Current progress of PM-localized protein functions in jasmonate pathway

ABSTRACT

Jasmonate (JA), a class of lipid-derived phytohormone, regulates diverse developmental processes and responses to abiotic or biotic stresses. The biosynthesis and signaling of JA mainly occur in various organelles, except for the plasma membrane (PM). Recently, several PM proteins have been reported to be associated with the JA pathway. This mini-review summarized the recent progress on the functional role of PM-localized proteins involved in JA transportation, JA-related defense responses, and JA-regulated endocytosis.

Introduction

Jasmonate (JA), including jasmonic acid and its oxylipin derivatives, are widely distributed throughout the plant kingdom.¹ The biosynthetic JA pathway is initiated from α-linolenic acid (18:3) (α-LeA) to 12-oxo-phytodienoic acid (OPDA), which is sequentially catalyzed by 13-lipoxygenase (13-LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) in the chloroplasts.^2–4 OPDA is then translocated to the peroxisomes and converted to jasmonic acid after reduction by 12-oxophytodienoate reductase (OPR) and β-oxidation by acyl-CoA-oxidase.^5,6 Jasmonic acid can be further metabolized into a series of derivatives including methyl jasmonate (MeJA) and biologically active jasmonoyl-isoleucine (JA-Ile) in the cytosol.⁷

JA is known to function as a critical regulator in various plant growth and developmental events, such as seed germination, root growth, anthocyanin accumulation, flowering time, male fertility, and leaf senescence.⁸ Meanwhile, JA plays a key role in plant resistance to abiotic and biotic stresses, including UV damage, insect attack, necrotrophic fungus infections, and wounding.^1,9 JA acts through a conserved signal transduction pathway localized in the nucleus. It comprises coronatine insensitive 1 (COI1), jasmonate ZIM-domain (JAZ) proteins, and downstream transcriptional factors (TFs). JA-Ile is perceived by F-box protein COI1 (part of the SCF^COI1 ubiquitin ligase complex), and then JAZ repressors are recruited for ubiquitination and degradation through the 26S proteasome.^10–14 Thus, the JAZ-repressed TFs could be released to regulate their corresponding JA-responsive genes.^15–19

Taken together, the main components involved in JA biosynthesis and signaling are distributed in different subcellular compartments including chloroplasts, peroxisomes, cytosol, and nuclei, with the exception of the plasma membrane (PM). The PM forms a selective barrier between eukaryotic cells and extracellular environments. Functional proteins specific to the PM, such as transporter, receptor, anchor, and enzyme, mediate intercellular communication through the transport of metabolites and transmission of external signals. Recently, several PM-located proteins were identified to act as upstream regulators or downstream targets of JA signaling. In this review, we summarized recent research advances on the PM-located proteins involved in the JA pathway.

The PM-localized proteins involved in JA transportation

The synthesis of many phytohormones has been found at different sites from their action sites. Thus, PM-localized transporters are essential to mediate intra- and intercellular phytohormone distribution. Most of them belong to the ATP-binding cassette (ABC) family (transporters of auxin, abscisic acid, cytokinins, and strigolactones) and the nitrate transport1/peptide transporter family (NPF) (transporters of abscisic acid and gibberellins).²⁰ Of which, a JA-responsive NFP member NPF2.10/GTR1 showed the transport activity of JA-Ile and jasmonic acid in Xenopus oocytes.²¹ After wounding, the accumulation of JA-Ile/jasmonic acid in undamaged leaves and the translocation of JA-Ile/jasmonic acid from wounded leaves to distal leaves were both impaired in the gtr1 mutant, indicating a possible role of GTR1 in JA transportation via phloem.²²

Recently, three members of the ABC G family (ABCG) clade of Arabidopsis, including jasmonate transporter 1 (AtJAT1/ABCG16), AtJAT3/ABCG6, and AtJAT4/ABCG20, were identified as the JA transporters.^23–25 AtJAT1, with dual localization at the PM and nuclear envelope (NE), regulates the nucleoplasmic homeostasis of JA (Figure 1(a)).²⁴ Under low cytosolic JA conditions, AtJAT1 at the NE is responsible for the nuclear influx of JA-Ile to maintain its concentration for degradation of JAZ proteins and activation of JA responses such as inhibition of root growth, enhanced resistance to Botrytis cinerea (B. cinerea), and male fertility.²⁴ Under high cytosolic JA conditions, AtJAT1 at the PM mediates the export of cytoplasmic jasmonic acid to decrease its retention and attenuate the JA signaling.²⁴ The dual function of AtJAT1 could act as a critical node for balancing the trade-off between growth and immunity by promoting activation and deactivation of JA signaling.

