Growth Control: A Saga of Cell Walls,ROS, and Peptide Receptors

Despite an increasingly detailed understanding of endogenous and environmental growth-controlling signals and their signaling networks, little is known on how these networks are integrated with the cell expansion machinery. Members of the CrRLK1L family control cell wall properties and cell expansion in a variety of developmental and environmental contexts. Two recent reports provide exciting new insights into the mode of action of these RLKs. One study shows that one family member, FERONIA (FER), is required for the production of hydroxyl radicals in the female gametophyte, which causes pollen tube rupture and sperm cell release during fertilization. Another study shows that FER is a receptor for a signaling peptide (Rapid Alkalinization Factor 1 [RALF1]) that triggers cell wall alkalinization and growth arrest, possibly through the inhibition of plasma membrane H⁺-ATPase activity. RALF1 belongs to a large gene family, with a wide range of expression patterns. Other CrRLK1L family members therefore may also be receptors for RALF-like peptides. These findings have important implications for our understanding of the control of cell wall integrity during growth and raise new intriguing questions.     

A temperature-sensitive FERONIA mutant allele that alters root hair growth

In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

A new, temperature-sensitive allele of FERONIA rapidly inhibits FER signaling and root hair tip-growth at elevated temperatures.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Inter‐relationships between the heterotrimeric Gβ subunit AGB1, the receptor‐like kinase FERONIA,and RALF1 in salinity response

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor‐like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G‐protein‐coupled‐receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1‐like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein β subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt‐induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na⁺ accumulation and decreasing K⁺ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1‐independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.     

Functional evaluation of a homologue of plant rapid alkalinisation factor (RALF) peptides in Fusarium graminearum

The cereal infecting fungus Fusarium graminearum is predicted to possess a single homologue of plant RALF (rapid alkalinisation factor) peptides. Fusarium mutant strains lacking FgRALF were generated and found to exhibit wildtype virulence on wheat and Arabidopsis floral tissue. Arabidopsis lines constitutively overexpressing FgRALF exhibited no obvious change in susceptibility to F. graminearum leaf infection. In contrast transient virus-mediated over-expression (VOX) of FgRALF in wheat prior to F. graminearum infection, slightly increased the rate of fungal colonisation of floral tissue. Ten putative Feronia (FER) receptors of RALF peptide were identified bioinformatically in hexaploid wheat (Triticum aestivum). Transient silencing of two wheat FER homoeologous genes prior to F. graminearum inoculation did not alter the subsequent interaction outcome. Collectively, our VOX results show that the fungal RALF peptide may be a minor contributor in F. graminearum virulence but results from fungal gene deletion experiments indicate potential functional redundancy within the F. graminearum genome. We demonstrate that virus-mediated over-expression is a useful tool to provide novel information about gene/protein function when results from gene deletion/disruption experimentation were uninformative.     

Peptomics,identification of novel cationic Arabidopsis peptides with conserved sequence motifs

Few plant peptides involved in intercellular communication have been experimentally isolated. Sequence analysis of the Arabidopsis thaliana genome has revealed numerous transmembrane receptors predicted to bind proteinacious ligands, emphasizing the importance of identifying peptides with signaling function. Annotation of the Arabidopsis genome sequence has made it possible to identify peptide-encoding genes. However, such annotational identification is impeded because small genes are poorly predicted by gene-prediction algorithms, thus prompting the alternative approaches described here. We initially performed a systematic analysis of short polypeptides encoded by annotated genes on two Arabidopsis chromosomes using SignalP to identify potentially secreted peptides. Subsequent homology searches with selected, putatively secreted peptides, led to the identification of a potential, large Arabidopsis family of 34 genes. The predicted peptides are characterized by a conserved C-terminal sequence motif and additional primary structure conservation in a core region. The majority of these genes had not previously been annotated. A subset of the predicted peptides show high overall sequence similarity to Rapid Alkalinization Factor (RALF), a peptide isolated from tobacco. We therefore refer to this peptide family as RALFL for RALF-Like. RT-PCR analysis confirmed that several of the Arabidopsis genes are expressed and that their expression patterns vary. The identification of a large gene family in the genome of the model organism Arabidopsis thaliana demonstrates that a combination of systematic analysis and homology searching can contribute to peptide discovery.     

