Subcellular localization and turnover of Arabidopsis phototropin 1

Abstract

We reported recently that internalization of the plant blue light receptor phototropin 1 (phot1) from the plasma membrane in response to irradiation is reliant on receptor autophosphorylation. Pharmacological interference and co-immunoprecipitation analyses also indicated that light-induced internalization of phot1 involves clathrin-dependent processes. Here, we describe additional pharmacological studies that impact the subcellular localization and trafficking of Arabidopsis phot1. Alterations in the microtububle cytoskeleton led to dramatic differences in phot1 localization and function. Likewise, inhibition of phosphatidic acid (PA) signaling was found to impair phot1 localization and function. However, action of PA inhibition on phot1 function may be attributed to pleiotropic effects on cell growth. While phot1 kinase activation is necessary to stimulate its internalization, autophosphorylation is not required for phot1 turnover in response to prolonged blue light irradiation. The implications of these findings in regard to phot1 localization and function are discussed.

A Possible Role for Microtubules in Phot1 Localization and Function

Phot1 is the principle photoreceptor for phototropism in Arabidopsis¹ and is tightly associated with the plasma membrane in darkness.² Upon irradiation, a fraction of phot1 is internalized from the plasma membrane into, as yet unidentified, cytosolic structures.^3–5 To date, it is not known whether light-induced receptor trafficking represents a mode of phot1 signaling or is associated with some other process such as receptor desensitization and adaptation.

Microtubules have been implicated in the differential growth response that underlies tropic responses in plants, including phototropism and gravitropism (reviewed in refs. ⁶ and ⁷). Treatment of etiolated transgenic Arabidopsis expressing functional phot1-GFP with 10 µM oryzalin for 1 hour caused partial receptor internalization from the plasma membrane in darkness (Fig. 1A). The high-intensity blue light laser used to excite GFP promoted further internalization of phot1 (Fig. 1B), whereas equivalent concentrations of oryzalin had no effect on the plasma membrane marker fusion GFP-Lti6b (data not shown), consistent with previous reports.⁸

Figure 1.

Pharmacological interference of phot1-GFP localization in etiolated Arabidopsis hypocotyls. (A) Oryzalin (10 µM) induces phot1-GFP relocalization in darkness (as shown by the white arrows). (B) High-intensity-laser confocal scanning with blue light further enhances phot1-GFP internalization in oryzalin treated seedlings. (C) 1-Butanol (0.8%) induces phot1-GFP relocalization in darkness. (D) 2-Butanol does not impact phot1-GFP localization in darkness.

Oryzalin causes depolymerization of microtubules and was found to impair the phototropic responsiveness of etiolated Arabidopsis seedlings relative to control samples treated with DMSO (Fig. 2A). These findings are intriguing given that phot1 is associated with microtubules in Arabidopsis in vivo.⁵ Microtubule depolymerization has also been reported to inhibit gravitropism and impact the phototropic response of maize coleoptiles.⁹ Additionally, oryzalin has been shown to influence the subcellular trafficking of PIN auxin efflux carriers, including PIN1,¹⁰ which is implicated in phototropism.^11,12 Further investigation of the mircotubule cytoskeleton will help to clarify its role in phototropism and other phototropin-mediated responses. Microtubules may aid cell elongation by possibly guiding the deposition of cellulose microfibrils into the cell wall (reviewed in ref. 7).

Figure 2.

Pharmacological interference of phot1 function. (A) Effects of oryzalin and 1-butanol on phot1-mediated phototropism to low intensity blue light (1 µmol m-2 s-1). Seedlings were grown on agar plates for 2.5 days then transferred to plates containing either oryzalin (10 µM), 1-butanol (0.8%), 2-butanol (0.8%) or DM SO (10 µM) for 3 hours prior to unilateral blue light irradiation for 10 hours. Each curvature value is the mean of at least 20 seedlings. Standard errors are shown. (B) Representative images of Arabidopsis seedlings treated with 1-butanol and 2-butanol.

Impact of Phospholipase D Signaling on Phot1 Localization and Function

Phospholipase D (PLD) hydrolyzes membrane lipids to generate the important second messenger phosphatidic acid (PA). PLD activity is widespread in plants and has been shown to be involved in a variety of processes including vesicle trafficking, seedling development, pollen tube growth and abscisic acid-induced stomatal closure (reviewed in ref. 13). 1-Butanol is an inhibitor of PA production and competes with water as a substrate for PLD transphosphatidylation activity, resulting in the formation of phosphatidylbutanol rather than PA. 2-butanol is commonly used as a negative control since it is not a substrate for PLD.