Figure 1.

Schematic model of several PM-localized proteins involved in JA pathway

(a) The PM-localized ABCG proteins involved in JA transportation. AtJAT1 regulates the nucleoplasmic homeostasis of JA. AtJAT3 and AtJAT4 function in long-distance translocation of wound-induced JA.(b) PM-localized H⁺-ATPase is involved in stomatal aperture regulation. AHA1-mediated H⁺ efflux in guard cells is the early event for MeJA-induced stomata closure. P. syringae type III effector AvrB acts through AHA1-mediated JA signaling to induce stomatal virulence.(c) The PM-localized proteins involved in regulation of JA-based defense against insect herbivory. GLR3.3 and GLR3.6 act as Glu sensors to trigger plant systemic defense responses including the JA pathway. The Ca²⁺ channel CNGC19 mediates plant defense upon insect herbivory through JA signaling. AHA1 negatively regulates wound-induced JA biosynthesis and signaling.The solid arrows are supported by literature, whereas dashed arrows indicate unclear function.

Another two ABCG clade proteins, AtJAT3 and AtJAT4, were recently reported to function in long-distance translocation of wound-induced JA (Figure 1(a)).²⁵ Unlike AtJAT1, AtJAT3 and AtJAT4 are located at the PM of phloem cells and prefer transporting jasmonic acid rather than JA-Ile.²⁵ Petiole-to-rosette grafting experiments showed that AtJAT3/4 were responsible for JA translocation from local leaves to distal systemic leaves.²⁵ Moreover, analysis of atjat3-1;4–1, glr3.3, and atjat3-1;4–1;glr3.3 mutants demonstrated that AtJAT3/4 cooperated with glutamate receptor 3.3 (GLR3.3) to regulate the wound-induced systemic response (WSR) including JA-Ile accumulation, JA-responsive gene expression (OPR3, JAZ5, JAZ7 and JAZ10), and defense against B. cinerea in the distal leaves.²⁵ Thus, AtJAT3/4 could mediate the long-distance transmission of wound-induced JA signals to activate WSR.

PM-localized H⁺-ATPase is involved in stomatal aperture regulation

The PM-localized proton-pumping ATPase (H⁺-ATPase), which mediates H⁺ influx to generate proton motive force across the PM, is necessary to the secondary transportation of most ions and metabolites in plants.²⁶ There are 11 isoforms of H⁺-ATPase in Arabidopsis.²⁷ Several isoforms have been shown to be involved in various plant developmental processes, including pollen development, embryo formation, synthesis of seed coat flavonoids, stomatal opening, nutrient uptake, and plant adaptation to abiotic stresses, such as salinity, P deficiency, and Al toxicity.^27–31

Stomatal aperture, which is regulated by several environmental signals, phytohormones, and pathogen infection, is determined by the shape of guard cells.^32–34 Turgor pressure, which changes the guard cell shape, is determined by transmembrane ion fluxes such as K⁺, Cl⁻, and malate ion.^33,34 Through noninvasive microtest technology (NMT) analysis, Yan et al. showed that treatment with exogenous MeJA-induced H⁺ efflux, Ca²⁺ influx, and K⁺ efflux across the PM and stomatal closure in a COI1-dependent manner.³⁵ Furthermore, the MeJA-induced Ca²⁺ and K⁺ flux as well as stomatal closing was strongly inhibited by pretreatment with vanadate (a specific ATPase inhibitor) or in the aha1-6 and aha1-7 mutant, indicating that AHA1-mediated H⁺ efflux occurs early in guard cells upon MeJA stimulation (Figure 1(b)).³⁵ Thus, the electrochemical proton gradient activated Ca²⁺ and outward K⁺ channels and then led to the closure of stomata.³⁵