RALF22 promotes plant immunity and amplifies the Pep3 immune signal

Rapid alkalinization factors(RALFs) in plants have been reported to dampen pathogenassociated molecular pattern(PAMP)-triggered immunity via suppressing PAMP-induced complex formation between the pattern recognition receptor(PRR) and its co-receptor BAK1. However, the direct and positive role of RALFs in plant immunity remains largely unknown. Herein, we report the direct and positive roles of a typical RALF, RALF22, in plant immunity. RALF22alone directly elicited a variety of typical immune re...     

The Secreted Peptide PIP1 Amplifies Immunity through Receptor-Like Kinase 7

In plants, innate immune responses are initiated by plasma membrane-located pattern recognition receptors (PRRs) upon recognition of elicitors, including exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). Arabidopsis thaliana produces more than 1000 secreted peptide candidates, but it has yet to be established whether any of these act as elicitors. Here we identified an A. thaliana gene family encoding precursors of PAMP-induced secreted peptides (prePIPs) through an in-silico approach. The expression of some members of the family, including prePIP1 and prePIP2, is induced by a variety of pathogens and elicitors. Subcellular localization and proteolytic processing analyses demonstrated that the prePIP1 product is secreted into extracellular spaces where it is cleaved at the C-terminus. Overexpression of prePIP1 and prePIP2, or exogenous application of PIP1 and PIP2 synthetic peptides corresponding to the C-terminal conserved regions in prePIP1 and prePIP2, enhanced immune responses and pathogen resistance in A. thaliana. Genetic and biochemical analyses suggested that the receptor-like kinase 7 (RLK7) functions as a receptor of PIP1. Once perceived by RLK7, PIP1 initiates overlapping and distinct immune signaling responses together with the DAMP PEP1. PIP1 and PEP1 cooperate in amplifying the immune responses triggered by the PAMP flagellin. Collectively, these studies provide significant insights into immune modulation by Arabidopsis endogenous secreted peptides.     

Dual targeting and function of a protease in mitochondria and chloroplasts

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP–GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.     

