Jasmonates and salicylic acid: Evolution of defense hormones in land plants.

The emergence of plant hormone signaling pathways is deeply intertwined with land plant evolution. In angiosperms, two plant hormones, salicylic Acid (SA) and Jasmonates (JAs), play a key role in plant defense, where JAs-mediated defenses are typically activated in response to herbivores and necrotrophic pathogens, whereas SA is prioritized against hemi/biotrophic pathogens. Thus, studying the evolution of SA and JAs and their crosstalk is essential to understand the evolution of molecular plant-microbe interactions (EvoMPMI) in land plants. Recent advances in the evolution of SA and JAs biosynthesis, signaling, and crosstalk in land plants illustrated that the insight gained in angiosperms does not necessarily apply to non-seed plant lineages, where the receptors perceive different ligands and the hormones activate pathways independently on the canonical receptors. In this review, recent findings on the two main defense hormones (JAs and SA) in non-seed plants, including functional studies in the bryophyte model Marchantia polymorpha, will be discussed.

Evolution of the jasmonate ligands and their biosynthetic pathways.

Different plant species employ different jasmonates to activate a conserved signalling pathway in land plants, where (+)-7-iso-JA-Ile (JA-Ile) is the ligand for the COI1/JAZ receptor in angiosperms and dn-cis-OPDA, dn-iso-OPDA and Δ4 -dn-iso-OPDA act as ligands in Marchantia polymorpha. In addition, some jasmonates play a COI1-independent role. To understand the distribution of bioactive jasmonates in the green lineage and how their biosynthetic pathways evolved, we performed phylogenetic analyses and systematic jasmonates profiling in representative species from different lineages. We found that both OPDA and dn-OPDA are ubiquitous in all tested land plants and present also in charophyte algae, underscoring their importance as ancestral signalling molecules. By contrast, JA-Ile biosynthesis emerged within lycophytes coincident with the evolutionary appearance of JAR1 function. We identified that the OPR3-independent JA biosynthesis pathway is ancient and predates the evolutionary appearance of the OPR3-dependent pathway. Moreover, we identified a negative correlation between dn-iso-OPDA and JA-Ile in land plants, which supports that in bryophytes and lycophytes dn-iso-OPDA represents the analogous hormone to JA-Ile in other vascular plants.

Conservation of the PBL-RBOH immune module in land plants.

The rapid production of reactive oxygen species (ROS) is a key signaling output in plant immunity. In the angiosperm model species Arabidopsis thaliana (hereafter Arabidopsis), recognition of non- or altered-self elicitor patterns by cell-surface immune receptors activates the receptor-like cytoplasmic kinases (RLCKs) of the AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) family, particularly BOTRYTIS-INDUCED KINASE1 (BIK1).1,2,3 BIK1/PBLs in turn phosphorylate the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to induce apoplastic ROS production.4,5 PBL and RBOH functions in plant immunity have been extensively characterized in flowering plants. Much less is known about the conservation of pattern-triggered ROS signaling pathways in non-flowering plants. In this study, we show that in the liverwort Marchantia polymorpha (hereafter Marchantia), single members of the RBOH and PBL families, namely MpRBOH1 and MpPBLa, are required for chitin-induced ROS production. MpPBLa directly interacts with and phosphorylates MpRBOH1 at specific, conserved sites within its cytosolic N terminus, and this phosphorylation is essential for chitin-induced MpRBOH1-mediated ROS production. Collectively, our work reveals the functional conservation of the PBL-RBOH module that controls pattern-triggered ROS production in land plants.

JAZ is essential for ligand specificity of the COI1/JAZ co-receptor.

