Protein phosphorylation in stomatal movement

Abstract

As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation.

Abbreviations

AAPK

ABA activated protein kinase

ABA

abscisic acid

ABI

abscisic acid insensitive

AHK5

Arabidopsis histidine kinases 5

AKS

ABA-responsive kinase substrates

BL

blue light

BLUS1

blue light signaling1

CBL

calcineurin-B like proteins

CIPK

CBL-interacting protein kinase

CPK

calcium dependent protein kinase

EPs

epidermal peels

GCPs

guard cell protoplasts

GHR1

guard cell hydrogen peroxide-resistant1

HAB1

homology to ABI1

HXK

hexokinase

HRB1

hypersensitive to red and blue 1

IHC

immunohistochemistry

KAT1

K⁺ channel in A. thaliana 1

LC-MS/MS

liquid chromatography–mass spectrometry

MeJA

methyl jasmonate

MPK

mitogen-activated protein kinase

MAP4K

mitogen-activated protein kinase kinase kinase kinase

NO

nitric oxide

OST1

open stomata 1

PA

phosphatidic acid

PHO1

phosphate1

PP1

protein phosphatase

PP7

protein phosphatase

PRSL1

PP1 regulatory subunit2-like protein1

PTPases

protein tyrosine phosphatases

QUAC1

quickly-activating anion channel 1

RBOH

respiratory burst oxidase homolog

ROS

reactive oxygen species

SLAC1

slow anion channel-associated 1

SnRK2.6

sucrose nonfermenting-1 (Snf1)-related protein kinase 2.6

Introduction

Plants are sessile organisms, and their survival depends on efficient perception and response to the constantly changing environment. A major interface between plants and their surroundings is represented by stomatal pores formed by pairs of highly specialized guard cells. To maximize CO₂ uptake for photosynthesis and at the same time minimize water loss, guard cells sense various signals and adjust the stomatal pore size accordingly.^1-3 How stomatal aperture is adjusted by the rapid movement of guard cells has fascinated plant biologists for decades.

Changes in stomatal aperture are accomplished by osmotic flux in guard cells.^3,4 Under conditions that favor stomatal opening, influx of ions such as K⁺ leads to lowered water potential and water uptake into the guard cells. The increased cell volume and turgor applies mechanical pressure on the cell wall, which in turn enlarges the stomatal aperture. Unfavorable conditions, such as drought and pathogen attack, induce outward movement of ions, such as K⁺ and Cl⁻, across the plasma membrane and reduce guard cell turgor, causing stomatal closure. Channels mediating the inward and outward movement of ions have been identified in guard cells in the past few decades.^3-7 However, mechanisms by which the environmental cues are transduced to activate/deactivate the channels are still not completely understood.

How abscisic acid (ABA) induces stomatal closure and how light induces opening have been extensively studied.^1,3,8,9 ABA is a plant hormone synthesized during drought, and it prevents water loss by inducing rapid stomatal closure.^10,11 ABA triggers a signaling network in guard cells that includes protein kinases and leads to activation of anion channels and the outward movement of anions, resulting in loss of turgor and reduction of the stomatal aperture.^12-17 Blue light triggers stomatal opening in most plants¹⁸ by a signaling network that involves protein kinases and results in the phosphorylation and activation of the H⁺-ATPase in the plasma membrane of the guard cell.^19–23 Loss of H⁺ leads to uptake of K⁺, increase in turgor, and increase of the stomatal aperture. Thus, the mechanisms for regulating stomatal closure and opening each involve distinct proteins kinases that regulate ion traffic across the plasma membrane.

Dynamic changes of phosphorylation of proteins in guard cell signaling have been detected (Table 1). At a large scale, the involvement of protein phosphorylation is supported by a transcriptomic study that identified 689 protein kinases and 113 protein phosphatases in Arabidopsis thaliana guard cell protoplasts (GCPs),²⁴ suggesting that an extensive array of phosphorylation/dephosphorylation events are involved in guard cell signaling. Genetic approaches have also been used to identify an increasing number of key kinases and phosphatases in guard cell signaling (Table 2).

Table 1.

