New functions of an old kinase MPK4 in guard cells

ABSTRACT

Mitogen-activated protein kinase (MPK) cascades play important roles in plant development, immune signaling and stress responses. MPK4 was initially identified as a negative regulator in systemic acquired resistance (SAR) because the levels of salicylic acid (SA) and reactive oxygen species (ROS) were higher in the Arabidopsis mpk4 mutant. MPK4 is highly expressed in guard cells, specialized epidermal cells forming stomatal pores on leaf surface that function at the frontline of bacterial pathogen invasion. In addition to biotic stresses, stomatal guard cells also mediate cellular responses to abiotic stimuli such as drought and CO₂ changes. MPK4 appears to play different roles in different plant systems. In this review, we briefly discuss the protein kinase MPK4 functions and focus on its signaling roles in different plant systems, especially in stomatal guard cells.

Introduction

Plants constantly face numerous environmental challenges. As sessile organisms, their survival relies on efficient signaling networks in response to the ever changing environment. MPK cascades are important components of plant signaling networks, which are important in plant development, abiotic and biotic stress responses.¹ Among the MPK family, MPK4 was initially identified as a negative regulator in SAR and highly expressed in guard cells, specialized cells forming stomatal pores on leaf surface.²

Stomatal guard cells play a crucial role in controlling gas exchange between plant shoots and atmosphere. They serve as a passage for CO₂ intake and water transpiration.³ Meanwhile, they also provide entry ports for bacterial pathogens.^4,5 In consequence, stomatal guard cells are at the frontline of sensing and responding to environmental factors (e.g., humidity, CO₂ changes and microorganism invasion) by rapidly adjusting stomatal aperture.

Stomatal movement depends on changing of guard cell size. Stomata will open when guard cells increase turgor and cell size as a result of accumulating osmolytes, such as K^+.6,7 Transport proteins located on the plasma membrane and vacuolar membrane move ions between apoplast, cytosol and vacuoles.⁸ Stomata opening is driven by a concerted action of P-type ATPases, K_in channels, and nitrate and chloride transporters on the plasma membrane,^9–11 together with V-type ATPases, pyrophosphatases, Na⁺/H⁺ exchange (NHX) transporters, aluminum-activated anion channels (ALMTs) and Cl⁻ channels (CLC) on the vacuolar membrane.^12–16 Stomatal closure also involves different proteins, including S-type anion channels, R-type anion channels and K_out channels on plasma membrane,^9,10,17 as well as ALMTs and two pore K⁺-selective (TPK) channels on vacuolar membrane.^15,18

Many of the membrane transporters are regulated by kinases in guard cells. For example, the guard cell slow anion channel (SLAC) 1 was activated through phosphorylation by the protein kinase OST1 (open stomata 1) and deactivated by the protein phosphatase ABI1 (ABA insensitive 1).¹⁹ As to MPK4, it was found to be highly abundant in guard cells, and it may play an important role in stomatal movement.²⁰ In this review, we discuss the functions of MPK4s in the context of different plant systems, highlight controversial findings and focus on the roles of the MPK4s in stomatal guard cell signaling.

MPK4 functions in plant immunity and other processes

MPK4 was initially identified as a negative regulator of plant immunity. The mpk4 knockout in Arabidopsis thaliana Landsberg ecotype (Ler) displayed lower levels of pathogens in leaves than wild type (WT) after infiltration with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) or spray with Peronospora parasitica.² The mpk4 mutant in Columbia ecotype (Col) also showed enhanced resistance to spray inoculation with Hyaloperonospora parasitica Noco2 (H.p. Noco2).²¹ Furthermore, Arabidopsis with constitutively active MPK4 (CA-MPK4) contained more pathogen after spraying with Pst DC3000. However, their resistance level is similar to WT when infiltrated with Pst DC3000,²² suggesting stomatal guard cell defense contributes to the difference in immunity between the CA-MPK4 plants and WT.

Consistent with AtMPK4 being a negative regulator of plant immunity, the mpk4 mutants over-accumulate SA an ROS, and exhibited high expression levels of pathogenesis-related (PR) genes and wound-induced genes, including chitinase, PR2, extensin, pectin methylesterase, glutathione S-transferase, ascorbate reductase and oxalate oxidase.^2,21,23 On the other hand, CA-MPK4 plants had reduced SA levels, which may contribute to the susceptibility to pathogen.²² Interestingly, a homolog of AtMPK4 from Brassica napus, BnMPK4 seems to function differently. Plant leaves expressing constitutively active BnMPK4 (BnMPK4^CA) showed lesions and accumulation of H₂O₂, but not the vector control, WT BnMPK4 or inactive BnMPK4, indicating BnMPK4^CA promotes ROS production and cell death.²⁴