The stomata are a major gateway for pathogenic bacteria invasion. Several bacterial pathogens have evolved serious virulence factors to promote stomatal opening.³² Zhou et al. demonstrated that AHA1 activity was positively regulated by the Pseudomonas syringae type III effector protein AvrB to induce stomatal opening and promote bacterial virulence in a RPM1-interacting 4 (RIN4)-dependent manner.³⁶ The canonical JA signaling pathway, including receptor, repressors, and downstream transcription factors, was shown to be involved in AvrB-induced stomatal opening. Both AvrB and AHA1 indirectly promoted the COI1-JAZ interaction and JAZ degradation, which might activate no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon (NAC) transcription factors to open stoma.³⁶ In conclusion, AvrB acts through AHA1-mediated JA signaling to induce stomatal virulence (Figure 1(b)).

The PM-localized proteins involved in regulation of JA-based defense against insect herbivory

Insect herbivory causes severe damage to plants. In response to insect herbivory and mechanical wounding signals, plants rapidly generate electrical and Ca²⁺ signals in local and systemic tissues, thereby activating defense signaling.³⁷ The JA pathway plays a vital role in the regulation of plant resistance against insect attack.⁹ Both electrical and Ca²⁺ signals could activate JA biosynthesis and signaling in the damaged site or distal/systemic to it.⁹

Mousavi et al. revealed that glutamate receptor (GLR)-type cation channels GLR3.3 and GLR3.6 mediated long-distance electrical signaling to activate the JA pathway in distal leaves.³⁸ Upon wounding, the glr3.3 glr3.6 double mutants showed a stronger reduction in both wound-induced surface potential changes and JAZ10 expression level compared with the WT plants.³⁸ Toyota et al. further found that leaf-to-leaf transmission of wounding-induced [Ca²⁺]cyt increases also depended on GLR3.3 and GLR3.6.³⁹ After wounding, the production of apoplastic glutamate (Glu) was promoted and then elicited defense signal propagation through GLR3.3/GLR3.6-induced systemic Ca²⁺ waves and JA-responsive gene expression such as OPR3, JAZ7, and JAZ10.³⁹ Consistent with this, the growth of Spodoptera littoralis larvae on glr3.3 and glr3.3 glr3.6 mutants was better than that on WT plants.⁴⁰ In addition, the activation of GLR3.1/GLR3.6 was also required for the systemic JA signaling in the root-to-shoot model.⁴¹ In conclusion, GLR3.3 and GLR3.6 act as Glu sensors to trigger plant systemic defense responses including the JA pathway (Figure 1(c)).

Apart from the GLRs, the PM-localized Ca²⁺-permeable channel CNGC19 is also involved in Ca²⁺-based herbivory defense.⁴² The cngc19 mutants showed attenuated Ca²⁺ flux and less resistance to Spodoptera litura feeding.⁴² The defection of Ca²⁺ elevation caused by the CNGC19 mutation led to a decrease in the herbivory-induced JA pathway.⁴² Relative to the WT plants, both the cngc19-1 and cngc19-2 mutants displayed a reduction of jasmonic acid/JA-Ile accumulation and JA-responsive gene expression including vegetative storage protein 2 (VSP2), Thionin 2.1 (Thi2.1), plant defensin 1.2 (PDF1.2), and LOX2.⁴² In addition, the upregulation of CNGC19 expression after simulated herbivory is COI1-dependent.⁴² Thus, reduced activation of the JA pathway contributes to decreased defense in cngc19 mutants upon herbivory (Figure 1(c)).

In addition to stomatal aperture regulation, AHA1 also functioned as a suppressor in JA signaling induced by wounding and insect herbivory. Long-distance transmission of slow wave potentials (SWPs) is critical for systemic wound response. Kumari et al. found that the SWP repolarization and cytosolic Ca²⁺ increase followed SWP depolarization both were negatively regulated by AHA1.⁴³ Furthermore, the reduced function aha1 allele showed higher JAZ10 transcript levels and enhanced jasmonic acid, JA-Ile, and OPDA accumulation in distal leaves upon wounding treatment.⁴³ Consistently, the Spodoptera littoralis larvae fed with aha1-7 mutant were significantly lighter than those fed with WT plants.⁴³ These data reveal that, when wounded, the activity of AHA1 is inhibited to induce leaf-to-leaf electrical signaling as well as JA biosynthesis and signaling (Figure 1(c)).