List of selected studies characterizing protein phosphorylation in guard cell signaling

Species	Material	Approach	Significant findings	Ref
V. faba	GCPs	32P label	Identification of AAPK, a homolog of OST1	2,78
V. faba	GCPs	32P label	ABA activates several protein kinases	15
V. faba	GCPs	32P label	A CDPK from guard cell phosphorylates KAT1, an inward K+ channel	79
V. faba	GCPs	32P label	Blue light promotes stomatal opening by activating H+-ATPase via phosphorylation	23
P. sativum	EPs	32P label	Activation of a MAPK is required in ABA signaling	80
V. faba	GCPs	32P label	ABA activate a 48 kDa protein kinase that phosphorylates K+ channel KAT1	16
C. communis	GCPs	PTPase inhibition	PTPases are involved in stomatal movement regulation	81
V. faba	GCPs	32P label	Blue light induces the phosphorylation of phototropins and H+-ATPase	21
A. thaliana	GCPs	32P label LC-MS/MS	Biochemical characterization of OST1	82
V. faba	GCPs	32P label	NO inhibits blue light induced stomatal opening by inhibition of H+-ATPase phosphorylation	83
V. faba	GCPs	32P label	A 61 kDa protein is phosphorylated and bound to 14–3–3 protein in response to ABA	17
A. thaliana	peptide	LC-MS/MS	OST1 interacts with and phosphorylates ROBH F	12
A. thaliana	EPs	inhibitor	K252a-sensitive protein kinases are involved in MeJA induced stomatal closure	84
A. thaliana	EPs	IHC	IHC method is suitable for phosphorylation detection in guard cells	69
A. thaliana	EPs	IHC	ABA inhibition of stomatal opening is impaired in mutant defective in 4 ABA receptors	85
A. thaliana	GCPs	32P label LC-MS/MS	ABA represses AKSs, K+ channel facilitating proteins, by phosphorylation	35

Table 2.

Genetics studies on involvement of kinases and phosphatases in guard cell signaling

Name	Species	Significant finding	Ref
Kinase
MP3	A. thaliana	MPK3 is required for ABA response and it may act downstream of ROS	86
MPK4	N. tabacum	MPK4 silenced plants show greater stomatal conductance and normal ABA response.	55
	N. attenuate	MAP4 silenced plants show enhanced stomatal opening and reduced CO2 sensitivity	56
	N. attenuata	MPK4 silenced plant showed impaired ABA response and higher stomatal conductance	57
	A. thaliana	MPK4 may regulate defense against pathogen by limiting the entry into apoplast	54
MPK9/12	A. thaliana	MPK9 and MPK12 silenced plants show impaired ABA and H2O2 response, and also abolished anion channels activation upon Ca2+	87
MPK12	A. thaliana	MPK12s in different Arabidopsis accessions contribute to different guard cell size and ABA response	51
MPK9/12		Yeast elicitor induced stomatal closure is abolished in mpk9/12 double mutant	88
CPK3/6	A. thaliana	ABA response is impaired in cpk3cpk6 double mutant	36
CPK6	A. thaliana	MeJA-induced stomatal closure is impaired in cpk6 mutant	38
	A. thaliana	Yeast elicitor induced stomatal closure is impaired in cpk6 mutant	37
	A. thaliana	CPK6 phosphorylates and activates SLAC1 in Xenopus Oocytes	60
CPK4/10/11	A. thaliana	Ca2+, but not ABA, induced stomatal closure is impaired in cpk4/11 double and cpk10 mutant	45
CPK10	A. thaliana	cpk10 mutant shows higher sensitivity to drought and CPK overexpression lines shows enhanced drought tolerance	89
CPK21	A. thaliana	Ca2+ activated kinase CPK21 phosphorylates SLAH3 and induces anion currents in Xenopus oocytes	11
CIPK23	A. thaliana	ABA response is enhanced in cipk23 mutant and CBL1/9 acts upstream of CIPK23 to regulate K+ transport in guard cells.	39
BLUS1	A. thaliana	Stomata of blus1 mutants do not open in response to BL and phosphorylation of BLUS1 by phototropins is required for stomatal opening	66
GHR1	A. thaliana	ABA and H2O2 induced stomatal closure is impaired in ghr1 mutant. GHR1 phosphorylate and activate SLAC1.	59
HXK	A. thaliana	Sucrose induced hexokinase promotes stomatal closure	90
AHK5	A. thaliana	ahk5 mutant shows impaired stomatal closure in response to H2O2 and a 2-component system is involved in stomatal closure response	91,92
Phosphatase
PP1	V. faba	PP1 is a positive regulator between phototropins and H+-ATPase in BL-induced stomatal opening	73
	V. faba	ABA induced PA inhibits light-induced stomatal opening via inhibition of PP1 activity	74
PP7	A. thaliana	PP7 dephosphorylates HRB1 and both pp7 and hrb1 mutants show abnormal stomatal movement	76
type A PP2C	A. thaliana	Inhibits the kinase activity of OST1	10,60
PP2A	A. thaliana	Inhibits the kinase activity of phototropin	72
PRSL1	V. faba	PRSL1 is a regulatory subunit of PP1	75

