Involvement of PP6-type protein phosphatase in hypocotyl phototropism in Arabidopsis seedlings

ABSTRACT

Recently, we reported that the D6 protein kinase subfamily, which belongs to the AGCVIII kinase family, is a critical component of hypocotyl phototropism in Arabidopsis seedlings. Furthermore, we demonstrated that AGC1-12, which is also a member of the AGCVIII kinase family, is involved in both the pulse-induced first positive phototropism and gravitropism in Arabidopsis hypocotyls. Those results indicated that phosphorylation control is an important mechanism in phototropic signaling. As phosphorylation regulation is controlled by both kinases and phosphatases, we investigated the roles of phosphatases in hypocotyl phototropism. Our physiological analysis, which was performed using Arabidopsis mutants, indicated that the flower-specific, phytochrome-associated protein phosphatase family, which functions as a catalytic subunit of protein phosphatase 6 (PP6), is involved in both the pulse-induced first positive phototropism and the time-dependent second positive phototropism, although it is not necessary for the continuous-light-induced second positive phototropism. These results suggest that not only kinases, but also phosphatases play critical roles in hypocotyl phototropism to control phosphorylation status and that PP6-type protein phosphatases may act antagonistically with AGCVIII protein kinases on the same targets, such as PIN-formed proteins.

The regulation of phosphorylation status is very important in the control of many cellular events. We reported recently that the D6 protein kinase (D6PK) family, which are plant-specific AGCVIII kinases, is involved in not only the second positive phototropism, but also the first positive phototropism in Arabidopsis hypocotyls.¹ Furthermore, we found that AGC1-12, another AGCVIII kinase, is also necessary for the first positive hypocotyl phototropism.¹ Those results indicate that the phosphorylation status is critical for phototropic responses, and suggest that phosphatases are involved in phototropic responses since they act antagonistically to protein kinases. However, the physiological roles of phosphatases in hypocotyl phototropism are not well understood. Therefore, we analyzed phototropic responses in Arabidopsis phosphatase mutants.

Two potent candidates were addressed in this study. The first was root curling in N-naphthylphthalamic acid 1 (RCN1/PP2AA1), which was identified previously as the A1 regulatory subunit of the Ser/Thr protein phosphatase 2A (PP2A). RCN1 interacts with phototropin 2 (phot2) which is one of photoreceptors responsible for hypocotyl phototropism induced by strong blue light. Moreover, PP2A dephosphorylates phot2 proteins, implying that PP2A is involved in the regulation of the blue-light-mediated phosphorylation of phot2 proteins.² The other candidate phototropism regulator examined here was protein phosphatase 6 (PP6), which is involved in light signaling. PP6 is a heterotrimeric complex containing the catalytic subunit flower-specific, phytochrome-associated protein phosphatase 1 (FyPP1) or FyPP3, the regulatory subunit SAPS domain-like protein (SAL1-3), and the PP2AA protein (RCN1/PP2AA1, PP2AA2, or PP2AA3).³ The FyPP genes were originally identified as phytochrome-interacting factors.⁴ The fypp1 fypp3 double mutant lacks PP6 activity and shows abnormalities of phosphorylation status and subcellular localization of PIN proteins.^3,5 Although PP6 also contains the PP2AA subunit as well as PP2A and appears to control polar auxin transport,³ the physiological function of PP6 in phototropic responses has not been clarified.

To evaluate the roles of the two candidates in hypocotyl phototropism, we first investigated the pulse-induced first positive phototropism (Figure 1A). Two-day-old dark-grown Arabidopsis seedlings were irradiated with a unilateral pulse of blue light at the optimum total fluence (0.0017 μmol m⁻² s⁻¹ for 1 min)⁶ and incubated under complete darkness for 3 h to develop phototropic curvatures. Under such conditions, wild-type hypocotyls showed curvatures of ~20° (Figure 1A, left panel). The pulse-induced phototropism was impaired in the fypp single mutants and in the fypp1 fypp3 double mutant, although such phototropic impairment was not observed in the rcn1-2 mutant. Red-light pretreatment enhanced the pulse-induced phototropism in the wild-type hypocotyls and in the rcn1-2 mutant (Figure 1A, right panel). Although such enhancement was also observed in the fypp mutants, phototropic responses were impaired in the fypp single mutants and were severely affected in the fypp1 fypp3 double mutant. These results indicate that the FyPP family, but not RCN1, is a critical component of the pulse-induced hypocotyl phototropism, and that FyPP1 and FyPP3 function redundantly in phototropic signaling.

Figure 1.

Hypocotyl phototropism in phosphatase mutants.