MPK4 plays important roles in both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). CA-MPK4 Arabidopsis exhibited a strong reduction of flg22-induced ROS burst, but no significant difference in flg22-induced callose deposition compared to WT.²² This result indicates although MPK4 plays an important role in PTI, it does not control all PTI responses. In terms of pathogen defense, CA-MPK4 Arabidopsis appeared susceptible to Pst DC3000 hrcC with impaired effector and Pst DC3000 expressing TIR-NB-LRR-type effector AvrRps4, but not to that expressing TIR-NB-LRR-type effectors AvrRpm1 and AvrRpt2, indicating that MPK4 partially functions in ETI.²² A homolog of AtMPK4 from Nicotiana tabacum, NtMPK4 was shown to function in jasmonic acid (JA) signaling pathway, but not the ethylene signaling pathway.²⁵ So it may indirectly regulate SA through the JA antagonism.

Although MPK4 seems to be an important negative regulator of plant immunity, it is a positive regulator in cytokinesis, reproduction and photosynthesis.^26–29 The mpk4 mutant in the Ler background showed dwarfism, reduced fertility and curled leaves,² while mpk4 mutant in the Col background presented more severe dwarfism than that in the Ler background²⁶. In addition, its abnormal roots formed with multiple-branched root hairs and had more protruding microtubules than WT plants.³⁰ On the other hand, the mpk4 in Ler background contained large cells with incomplete cell walls and multiple nuclei in roots and cotyledons,²⁶ it also had anthers containing few large pollen grains with multiple nuclei caused by incomplete meiosis of microspore mother cells (MMCs).²⁷ These data indicate that MPK4 is required for meiotic and somatic cytokinesis. MPK4 may also be involved in photosynthetic electron transport and chloroplast ROS metabolism based on the evidence that the mpk4 mutant chloroplasts yielded less quantum energy from photosystem II, but more in non-regulated energy dissipation and more ¹O₂ in thylakoids than WT.²⁹ In the mpk4 mutant, 538 genes were found to be upregulated and 238 downregulated compared to WT. These differential genes fell into different biological processes, including stress response, SA signaling and ROS metabolism.³¹

MPK4 signaling cascade

MPK4 is activated by upstream kinases in response to developmental cues, pathogen invasion or other environmental changes, and then interacts with and phosphorylates downstream substrates.³² AtMKK2 and AtMEK1 (AtMKK1) are two MAP kinase kinases (MAPKKs), which share 65% sequence identity.³³ In a yeast two-hybrid (Y2H) assay, AtMEK1 and AtMKK2 specifically interacted with AtMPK4.³³ In addition, constitutively active MKK2 can strongly activate MPK4 by phosphorylation.³⁴ AtMEK1 can also activate AtMPK4 in vitro through phosphorylation on Threonine (Thr) and Tyrosine (Tyr) residues.³⁵ Upstream of the MAPKKs, AtMEKK1 is a MAP kinase kinase kinase (MAPKKK) involved in the MAPK cascades.³⁶ Y2H results showed that AtMEKK1 specifically interacted with AtMEK1 and AtMKK2, as well as AtMPK4 ³³. AtMPK4 binds to regulatory domain of AtMEKK1, while AtMEK1 and AtMKK2 can bind to catalytic domain of AtMEKK1, indicating that they may form a protein complex.³³ It is known that the MEKK1-MKK1/MKK2-MPK4 kinase cascade is involved in plant immunity and ROS signaling,^21,23,31 how this signaling cascade functions in stomatal guard cell immunity and stomatal response to environmental changes awaits further investigation.

Several proteins have been identified as substrates of MPK4 as they can be activated by MPK4 through phosphorylation (Table 1). They include MAP kinase 4 substrate 1 (MKS1), topoisomerase II 1 (PAT1), microtubule-associated protein 65 (MAP65) and Arabidopsis SH4-related 3 (ASR3).^30,37–39 Recombinant MKS1 purified from E.coli can be phosphorylated by HA-tagged MPK4 isolated from plants. Co-immunoprecipitation (Co-IP) using pep22, a monoclonal antibody against MKS1 demonstrated that MKS1 interacted with MPK4 in vivo. Result of a green fluorescence protein (GFP)-fusion experiment showed that MPK4 and MKS1 co-localized in the nuclei, which is consistent with their interaction in vivo.³⁷ In addition to MKS1, a phosphorylated His₆-PAT1 (a mRNA decay factor) was detected to associate with activated MPK4 in an in vitro pull-down experiment.³⁸ In another in vitro pull-down assay, ASR3 (a negative regulator of PTI) was found to directly interact with MPK4 and activated MPK4 can phosphorylate ASR3.³⁹ Moreover, a MAP65 involved in microtubule organization was shown to interact with MPK4 by co-immunoprecipitation analysis.³⁰ Another two MAPs, IQD31 and IQD32, were found being absent in mpk4 by phosphoproteome.⁴⁰ In addition, another 15 phosphopeptides were specifically undetected in the mpk4, including mRNA splicing factor SCL30, chromatin protein SKIP and ATP-dependent RNA helicase DHX8/PRP22,⁴⁰ indicating they are potential MPK4 substrates.