In this review, we highlight the role of protein phosphorylation in stomatal movement. In particular, we focus on the ABA-induced stomatal closure and blue light-induced opening processes. We also discuss kinases recently identified to be involved in stomatal movement and that reveal the complexity of the signaling events in guard cells.

Protein Phosphorylation as a Major Mechanism in Guard Cell ABA Signaling

Overview of ABA signaling in guard cells

The current model of ABA signaling in guard cells is illustrated in Figure 1, which highlights protein phosphorylation events. Identification of the key components in early ABA signaling is one of the most exciting breakthroughs in modern plant biology. The core pathway from A. thaliana has been successfully reconstituted based on knowledge of protein-protein interaction.¹⁰ In the absence of ABA, the protein kinase open stomata 1 (OST1, named for the phenotype of the null mutant upon ABA treatment; also called sucrose nonfermenting-1 (Snf1)-related protein kinase 2.6 or SnRK2.6) is bound to a type A protein phosphatase 2C (abscisic acid insensitive 1 or 2 (ABI1 or ABI2), or homology to ABI1 (HAB1). The activation loop of OST1 is bound to, and thus blocked by, the active site of the PP2C. When ABA is present, it binds to the soluble ABA receptor, pyrabactin resistance/pyrabactin-like/regulatory components of ABA receptor (PYR/PYL/RCAR), which subsequently induces a conformational change of the receptor and promotes its binding to PP2C. This binding inhibits the activity of PP2C and releases OST1 to phosphorylate downstream targets. The phosphorylated proteins include transcription factors and ion channels that play essential roles in triggering and maintaining the stomatal closure.^8,25

Figure 1.

Simplified diagram of guard cell ABA and blue light signaling highlighting protein phosphorylation events. Solid lines represent direct molecular interaction, and dashed lines represent indirect regulation. Arrowheads represent activation and closed arrows indicate inhibition. Kinases are shaded in green and phosphatases in red. Left guard cell: ABA-induced stomatal closure. In the absence of ABA, PP2C binds and represses OST1, a master positive regulator in guard cell ABA signaling. ABA binds to the receptor ABA-R_ABA (ABA receptor), which in turn binds to PP2C. OST1 is released from PP2C and gains kinase activity. Phosphorylation of SLAC1, KAT1 and RBOH by OST1 promotes anion efflux, inhibits potassium influx, and generates ROS burst, respectively. An emerging yet untested idea is that other kinases such as GHR1, CPK6, CPK21 and CPK23 may function in parallel with OST1 in guard cells. Generation of ROS triggers an increase in the concentration of cytosolic calcium, an important second messenger decoded by CDPK and CIPKs. MAPKs are also activated by ROS burst. CDPKs and MPKs may interact with transcription factors and modify ion channel activities in guard cells. Net efflux of ions leads to loss of water and turgor in guard cells, thus closure of the stomatal pores. Right guard cell: blue light induced stomatal opening. Blue light activates the receptors PHOT1 and PHOT2 via phosphorylation. PHOTs promote H⁺-ATPase activity indirectly through proteins including PP1, PP7 and HRB1, as well as BLUS1. Blue light could also be perceived by CRYs, and photoperiodic components were involved in regulating H⁺-ATPase. H⁺-ATPase promotes outward movement of proton, leading to hyperpolarization of guard cell membrane. Subsequent K⁺ influx induces inward water movement and increase in turgor, thus stomatal opening.