Jasmonates are phytohormones that regulate defense and developmental processes in land plants. Despite the chemical diversity of jasmonate ligands in different plant lineages, they are all perceived by COI1/JAZ co-receptor complexes, in which the hormone acts as a molecular glue between the COI1 F-box and a JAZ repressor. It has been shown that COI1 determines ligand specificity based on the receptor crystal structure and the identification of a single COI1 residue, which is responsible for the evolutionary switch in ligand binding. In this work, we show that JAZ proteins contribute to ligand specificity together with COI1. We propose that specific features of JAZ proteins, which are conserved in bryophytes and lycophytes, enable perception of dn-OPDA ligands regardless the size of the COI1 binding pocket. In vascular plant lineages beyond lycophytes, JAZ evolved to limit binding to JA-Ile, thus impeding dn-OPDA recognition by COI1.

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.

A small molecule antagonizes jasmonic acid perception and auxin responses in vascular and nonvascular plants.

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein Coronatine Insensitive 1 (COI1) and a Jasmonate (JA) ZIM-domain (JAZ) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signaling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation, and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1-JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signaling, J4 works as an antagonist of the closely related auxin signaling pathway, preventing Transport Inhibitor Response1/Aux-indole-3-acetic acid interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression, and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways. One-sentence summary: A chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and nonvascular plants.

Plant Evolution: Divergent Plants, Divergent Functions for C1HDZ Orthologs.

Ortholog identification inferred by phylogenetic analyses does not always correlate with functional conservation. The recent functional characterization of the C1HDZ transcription factor in the early-diverging land plant Marchantia polymorpha reveals its role in biotic stress responses, contrary to its orthologs in flowering plants.

An Ancient COI1-Independent Function for Reactive Electrophilic Oxylipins in Thermotolerance.

The jasmonate signaling pathway regulates development, growth, and defense responses in plants. Studies in the model eudicot, Arabidopsis thaliana, have identified the bioactive hormone (jasmonoyl-isoleucine [JA-Ile]) and its Coronatine Insensitive 1 (COI1)/Jasmonate-ZIM Domain (JAZ) co-receptor. In bryophytes, a conserved signaling pathway regulates similar responses but uses a different ligand, the JA-Ile precursor dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), to activate a conserved co-receptor. Jasmonate responses independent of JA-Ile and COI1, thought to be mediated by the cyclopentenone OPDA, have also been suggested, but experimental limitations in Arabidopsis have hindered attempts to uncouple OPDA and JA-Ile biosynthesis. Thus, a clear understanding of this pathway remains elusive. Here, we address the role of cyclopentenones in COI1-independent responses using the bryophyte Marchantia polymorpha, which is unable to synthesize JA-Ile but does accumulate OPDA and dn-OPDA. We demonstrate that OPDA and dn-OPDA activate a COI1-independent pathway that regulates plant thermotolerance genes, and consequently, treatment with these oxylipins protects plants against heat stress. Furthermore, we identify that these molecules signal through their electrophilic properties. By performing comparative analyses between M. polymorpha and two evolutionary distant species, A. thaliana and the charophyte alga Klebsormidium nitens, we demonstrate that this pathway is conserved in streptophyte plants and pre-dates the evolutionary appearance of the COI1-dependent jasmonate pathway, which later co-opted the pre-existing dn-OPDA as its ligand. Taken together, our data indicate that cyclopentenone-regulated COI1-independent signaling is an ancient conserved pathway, whose ancestral role was to protect plants against heat stress. This pathway was likely crucial for plants' successful land colonization and will be critical for adaption to current climate warming.

Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts.

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.

Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions.

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

Jasmonate-Related MYC Transcription Factors Are Functionally Conserved in Marchantia polymorpha.