The endocytosis of PM-localized proteins regulated upon JA treatment

An important determinant for controlling the fundamental properties of PM proteins is thought to be the spatiotemporal organization of these proteins. PM proteins in diverse types of plants undergo endocytosis to regulate cellular responses upon environmental stimulation.⁴⁴ In the context that JA plays an important role in various plant developmental processes and stress responses, the JA-modulated endocytosis of specific PM proteins has been recently reported.

PIN-FORMED 2 (PIN2), a member of the auxin efflux carriers in the roots of Arabidopsis, is involved in the regulation of auxin redistribution induced by gravity. At a low concentration of MeJA (5 μM), the endocytosis of PIN2 was inhibited in an auxin biosynthesis- and signaling-dependent manner.⁴⁵ Thus, the PIN2 protein was relatively stable on the PM. However, upon treatment with a higher concentration of MeJA (50 μM), the abundance of PIN2 proteins at the PM was obviously reduced through the endocytosis pathway independent of auxin.⁴⁵ The amount of PIN1 proteins (another member of the auxin efflux carriers) at the PM also decreased after treatment with 50 μM MeJA.⁴⁵ In line with the negative effect of a high concentration of MeJA on PM-resident PIN2 proteins, the asymmetric distribution of auxin-inducible DR5::GFP reporter in root tips was also impaired by MeJA treatment, thereby leading to the misregulation of root gravitropic responses.⁴⁵ Thus, JA affects root gravitropism by altering the intracellular trafficking of PIN family proteins.

In Arabidopsis, heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits function in the regulation of various plant growth and defense processes by coupling to the seven-transmembrane (7TM) regulator of the G protein signaling (RGS) protein AtRGS1.⁴⁶ It has been reported that D-glucose or flg22 could stimulate the endocytosis of AtRGS1 to activate the G-protein-coupled signaling.^47–49 A recent study showed that JA-induced endocytosis of AtRGS1 was also involved in G-protein-mediated JA responses.⁵⁰Gα and Gβ subunit mutants were hyposensitive to MeJA treatment in primary root development, anthocyanin accumulation, and defense gene expression, indicating the possible role of G proteins in JA signaling.⁵⁰ Furthermore, upon MeJA treatment, AtRGS1 proteins were internalized in a phosphorylation- and C-terminus-dependent manner.⁵⁰ Subsequently, AtRGS1 dissociated from G protein alpha subunit 1 (AtGPA1) and released its inhibition of AtGPA1 to trigger the downstream G protein effectors.⁵⁰ In addition, the diffusion dynamics of AtRGS1, such as the motion ranges and diffusion coefficients, also changed in response to MeJA treatment.⁵⁰ Taken together, JA may transmit signals to G proteins through the endocytosis of AtRGS1.

In addition, some PM proteins in other plant species such as Malus domestica hypersensitive-induced reaction 4 (MdHIR4) and Capsicum annuum membrane-located receptor-like protein 1 (CaMRP1) was also involved in JA pathway.^51,52 However, the relationship of the PM location and their biological function is not clear.

Conclusion

In summary, several PM-localized proteins functioned in the JA pathway. Based on the current research, three ABCG proteins were identified as the JA transporters for intracellular transport and long-distance translocation of JA, respectively. Whether other ABCGs or transporter families localized in the PM or endomembrane also have a transport activity for JA still remains to be elucidated. Furthermore, some PM-localized receptors and ion transporters/channels were found to regulate the canonical JA signaling pathway, thereby affecting plant defense responses upon pathogen infection or insect herbivory. These findings indicate that PM-localized proteins associated with the systemic wound responses such as Ca²⁺ waves and SWPs might also involved in regulation of JA biosynthesis or signaling. The in-depth functional characterization of these candidates will provide new insight into the JA pathway and improve our understanding of plant defenses. In addition, there were a few reports about the endocytosis of PM-localized proteins regulated by JA. The proper localization is crucial for transport processes and signal transduction mediated by PM-localized proteins. Thus, a serious of cell biological approaches should be used to uncover the cytological behavior of PM-localized proteins in response to JA.

Funding Statement

This work was supported by the National Natural Science Foundation of China [31871424].