Treatment of etiolated Arabidopsis seedlings with 0.8% 1-butanol for 1 hour resulted in marked internalization of phot1-GFP from the plasma membrane (Fig. 1C). By contrast, 2-butanol was without effect (Fig. 1D). As well as inhibiting PLD activity, 1-butanol has been shown to cause microtubule depolymerization and affect actin filament structure.¹⁴ As found for oryzalin, an equivalent concentration of 1-butanol impaired phototropism in etiolated Arabidopsis seedlings, whereas 2-butanol had no effect (Fig. 2A). Oryzalin and 1-butanol may therefore exert their effects on phot1 function and localization through alterations in the cytoskeleton. Some isoforms of PLD are known to control microtubule orientation by promoting their interaction with the plasma membrane.¹⁵ However, 1-butanol treatment has been reported to inhibit the growth of Arabidopsis seedlings.¹⁴ Indeed, less signs of de-etiolation (cotyledon opening and greening) were observed in seedlings treated with 1-butanol following 10-hour exposure to unilateral blue light compared to samples treated with 2-butanol (Fig. 2B). While these findings could account for the reduced phototropism observed, the rapid effects of 1-butanol on phot1-GFP localization are still of interest.

Phot1 Turnover by Blue Light is not Mediated by Receptor Autophosphorylation

One possible function of phot1 internalization is that its serves to attenuate receptor signaling. In this regard, the enhanced internalization observed for phot1-GFP upon treatment with either oryzalin or 1-butanol (Fig. 1) may partly account for reduced phototropic responsiveness (Fig. 2). Autophosphorylation of phot1 within the kinase activation loop is required to stimulate its trafficking from the plasma membrane.⁵ We therefore examined whether inhibitor treatment had any effect on phot1 autophosphorylation. The effects of 1-butanol were investigated since it had a more pronounced effect on phot1 localization (Fig. 1). However, 1-butanol did not appear to alter the phosphorylation status of phot1 relative to control treated samples, as monitored by its change in electrophoretic mobility upon irradiation (Fig. 3A). These data suggest that enhanced internalization of phot1-GFP in response to 1-butanol does not result from increased receptor kinase activity in the absence of light.

Figure 3.

Phot1 autophosphorylation and receptor turnover by blue light. (A) Western blot of phot1 in protein extracts isolated from 3-day-old etiolated Arabidopsis seedlings treated with 1-butanol or 2-butanol for 2.5 hours. Seedlings were either kept in darkness (D) or irradiated with blue light (BL, 50 µmol m-2 s-1) for a further 30 minutes prior to harvest. The dashed line indicates the lowest mobility edge of phot1. (B) Prolonged exposure (16 hours) to blue light (50 µmol m-2 s-1) results in phot1 degradation in wild-type seedlings and in seedlings expressing a kinase inactive mutant (D806N) of phot1. Ponceau S staining of the total protein extracts is shown below as a loading control.

Prolonged exposure of phot1 to blue light results in its degradation² that most likely reflects a long-term adaptation process. Retrieval of phot1 from the plasma membrane may therefore lead to signal attenuation through its targeted degradation. Whilst the phosphorylation status of phot1 impacts its subcellular localization,⁵ there is currently no evidence linking phot1 autophosphorylation to receptor turnover. Surprisingly, a mutant allele of PHOT1 carrying a single point mutation D806N that is known to abolish phot1 kinase activity^16,17 still showed phot1 turnover in response to long-term blue light exposure despite being expressed at a lower level than wild type (Fig. 3B). These findings suggest that turnover of phot1 is initiated by some means other than receptor autophosphorylation and internalization.

Future Perspectives

The functional significance of phot1 internalization by blue light is still not fully understood, as is that of its paralogue phototropin 2.^18–20 Yet, this unique mode of plant photoreceptor trafficking offers a non-invasive system to study subcellular protein movements from the plasma membrane. So far, the subcellular trafficking of phototropins has been studied through the use of translation fusions to GFP, the excitation of which overlaps with photoactivation of these flavoprotein photoreceptors. Thus, tagging of phototropins with fluorescent reporters that are excited at longer wavelengths will extend the conditions under which these receptors can be imaged without simultaneously causing their photoactivation and movement.

Abbreviations

GFP

green fluorescent protein

DMSO

dimethyl sulfoxide

PLD

phospholipase D

PA

phosphatidic acid

Addendum to: Kaislerli E, Sullivan S, Jones MA, Feeney KA, Christie JM. Domain swapping to assess the mechanistic basis of phot1 receptor kinase activation and endocytosis by blue light. Plant Cell. 2009;10:3226–3244. doi: 10.1105/tpc.109.067876.