Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction

Highlights

• Red light triggers an acute cytosolic Ca²⁺ increase through photoreceptor phyB

• CPK6/12 interact with and phosphorylate phyB depending on both Ca²⁺ and light

• CPK6/12 regulate phyB nuclear import to promote cotyledon opening and expansion

• S80/S106 phosphorylation is required for and generally controls phyB nuclear import

Summary

Although Ca²⁺ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca²⁺ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca²⁺ increases via phyB, which are sensed by Ca²⁺-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca²⁺ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyB^S80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyB^S80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca²⁺ increases to specific responses in sensory stimulus processing.

Introduction

Ca²⁺ is the most versatile intracellular second messenger and is ubiquitously involved in numerous stimulus-specific biological processes. How the paradoxical versatility and specificity of Ca²⁺-mediated signaling are achieved has been a long-standing puzzle.^1,2,3 In plants, changes in the cytosolic calcium concentration ([Ca²⁺]_cyt) have been implicated in the transmission of diverse responses to environmental stresses. For instance, a variety of stimuli, such as soil salinity, touch, drought, extreme temperature, or herbivore attack, evoke an acute, transient [Ca²⁺]_cyt increase, which is sensed and relayed into distinct physiological outputs.^{4,5,6,7,8,9,10} The fundamental question of how the specificity of a particular signaling pathway is defined by [Ca²⁺]_cyt transients then arises.

Light is a critical environmental factor that exerts a wide range of biological effects. Decades ago, red light has been reported to induce [Ca²⁺]_cyt increases in algae and in protoplasts.^11,12,13 Early biochemical evidence further shows that microinjecting Ca²⁺ into tomato hypocotyl cells promotes light-responsive gene activation,^14,15 implicating Ca²⁺ in light signaling. Although it has been well established that Ca²⁺ functions as an obligatory intermediate in the cascade of visual transduction and photoreceptor light adaptation in animals,¹⁶ the lack of putative target proteins of Ca²⁺ or any genetic evidence means that the mechanism underlying Ca²⁺ signaling in plant phototransduction remains unidentified.

Plants utilize light as a source of both energy and information cues about their surrounding environment. After geminating in subterranean darkness, plant seedlings undergo etiolated growth. Light triggers a dramatic transition from skotomorphogenic to photomorphogenic development, termed de-etiolation, when plants emerge from the soil.^17,18 The light signals initiating this vital transition are primarily perceived by photoreceptor phytochromes (phys, phyA through phyE in Arabidopsis).^19,20,21,22 As the dominant red light photoreceptor, phyB is characterized by switching between two photoreversible conformers.^{23,24,25,26,27} In dark-grown seedlings, phyB proteins accumulate in the cytoplasm in their biologically inactive red-light-absorbing form (Pr).^23,24,28 Upon light activation, phyB proteins are photoconverted into their biologically active far-red-light-absorbing form (Pfr) and rapidly translocate into the nucleus, where they directly interact with and induce the degradation of transcription factors (TFs) to alter gene expression.^{29,30,31,32,33,34} Thus, the light-dependent nuclear import of photoreceptors is an early determinant step in phy signal transduction that delivers light information from its perception in the cytoplasm directly to nuclear events.

Here, we show that red light exposure evokes a robust [Ca²⁺]_cyt increase in seconds, which is stimulated by photoreceptor phyB and sensed by two calcium-dependent protein kinases, CPK6 and CPK12 (CPK6/12). We find that CPK6/12 directly interact with and phosphorylate phyB upon activation by Ca²⁺ and light, respectively. Phosphorylation of phyB at S80 and S106 residues generally determines the nuclear translocation of phyB, in which CPK6/12 play a predominant role during initial light exposure of etiolated seedlings. This phyB-Ca²⁺-CPK6/12-phyB regulatory loop triggers stimulus-specific responses by coordinating cytosolic Ca²⁺ signaling and light information into the phosphorylation and nuclear import of phyB, thus providing insights into the mechanisms of phyB nuclear translocation and the action of calcium in plant light signaling.