OST1, a molecular switch that triggers stomatal closure by phosphorylation

OST1 is a serine/threonine protein kinase identified in A. thaliana. The knock-out mutant ost1 has cooler leaf temperature because it has constantly open stomata.¹³ Both slow and rapid types of anion currents in guard cells induced by ABA are largely suppressed in ost1 mutant.^26,27 In contrast, ABA-induced anion current efflux is enhanced in transgenic A. thaliana plants with OST1 overexpression.²⁸ In addition, OST1 overexpression lines show hypersensitivity in ABA-induced stomatal closure, activation of Ca²⁺ channel and inhibition of inward K⁺ channel, suggesting OST1 is a limiting factor in guard cell ABA response.²⁸ The OST1 homolog in Vicia faba, an ABA-activated protein kinase (AAPK), is also a positive regulator in in guard cell ABA signaling.² The mechanism of activation of OST1 upon its release from binding to PP2C is generally believed to be phosphorylation of its activation loop.^29,30 OST1 could autophosphorylate and self-activate or be phosphorylated by another protein kinase.³¹ How an activated OST1 triggers stomata closure has been revealed recently to involve phosphorylation of a set of substrates. Consistent with the central role and extensive interaction network of OST1, recent work identified many potential OST1 substrates using quantitative phosphoproteomics approach.^32,33

One of OST1's targets is slow anion channel-associated 1 (SLAC1), a major S-type anion channel in guard cells.¹⁴ Activation of SLAC1 by phosphorylation leads to anion efflux through the guard cell plasma membrane, resulting in the outward K⁺ channel activation and K⁺ release. Another known OST1 target is K⁺ inward rectifying channels in A. thaliana (KAT1). In the presence of ABA, OST1 inhibits KAT1 by phosphorylation, thus preventing influx of K⁺.³⁴ OST1 also inhibits KAT1 by phosphorylating and thus deactivating ABA-responsive kinase substrates (AKSs), transcription factors that enhance the expression of KAT1 genes.³⁵ Activation of SLAC1 and inhibition of KAT1 in turn promote anion efflux and suppress cation influx, respectively. The net movement of ions out of guard cells contributes to decrease in turgor pressure and shrinking of the stomatal aperture. In addition to SLAC1, recent studies showed that OST1 can also interact with and activate a quickly-activating anion channel (QUAC1) in A. thaliana guard cells, most likely through phosphorylation.²⁷ Another very important target of OST1 is the plasma membrane localized respiratory burst oxidase homolog (RBOH), an NADPH oxidase that generates reactive oxygen species (ROS).¹² Recent data show that OST1 interacts with both RBOH D and F, and Ser¹⁷⁴ of RBOH F has been identified as the phosphorylation site.^12,28 ROS, including H₂O₂, plays an essential role in ABA signaling.⁹ ROS burst in turn activates Ca²⁺ channels in the plasma membrane and vacuolar membrane, leading to Ca²⁺ release into the cytosol. Ca²⁺ signaling is one of the earliest ABA responses, and specific Ca²⁺ oscillations in the cytosol trigger specific downstream responses.^3,9

CDPK and CIPK act downstream of Ca²⁺ signal and activate anion channels

Calcium-dependent protein kinases (CDPKs) act as a group of sensors to decode intracellular Ca²⁺ transients. For example, CPK3 and CPK6 may function as Ca²⁺ sensors and positive transducers in stomatal ABA signaling.³⁶ In guard cells of cpk3cpk6 double mutant, both ABA and Ca²⁺ activation of SLAC1 is compromised compared to that of wild type. Consequently, stomatal closing in response to ABA and Ca²⁺ was also inhibited in cpk3cpk6 mutant. More recently, CPK6 has also been shown as a positive player in methyl jasmonate and yeast elicitor induced stomatal closure, by activating the anion channels in guard cells.^37,38 This is also supported by the observation that SLAC1 expressed in Xenopus spp. oocytes can be phosphorylated and thus activated by CPK6.²⁵