Two-day-old dark-grown seedlings of the fypp1 mutant, fypp3 mutant, fypp1 fypp3 double mutant, and rcn1-2 mutant were used. (A) Pulse-induced first positive phototropism. The seedlings were pretreated with (right panel) or without (left panel) an overhead red light (RL) at 20 μmol m⁻² s⁻¹ for 2 min. Two hours after the pretreatment, the hypocotyls were stimulated with a unilateral blue light (BL) at the optimum fluence (left panel, 0.0017 μmol m⁻² s⁻¹ for 1 min; right panel, 0.017 μmol m⁻² s⁻¹ for 1 min). The hypocotyl curvatures were determined 3 h after the onset of blue light. The data shown are the means ±SE from 13–16 seedlings. The asterisk indicates a statistically significant difference between the wild-type and the mutant hypocotyls (Student’s t test, *P < 0.05, **P < 0.01 and ***P < 0.001). (B) Time-dependent and continuous-light-induced second positive phototropism. Two hours after the red-light pretreatment, the hypocotyls were stimulated with a unilateral blue light at 0.17 μmol m⁻² s⁻¹ for 27 min (left panel) and 3 h (right panel), respectively. The hypocotyl curvatures were determined 3 h after the onset of blue light. The data shown are the mean ± SE from 9–16 seedlings.

Next, we investigated the second positive hypocotyl phototropism (Figure 1B). To induce time-dependent second positive phototropism, dark-grown seedlings that had been pretreated with an overhead red light were irradiated with a unilateral blue light at 0.17 μmol m⁻² s⁻¹ for 27 min and incubated under complete darkness for ~2.5 h (Figure 1B, left panel). Under these conditions, the wild-type hypocotyls showed time-dependent phototropism (curvature >70°). The rcn1-2 mutant did not exhibit significant impairment of the phototropic responses. The fypp1 and fypp3 single mutants, however, displayed significant phototropic defects, which were further enhanced in the fypp1 fypp3 double mutant (Figure 1B, left panel). These results suggest that the FyPP family is an essential component not only of pulse-induced phototropism, but also of the time-dependent second positive phototropism. Finally, we investigated the continuous light-induced second positive phototropism. Pretreatment of wild-type seedlings with red light followed by stimulation with a unilateral blue light at 0.17 μmol m⁻² s⁻¹ for 3 h led to the bending of the hypocotyl to a nearly horizontal position (Figure 1B, right panel). Neither the rcn1-2 nor the fypp single and double mutants show significant defects in hypocotyl phototropism. These results suggest that both RCN1 and the FyPP family are not necessary for continuous light-induced phototropism.

Our study suggests that not only protein kinases, but also protein phosphatases play critical roles in phototropic signaling and that phosphatases may act antagonistically with protein kinases on the same targets. The present study demonstrated that phototropic responses were partially attenuated in fypp mutants (Figure 1). FyPP is the catalytic subunit of PP6 protein phosphatases and our results suggest that PP6 functions as a positive regulator of phototropic signaling. The FyPP family is reported to target and dephosphorylate PIN proteins in controlling their membrane localization.^3,7 Our previous analysis using transgenic plants harboring the cauliflower mosaic virus 35S-promoter-driven PID gene suggests that this disturbance, which may result in stronger or aberrant PIN protein phosphorylation, impacts hypocotyl phototropism negatively.⁸ Therefore, the proper control of the phosphorylation status of PIN proteins by the AGCVIII family and PP6 proteins must be important for phototropic responses.

Our analysis also showed that hypocotyl phototropism, which is induced by relatively weak blue light, is not impaired in the rcn1-2 mutant (Figure 1). This is consistent with the results of a previous study, which indicated that RCN1/PP2AA1 does not participate in phot1-mediated phototropic responses.² Although PP6 appears to be in a heterotrimeric complex with FyPP1/3, SAL, and PP2AA,³ other proteins, such as PP2AA, PP2AA2, and/or PP2AA3, may be able to control the FyPP1/3 enzymatic activities in phot1-mediated phototropic responses. Alternatively, it is also possible that FyPP functions in a dimer complex with SAL as found for other eukaryotes, implying that PP2A scaffolding subunits may not necessarily be required here. On the other hand, it is still not known how phot-mediated phototropic signal transduction participates in the functional regulation of the PP6 proteins to induce phototropic responses. Alternatively, phot may not control the function of phosphatases directly, and it is possible that early phototropic signaling is not mediated by such phosphatases. Further studies are necessary to clarify the functional regulation and physiological significance of the phosphorylation status in phototropic responses.

Abbreviations

AGC

protein kinase A, cGMP-dependent protein kinase and protein kinase C

D6PK

D6 protein kinase

FyPP

flower-specific, phytochrome-associated protein phosphatase

phot

phototropin

PIN

PIN-formed

PID

PINOID

PP2A

protein phosphatase 2A

PP6

protein phosphatase 6

RCN

root curling in N-naphthylphthalamic acid

SAL

SAPS domain-like protein

Funding Statement

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No. 24657027 and No. 17K07451 to K.H.; No. 17H03694 to T.S.), and a Grant-in-Aid for Scientific Research on Innovative Areas (No. 16H01231) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to T.S.). This work was also supported (in part) by a Grant for Basic Science Research Projects from the Sumitomo Foundation (to K.H.), the Itoh Science Foundation (to T.S.) and the Sasaki Environmental Technology Foundation (to T.S.).

Acknowledgments

We thank the Arabidopsis Biological Resource Center for providing the rcn1-2 (Salk_059903) mutant. We also thank Professor H. Wang (Yale University) for providing the fypp1 (CS874166) and fypp3 (CS877364) single mutants, as well as the fypp1 fypp3 double mutant.