and subsequent conformational changes of LOV2 activate the protein kinase domain.⁴ In Arabidopsis thaliana, this intramolecular signaling pathway induces the hypocotyl phototropic response.⁵

phy3 is an unconventional photoreceptor in the fern Adiantum capillus-veneris^6,7 consisting of an almost full-length phototropin region and an N-terminal extension of a phytochrome chromophore-binding domain. This chimeric photoreceptor mediates both red- and blue-light-induced phototropic responses, and has two different light-sensing modules; a light-sensing module of phytochrome (phy-module) and a light-sensing module of phototropin (phot-module).^8,9 Each light-sensing module contains the invariant cysteine residue required for the covalent attachment of the chromophore (cysteine 303: C303 of a phy-module; C712 in LOV1 and C966 in LOV2 of a phot-module). To assess the role of each light-sensing module in the phototropic response, we expressed cysteine-substituted phy3 variants (Cys303 to Ser: phy3-C303S; Cys712 to Ala: phy3-C712A; Cys966 to Ala: phy3-C966A; both Cys712 and 966 to Ala: phy3-C712AC966A) in phototropin-deficient Arabidopsis (phot1 phot2) and monitored the phototropic response in the transgenic seedlings.^9,10 Recently, we reported that blue light is sensed by both the phy-module and phot-module of phy3, and that each light-sensing module can independently lead to a blue light-dependent phototropic response in transgenic phototropin-deficient Arabidopsis seedlings.¹⁰ Irradiation with 5 μmol m⁻² s⁻¹ blue light resulted in comparable hypocotyl bending in plants expressing the cysteine-substituted phy3 variants and in those expressing intact phy3. These results demonstrate that, at 5 μmol m⁻² s⁻¹ blue light, the phy-module and phot-module work redundantly to induce hypocotyl phototropic bending. By contrast, transgenic seedlings expressing phy3-C712AC966A showed little phototropic response upon irradiation with 1 μmol m⁻² s⁻¹ blue light. This defect in the blue light-dependent phototropic response is likely due to the loss of phot-module function, and we hypothesized that the phot-module of phy3 plays a primary role in phototropism under relatively low light conditions.¹⁰ To test this hypothesis, we evaluated the phy3-C303S-mediated phototropic response at 1 μmol m⁻² s⁻¹ blue light (Fig. 1). At this fluence rate of blue light, phy3-C303S transgenic seedlings exhibited a phototropic response. Therefore, the phot-module of phy3 captures and transmits blue light, thereby inducing hypocotyl bending, when irradiated with 1 μmol m⁻² s⁻¹ of blue light, as hypothesized above. In addition, at the same fluence rate of blue light, phy3-C712A-expressing transgenic plants showed almost the same hypocotyl curvature as control plants expressing intact phy3; however, phy3-C966A-expressing seedlings did not show a notable phototropic response (Fig. 1). These results are in agreement with a previous report showing that the LOV2 domain of Arabidopsis phot1 is essential for hypocotyl phototropism and that phot1 predominantly senses light via LOV2.¹¹ Thus, the region of phy3 homologous to phototropin appears to operate via a similar intramolecular signaling pathway as do Arabidopsis phototropins.

Figure 1.

Phototropic responses of transgenic Arabidopsis seedlings expressing wild-type or mutated forms (phy3-C303S, phy3-C712A, phy3-C966A, and phy3-C712AC966A) of Adiantum phy3, and phot1 phot2 of Arabidopsis. The seedlings were grown in darkness for 3 days and irradiated with unilateral blue light at 1 μmol m⁻² s⁻¹ (A) or 5 μmol m⁻² s⁻¹ (B) for 16 h. At the end of the irradiation period, phototropic curvatures were measured. Each bar indicates the mean of at least 20 measurements with standard deviations.

As described above, phy3-C303S-expressing plants showed a clear phototropic response when irradiated with 1 μmol m⁻² s⁻¹ blue light, but the hypocotyl curvature was somewhat smaller than that of plants expressing intact phy3. This result suggests that phot-module activity alone is not sufficient for inducing the phy3-mediated blue light-dependent phototropic response at 1 μmol m⁻² s⁻¹. Furthermore, transgenic seedlings expressing phy3-C712AC, which retain both the phy-module and LOV2-module, showed an almost similar phototropic response to those expressing intact phy3 at 1 μmol m⁻² s⁻¹ blue light. These results imply that, to obtain full phy3 activity at this fluence rate of blue light, both the phy-module and phot-module must be present in a single phy3 photoreceptor molecule. Previously, we reported that simultaneous irradiation with red and blue light had a synergistic effect on phy3 activity.⁹ Concurrent irradiation with red and blue light significantly increased the hypocotyl curvature of phy3-expressing transgenic seedlings compared with irradiation with either red or blue light alone. This synergistic effect is prominent under weak light conditions and is thought to be due to the intramolecular co-action of the phy-module and phot-module in phy3. Taken together, it is conceivable that the co-action of two different light-sensing modules in a single photoreceptor molecule allows phy3 to respond to weak light signals. Hence, I propose that this intramolecular co-action of light-sensing modules in phy3 renders the environmental light monitoring systems of ferns hypersensitive to light. Ferns are thought to have acquired this unique photoreceptor gene through horizontal gene transfer from hornworts.¹² Therefore, by acquiring the hypersensitive photoreceptor phy3 during evolution, ferns adapted to low-light conditions and were able to thrive in the shadow of angiosperms.

Funding

This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 20570041, 23570061) to T.K.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

I thank K. Hara for technical assistance.