In guard cells, beside CDPKs, calcineurin-B like proteins (CBLs) together with CBL-interacting protein kinases (CIPKs) also act downstream of Ca²⁺ to relay the signal.³⁹ For instance, CBL1 and CBL9 together with CIPK23 function in stomatal ABA responses.³⁹ More recently, CBL1 and CBL9 have also been shown to interact with CIPK26, which interacts and phosphorylates RBOH F in plant cells.⁴⁰ However, this interaction has not been confirmed in guard cells. Another CBL-CIPK interacting pair in guard cells is CBL1 and CIPK1, which dissociate with increased cytosolic Ca²⁺ concentration.⁴¹ Different combinations of CBLs and CIPKs as well as many CDPKs downstream of Ca²⁺ may represent one mechanism by which the Ca²⁺ signatures are recognized.

The role of CDPKs is not limited to decoding Ca²⁺ signatures, downstream of the ROS burst. Evidence also shows that CDPK could activate RBOH by phosphorylation, resulting in ROS production. For example, CPK4 and CPK5 in potato phosphorylate RBOH B and promote an ROS burst.⁴² Similarly, phosphorylation of RBOH D and F by A. thaliana CPKs was detected using selected reaction monitoring (SRM) mass spectrometry.^43,44 Therefore, activation of CDPKs downstream of ROS and Ca²⁺ could in turn promote ROS production by phosphorylation of RBOH, thus providing an amplification feedback regulation of the signaling pathway. Such a positive feedback is supported by a recent study in which OST1 overexpression lines show higher ROS production and increased cytosolic Ca²⁺ oscillation in guard cells, while ROS production is negligible in ost1 mutant.²⁸ However, RBOH phosphorylation by CDPKs in guard cells has not been reported yet.

While understanding of the multi-faceted roles of CDPKs is emerging, the specificity of different CDPK members is also been realized. For instance, although A. thaliana mutants cpk10, cpk4cpk11 and cpk7cpk8cpk32, showed impaired stomatal response to Ca²⁺ oscillation, they showed normal ABA-induced stomatal closure.⁴⁵ This indicates that these CDPKs work specifically to decode Ca²⁺ and that Ca²⁺-independent ABA pathways in guard cells exist.

MAP kinase amplification of signal between ROS and ion channels

Mitogen activated protein kinases (MPKs) are activated in response to unfavorable environmental conditions to trigger stomatal closure.^9,46 For example, biochemical studies showed that an inhibitor of mammalian p38 MAP kinase blocks ABA- and H₂O₂-induced stomatal closure in V. faba guard cells.⁴⁷ In a subsequent study, MEK1/2, an upstream kinase in the MAP kinase cascade, was also shown to be activated by ABA and H₂O₂ in guard cells.⁴⁸ Consistent with involvement of MPKs in ABA signaling in guard cells, 14 MAP kinase transcripts in A. thaliana GCPs were identified in a microarray study, and MPK3 and MPK7 showed transcriptional changes in response to ABA.²⁴

A few other studies have addressed the function of MPKs in guard cell signaling using genetics. Transgenic A. thaliana in which MPK3 expression is reduced specifically in guard cells showed normal ABA-induced stomatal closure, but closure was partially reduced in response to H₂O₂ and opening was only partially inhibited by ABA treatment.⁴⁹ With a similar approach, both MPK9 and MPK12 were silenced in A. thaliana using RNA interference (RNAi). The stomata of the RNAi lines failed to close in response to ABA and H₂O₂.⁵⁰ Collectively, these studies suggest that MPKs may act downstream of ROS and Ca²⁺ in guard cell signaling. Interestingly, a single amino acid substitution in MPK12 among different accessions of A. thaliana results in different stomata sizes and responses to ABA, highlighting the contribution of MPK12 in stomatal development and function.⁵¹ MPK4 is also strongly expressed in guard cells.^52–54 The role of MPK4 in Nicotiana tabacum was studied using NtMPK4-silenced tobacco plants.⁵⁵ Under normal growth conditions, stomatal density was similar in wild type and MPK4-silenced plants. However, stomatal aperture of the NtMPK4-silenced lines was larger. The larger aperture in MPK4-silenced tobacco leads to greater stomatal conductance, which in turn, gives rise to higher transpiration rate and lower leaf temperature. In addition, another study found that the MPK4-silenced tobacco does not close stomata at high CO₂ conditions.⁵⁶ Moreover, NaMPK4-silenced Nicotiana attenuata also showed higher transpiration rates and enhanced photosynthesis.⁵⁷ Interestingly, higher kinase activity in transgenic A. thaliana overexpressing a constitutively active MPK4 facilitates the entry of bacteria into the apoplast.⁵⁴ Taken together, these results underscore the importance and complexity of MPKs in guard cell signaling. The lack of information on the direct kinase interactors calls for further research toward a clear understanding of the role of MPKs in guard cells signaling.