Table 1.

List of known MPK4 substrates, their functions and methods used in the studies.

Substrate	Function	Method	Reference
MEK1/MKK2	Immunity & ROS signaling	Y2H	21,23,31,33
MKS1	Plant immunity	Co-IP & GFP-fusion	37
MAP65	Microtubule-associated protein	Co-IP	30
PAT1	mRNA decay factor	in vitro pull-down	38
ASR3	Negative regulator of PTI	in vitro pull-down	39
IQD31/IQD32	Microtubule-associated proteins	Phosphoproteomics	40
SCL30	RNA splicing factor	Phosphoproteomics	40
SKIP	Chromatin protein	Phosphoproteomics	40
DHX8/PRP22	ATP-dependent RNA helicase	Phosphoproteomics	40

MPK4 functions in guard cells

MPK4 expression pattern is largely confined to guard cells.² The difference in resistance to pathogen inoculation by spraying, but not by infiltration indicates that MPK4 may be involved in stomata-based immunity.²² However, there are different observations about its functions in guard cells.

First, stomatal aperture. The stomatal aperture of CA-MPK4 Arabidopsis showed no difference from that of WT after treating with Pst DC3000 and flg22 for 1 h and 2.5 h, suggesting that MPK4 is not involved in the regulation of stomatal aperture.²² However, in tobacco, NtMPK4-silenced plants displayed higher stomatal conductance and lower leaf temperature than WT, indicating that NtMPK4 is involved in regulation of stomatal closure.²⁵ NtMPK4-silenced plants were found insensitive to CO₂ level changes and have impaired CO₂- or dark-induced activation of S-type anion channels involved in stomatal closure.⁴¹ Second, stomatal development. NtMPK4-silenced plants were also found having larger guard cell length than WT, suggesting NtMPK4 might be involved in guard cell development,⁴¹ but there is no direct evidence to show that NtMPK4 and AtMPK4 are involved in guard cell development. Third, ABA signaling. The mpk4 mutant in Ler background had normal response to ABA.² Transpirational water loss in NtMPK4-silenced plants and WT plants was found similar after ABA treatment, suggesting that the NtMPK4-regulated stomatal closure is an ABA-independent process.²⁵ Interestingly, NaMPK4 from a wild tobacco species N. attenuata is required in ABA- and H₂O₂-induced stomatal closure. NaMPK4-silenced plants showed higher ABA accumulation and water loss after drought treatment, and slower response to H₂O₂ treatment than WT.²⁰ Similarly, BnMPK4 can also be activated by H₂O₂ and ABA as phosphorylated MPK4 was observed in plant cells after the treatment with H₂O₂ and ABA.²⁴ Fourth, ROS signaling. CA-MPK4 Arabidopsis exhibited reduced ROS accumulation after flg22 treatment, compared with WT,²² but BnMPK4^CA over-accumulated ROS to further trigger cell death.²⁴

Such differences observed in different studies may be attributed to the following: 1) different plant species used; 2) different developmental stages of plant materials and treatment conditions; 3) different methods used to assay stomatal movement. Therefore, caution needs to be taken when comparing the different studies to ensure fair comparisons and proper conclusions.

Concluding remarks

MPK4 has been well studied in different aspects,^20,22,24 but its role in stomatal movement seems controversial in different species in spite of the high sequence homology among the different MPK4s.^22,25 The molecular mechanisms underlying MPK4 functions in the guard cells are still not known. With the highly efficient guard cell preparation technique,⁴² modern proteomics, phosphoproteomics and redox proteomics approaches will greatly facilitate the discovery of MPK4 interacting proteins and substrates, as well as posttranslational regulations (e.g., redox) in stomatal guard cells. Together with the powerful genetic tools and resources available for the reference plant A. thaliana, new functions of MPK4 signaling cascades in stomatal guard cells are to be discovered in due course.

Funding Statement

This work was supported by the National Science Foundation [1412547];

Abbreviations

MPK

Mitogen-activated protein kinase

ROS

reactive oxygen species

SAR

systemic acquired resistance

Acknowledgments

We are grateful to Lisa David for corrections to the English. This work has been funded through a National Science Foundation grant (MCB 1412547) to S. Chen.

Disclosure statement

No potential conflict of interest was reported by the authors.