The lipid-derived phytohormone jasmonoyl-isoleucine regulates plant immunity, growth and development in vascular plants by activating genome-wide transcriptional reprogramming. In Arabidopsis (Arabidopsis thaliana), this process is largely orchestrated by the master regulator MYC2 and related transcription factors (TFs). However, the TFs activating this pathway in basal plant lineages are currently unknown. We report the functional conservation of MYC-related TFs between the eudicot Arabidopsis and the liverwort Marchantia polymorpha, a plant belonging to an early diverging lineage of land plants. Phylogenetic analysis suggests that MYC function first appeared in charophycean algae and therefore predates the evolutionary appearance of any other jasmonate pathway component. M. polymorpha possesses two functionally interchangeable MYC genes, one in females and one in males. Similar to AtMYC2, MpMYCs showed nuclear localization, interaction with JASMONATE-ZIM-DOMAIN PROTEIN repressors, and regulation by light. Phenotypic and molecular characterization of loss- and gain-of-function mutants demonstrated that MpMYCs are necessary and sufficient for activating the jasmonate pathway in M. polymorpha, but unlike their Arabidopsis orthologs, do not regulate fertility. Therefore, despite 450 million years of independent evolution, MYCs are functionally conserved between bryophytes and eudicots. Genetic conservation in an early diverging lineage suggests that MYC function existed in the common ancestor of land plants and evolved from a preexisting MYC function in charophycean algae.

A Single JAZ Repressor Controls the Jasmonate Pathway in Marchantia polymorpha.

JAZ proteins are negative regulators of jasmonate responses, acting both as repressors of transcription factors and as co-receptors of JA-Ile. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn-OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-Ile is not the sole COI1/JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes. Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJAZ in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA responses. Our results show that, consistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

An OPR3-independent pathway uses 4,5-didehydrojasmonate for jasmonate synthesis.

Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3). Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role independent of JA-Ile and its receptor COI1; however, this hypothesis has been challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-didehydro-JA in wounded wild-type and opr3-3 plants. OPR2 was found to reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of OPR3, OPDA enters the ß-oxidation pathway to produce 4,5-ddh-JA as a direct precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA biosynthesis.

The Venus Flytrap Dionaea muscipula Counts Prey-Induced Action Potentials to Induce Sodium Uptake.

Carnivorous plants, such as the Venus flytrap (Dionaea muscipula), depend on an animal diet when grown in nutrient-poor soils. When an insect visits the trap and tilts the mechanosensors on the inner surface, action potentials (APs) are fired. After a moving object elicits two APs, the trap snaps shut, encaging the victim. Panicking preys repeatedly touch the trigger hairs over the subsequent hours, leading to a hermetically closed trap, which via the gland-based endocrine system is flooded by a prey-decomposing acidic enzyme cocktail. Here, we asked the question as to how many times trigger hairs have to be stimulated (e.g., now many APs are required) for the flytrap to recognize an encaged object as potential food, thus making it worthwhile activating the glands. By applying a series of trigger-hair stimulations, we found that the touch hormone jasmonic acid (JA) signaling pathway is activated after the second stimulus, while more than three APs are required to trigger an expression of genes encoding prey-degrading hydrolases, and that this expression is proportional to the number of mechanical stimulations. A decomposing animal contains a sodium load, and we have found that these sodium ions enter the capture organ via glands. We identified a flytrap sodium channel DmHKT1 as responsible for this sodium acquisition, with the number of transcripts expressed being dependent on the number of mechano-electric stimulations. Hence, the number of APs a victim triggers while trying to break out of the trap identifies the moving prey as a struggling Na(+)-rich animal and nutrition for the plant.

Rational design of a ligand-based antagonist of jasmonate perception.

(+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile) regulates developmental and stress responses in plants. Its perception involves the formation of a ternary complex with the F-box COI1 and a member of the JAZ family of co-repressors and leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the COI1-JAZ co-receptor with higher affinity than JA-Ile. On the basis of the co-receptor structure, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists. One derivative, coronatine-O-methyloxime (COR-MO), has strong activity in preventing the COI1-JAZ interaction, JAZ degradation and the effects of JA-Ile or COR on several JA-mediated responses in Arabidopsis thaliana. Moreover, it potentiates plant resistance, preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for plant biology research, our results underscore its biotechnological potential for safer and sustainable agriculture.