SLAC1 and beyond

Activation of anion channels at the plasma membrane of guard cells is a critical step in stomatal closure.⁹ The slow anion channel SLAC1 can be phosphorylated and activated not only by OST1, but also by CDPKs,^25,36,58 as well as by a newly identified kinase, guard cell hydrogen peroxide-resistant1 (GHR1).⁵⁹ Furthermore, activation of anion channels by these kinases is depressed by different members of the PP2C family.^25,58,59 For example, while CPK6-activated SLAC1 activity is depressed by both ABA-insensitive1 (ABI1) and ABI2, GHR induced ion flux is inhibited by ABI2, not ABI1.^25,59 Thus, OST1, CDPKs and GHR1 may represent parallel branches of ABA signaling in guard cells. While PP2C binds to OST1 physically to inactive the kinase activity, whether the activity of CPKs and GHR1 is controlled by PP2C has not been determined in guard cells yet. Phosphorylation of SLAC1, the point of convergence of these pathways, at different sites could serve as a regulation mechanism. For example, while Ser120 is a critical phosphorylation site of OST1,²⁶ phosphorylation of Ser59 is crucial in CPK6 mediated signaling.⁶⁰ Residues of SLAC1 phosphorylated by CPK 21, CPK23 and GHR1 have not been reported yet. However, neither CPKs nor GHR1 can replace OST1 in ABA activation of SLAC1, as ABA-induced activation of slow anion channels and thus stomatal closure is abolished in ost1 mutant.²⁸ Thus, OST1 plays a dominant role in anion channel activation, and CPKs as well as GHR1 are required for fully functional ABA-induced anion efflux.

In addition to SLAC1, its homolog may also mediate anion currents in guard cell signaling. For instance, SLAC1 homolog 3 (SLAH3) is also expressed in guard cells and when coexpressed with CPK21 in Xenopus oocytes, nitrate-induced anion currents were observed.¹¹ However, the contribution of SLAH3 in stomatal closure is not clear. Phosphorylation site mapping of SLAH3 and subsequent single amino acid mutation will help to understand the relevance of phosphorylation in channel activity regulation.

Blue Light Induction of Stomata Opening via Protein Phosphorylation

Light promotion of stomatal opening

Light, as the energy source and signal information carrier, is one of the most important environmental cues that regulate plant growth and development. During the day time, opening of stomata is largely due to synergistic response to red and blue light in guard cells.¹ Compared to blue light, induction of stomata opening by red light requires high intensity and longer illumination.¹ Currently, very little is known about the red light signaling in guard cells, although studies showed the involvement of phytochromes, the red light receptors.⁶¹ Phytochromes have both auto-phosphorylation and trans-phosphorylation activities,⁶² but whether this activity is important for the regulation of stomatal opening by red light is unknown. Another unresolved question in red light-regulated stomatal movement is whether photosynthesis in the guard cells or the adjacent mesophyll cells has a role.⁶³ This is partially due to the fact that the action spectrum of photosynthesis and red light response overlaps. Recently, photosynthesis induced redox state change of electron transport chain components, especially plastoquinone, is suggested as a signal to control stomatal movement.⁶⁴

In contrast to the red-light pathway much more is known about blue light-induced stomata opening. In guard cells, blue light is perceived by both phototropins and cryptochromes.³ While cryptochromes may play a role in stomatal movement in response to relatively high fluence rates of blue light,¹⁸ phototropins have been established as major blue light receptors in guard cells.²² Blue light activates these receptor serine/threonine kinases by auto-phosphorylation, and the kinase activity of phototropins is essential for activation of plasma membrane H⁺-ATPase. The activated H⁺-ATPase pumps H⁺ outside of the plasma membrane, allowing influx of K⁺ and increase of turgor in the guard cells. In addition, blue light inhibits anion efflux by inhibition of anion channels activity at the plasma membrane via phototropin.⁶⁵

Activation of blue light receptor phototropins via phosphorylation

Phototropins have two light, oxygen, and voltage (LOV) domains at the N-termini to perceive the blue light, and a kinase domain at the C-termini for signal transduction.⁶⁵ While stomata from single mutants of phot1 or phot2 in A. thaliana still open under blue light, the stomata of the double mutant phot1 phot2 is not responsive.²² Additionally, phosphorylation and activation of H⁺-ATPase in response to blue light is completely abolished in the double mutant.²²

Both genetic and biochemical data showed that phosphorylation and activation of H⁺-ATPase is mediated by phototropins.^21,22 However, phototropins do not directly phosphorylate H⁺-ATPases although both are associated with the plasma membrane. A promising mediator candidate, the MAP4K blue light signaling1 (BLUS1), was identified.⁶⁶ The mutant blus1 does not open stomata under blue light. BLUS1 is phosphorylated directly by PHOT1 and the phosphorylation status of BLUS1 is required for H⁺-ATPase activation. Although BLUS1 encodes a novel Ser/Thr protein kinase, direct phosphorylation of H⁺-ATPase by BLUS1 has not been established. Identification of the steps linking the receptor kinases to regulation of the proton pumps remains elusive.

The phosphorylation status, thus activity of guard cell H⁺-ATPase, can also be regulated by the other group of blue light receptors, cryptochromes. This pathway is mediated by photoperiodic components such as FLOWERING LOCUS T (FT) and CONSTANS (CO).^67,68 For example, phosphorylation of H⁺-ATPase in response to blue light is abolished in the FT knockout mutant.⁶⁷ However, how the phosphorylation status of H⁺-ATPase is modified by the photoperiodic components is an open question.

Activation of H⁺-ATPase by phosphorylation in blue light-induced stomatal opening

The key event in blue light-induced stomata movement is the activation of H⁺ pump at the plasma membrane, resulting in accumulation of K⁺ in guard cells and thus opening the aperture.²³ The switch to turn on the pump activity was shown to be phosphorylation of the H⁺-ATPase upon blue light perception in GCPs. The modified H⁺-ATPase is further stabilized by binding to a 14–3–3 protein.²³ The phosphorylation sites were mapped to serine and threonine residues at the C-terminus using phosphoamino acid analysis, suggesting a serine/threonine protein kinase was involved.²³ It is also noteworthy that H⁺-ATPase maybe the limiting factor in stomatal light response. While overexpression of PHOT2 or KAT1 has no effect on stomatal light response, transgenic plants overexpressing H⁺-ATPase showed enhanced stomatal opening in response to light and thus higher photosynthesis activity.¹⁹ It thus seems likely that regulation of H⁺-ATPase is a critical step in stomatal movement control.

Regulation of the phosphorylation status of H⁺-ATPase could also provide a possible cross-talk point between ABA and blue light signaling pathways in guard cells (Fig. 2). ABA inhibits blue light-induced stomatal opening. The blue light-induced phosphorylation of H⁺-ATPase in epidermal peels was completely abolished with physiological concentration of ABA, and this inhibition effect was not observed in ABA signaling mutant abi1–1, abi2–1 and ost1–2.^69,70 This indicates that ABA inhibits light induced stomatal opening by inhibiting phosphorylation of H⁺-ATPase via the classical PP2C-OST1 pathway. Another line of evidence comes from the dominant A. thaliana mutant open stomata2 (ost2), which encodes a constitutively active H⁺-ATPase at the plasma membrane and thus abolishes the response of stomata to ABA. In contrast, stomatal closing in response to high CO₂ and dark was affected to a smaller extent,⁷¹ suggesting OST2 mainly functions in the guard cell ABA pathway.

Figure 2.

ABA inhibition of blue light-induced stomatal opening. Blue light-induced phosphorylation of H⁺-ATPase is inhibited by ABA, perhaps through production of PA, which inhibits PP1. ABA also activates OST1, which could either inhibit K⁺_in channel KAT1 by phosphorylation directly, or inhibit the K⁺_in channel facilitating protein AKS by phosphorylation.

Involvement of phosphatases in blue light-induced stomatal opening

While autophosphorylation of phototropins triggers the signaling events of blue light-induced stomata opening, dephosphorylation exerts the opposite effect. A candidate phosphatase was identified as a Ser/Thr protein phosphatase 2A (PP2A) through a yeast two hybrid experiment using full-length PHOT2 as a bait.⁷² Further study showed a higher phosphorylation level of PHOT2, and thus enhanced blue light-induced stomata opening in plants with reduced PP2A activity.⁷² Another phosphatase, protein phosphatase 1 (PP1), was suggested as a positive regulator between phototropins and H⁺-ATPase in guard cell light response.⁷³ Blue light-induced stomatal opening is impaired whereas the PP1 activity is reduced or inhibited. Moreover, one possible mechanism in ABA inhibition of light-induced stomatal opening is through production of phosphatidic acid (PA) in guard cells, which in turn inhibits PP1.⁷⁴ Recently, pp1 regulatory subunit2-like protein1 (PRSL1) was identified as a regulatory subunit of PP1 in blue light-induced stomatal opening.⁷⁵ In addition, protein phosphatase 7 (PP7) regulates the phosphorylation status of hypersensitive to red and blue 1 (HRB1), thus inhibiting the ability to form complex with other proteins to modulate stomatal movement.⁷⁶ Thus, reversible phosphorylation and dephosphorylation by specific kinases and phosphatases is extensive in guard cell signaling. Further studies aimed at finding the molecular links between the light and kinase/phosphatase activation, as well as components downstream of (de)phosphorylation will provide a better understanding.

Future perspectives

Phosphorylation allows for rapid regulation of protein function, such as ion channel activities in guard cells. Our understating on the role of phosphorylation in stomatal movement is expanding as more kinases and phosphatases involved are identified. A major unanswered question is what the direct substrates of the kinases are in guard cells. Substrates such as SLAC1 can be phosphorylated by multiple protein kinases. Unraveling the roles of kinases that phosphorylate the same protein at the same or different phosphorylation sites is critical. In addition, phosphorylation status of a protein represents a balance between kinase and phosphatase activities. However, the identity and function of most phosphatases in guard cell signaling remain largely elusive. Another issue to be resolved is how to detect in situ phosphorylation in the guard cells. Previous phosphorylation detection has been mainly performed using guard cell protoplasts, the preparation of which inevitably introduces stresses because of mechanical blending and enzyme digestion. This in turn may change in vivo protein phosphorylation status.

Approaches to understanding protein phosphorylation in vivo are emerging. Recently, an immunohistochemical detection method of H⁺-ATPase phosphorylation in guard cells was developed.⁶⁹ Another strategy is to identify the phosphorylation sites and determine stoichiometry and dynamics in the signaling processes. Recent advances in mass spectrometry have not only been applied to map the phosphorylation sites of proteins at high sensitivity and accuracy, but also to monitor the global protein phosphorylation changes in response to external stimuli in high spatial and temporal resolution.⁷⁷ Finally, applying the knowledge of protein phosphorylation to crop improvement for enhanced water usage, productivity and stress tolerance is the ultimate goal. We foresee that modification of the phosphorylation status of key components in guard cell signaling will provide important avenues to achieve this goal.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Drs. Biswa Misra and Mi-Jeong Yoo for critical reading of the manuscript. Dr. Mengmeng Zhu is thanked for providing the guard cell image used in Figure 1 as background.

Funding

This research was supported by grants from the National Science Foundation (MCB 0818051 to S Chen, and MCB 1412547 to S Chen and A Harmon).