FERONIA and Her Pals: Functions and Mechanisms

Current research into the FERONIA family of receptor kinases highlights both questions and opportunities for understanding signaling strategies in plant growth and survival.FERONIA and 16 closely related proteins form a distinct clade within the Arabidopsis (Arabidopsis thaliana) superfamily of receptor-like kinases (RLKs), transmembrane proteins with an extracellular domain for signal perception and a cytoplasmic domain that phosphorylates target molecules and induces cellular responses to incoming signals. Several members of this family, such as THESEUS1 and ANXUR1,2, are known to play distinct roles in growth and reproduction; FERONIA is unique in being critically involved in both plant growth and reproduction. The FERONIA family of proteins from Arabidopsis is distinguished from other RLKs by having extracellular protein motifs that share homology with malectin, an animal protein with the capacity to bind dimeric and oligomeric Glc. The possibility that these malectin-like motifs might interact with carbohydrates has generated widespread speculations that these receptor kinases could act as cell-wall sensors, communicating perturbations at the frontline of cell-cell and plant-environment interaction to the cytoplasm to induce responses. Here, we discuss emerging understanding of the functional roles and signaling mechanisms of FERONIA and its related proteins. We also highlight pressing questions, as well as the functional potential of the broader malectin-like domain-containing RLK family that exists across the plant kingdom. We believe FERONIA and her pals provide a rich ground for research with many emerging opportunities for uncovering novel insights into how plants strive for growth and survival.FERONIA/SIRÈNE was first identified genetically more than ten years ago as a key regulator of female fertility in Arabidopsis (Rotman et al., 2003; Huck et al., 2003). It was later determined to be a receptor kinase (Escobar-Restrepo et al., 2007) and one of 17 closely related receptor-like kinases (RLKs) in Arabidopsis (Fig. 1; Hèmaty and Höfte, 2008; Boisson-Dernier et al., 2011; Cheung and Wu, 2011). The name FERONIA (after an Etruscan goddess of fertility) will be used from hereon. Arabidopsis has more than 600 RLKs (Shiu and Bleecker, 2003). Several discoveries made at about the same time led to an extraordinary level of interest in FERONIA and related RLKs. These include: (1) a member of this group, THESEUS1 (named after the Greek mythological figure that slew Procustes the brigand) is a critical regulator of cell growth and appears to function as a surveyor of cell-wall status (Hèmaty et al., 2007); (2) FERONIA functions broadly throughout development and is fundamental to cell and plant growth (Guo et al., 2009; Deslauriers and Larsen, 2010; Duan et al., 2010); and (3) a closely related pair of these RLKs, ANXUR1 and ANXUR2 (named after the consort of FERONIA), is essential for male fertility (Boisson-Dernier et al., 2009; Miyazaki et al., 2009). Last but not least was the report of malectin, a novel protein from animals with the capacity to bind dimeric and oligomeric Glc-binding protein (Schallus et al., 2008) and the realization that FERONIA and related RLKs contain malectin-like motifs (PFAM CL0468) in their extracellular domains (Fig. 1). This led to widespread speculations that FERONIA and related RLKs might interact with carbohydrate moieties and function as sensors of perturbations in the cell wall, communicating conditions at the cell surface to induce appropriate cellular responses (Hèmaty and Höfte, 2008; Boisson-Dernier et al., 2011; Cheung and Wu, 2011; Lindner et al., 2012). FERONIA and related malectin-like domain-containing RLKs are often referred to as the CrRLK1-like RLKs (see e.g. Nibau and Cheung, 2011), after its founding member identified in Catharanthus roseus, CrRLK1 (Schulze-Muth et al., 1996), but for which no functional work has been reported. THESEUS1 was the first member of the group for which a clear functional role was demonstrated, and FERONIA is the most prevalently studied among these RLKs. To provide a functional context for our discussion here, we will refer to the FERONIA-related RLKs in Arabidopsis as the THESEUS1/FERONIA-related RLK family. We update current knowledge about these RLKs from Arabidopsis and highlight pressing questions and emerging opportunities from these and related malectin-like domain-containing RLKs, which are present throughout the plant kingdom (Hèmaty and Höfte, 2008; Antolin-Llovera et al., 2014; Nguyen et al., 2015).Open in a separate windowFigure 1.FERONIA protein domain structure and phylogenetic tree of the Arabidopsis THESEUS1/FERONIA receptor kinase family. A, Deduced FERONIA structural domains. SS, ECD, TM are, respectively, signal peptide, extracellular domain, transmembrane domain. MALA and MALB are tandem malectin-like domains. exJM, extracellular juxtamembrane region. Numbers indicate amino acid residues. B, The THESEUS1/FERONIA protein family.

ADVANCES

• FERONIA and related RLKs have extracellular motifs homologous with the diglucose-binding protein malectin, so potentially interact with cell wall carbohydrates and mediate wall-related activities.
• FERONIA controls growth and female fertility, mediates hormone- and pathogen-induced responses, and is required for a normal cell wall.
• FERONIA is a receptor for RALF1, a peptide regulatory factor, which affects phosphorylation of FERONIA and the key cell growth regulator H⁺-ATPase.
• FERONIA-related THESEUS1 suppresses growth in cellulose-deficient mutants, suggesting a role as surveyor of wall conditions.
• FERONIA homologs ANXUR1 and ANXUR2 ensure pollen tube integrity and male fertility.
• FERONIA, ANXUR1, and ANXUR2 signaling collectively involves a GPI-AP, a MLO protein, the RHO GTPase switch, NADPH oxidases, and a receptor-like cytoplasmic kinase; ROS and Ca²⁺ are key elements in their functions.

Extracellular homology with malectin (Schallus et al., 2008; Fig. 1A) distinguishes the THESEUS1/FERONIA-related RLKs from other members of the Arabidopsis RLK family. Malectin, named after its in vitro ability to bind maltose (Glc α1-4 Glc), is a conserved animal protein located in the lumen of the endoplasmic reticulum where it is involved in protein quality control in the early steps of secretion (Schallus et al., 2008; Qin et al., 2012). A majority of the Arabidopsis THESEUS1/FERONIA family (Fig. 1B) has tandem malectin-like motifs (Fig. 1A; Boisson-Dernier et al., 2011) in their extracellular domains. We focus our discussion on FERONIA, THESEUS1, and three other members of the family, ANXUR1, ANXUR2, and ERULUS/[Ca²⁺]_cyt-associated Protein Kinase1, for which clear biological roles have been demonstrated (Miyazaki et al., 2009; Boisson-Dernier et al., 2009; Bai et al., 2014). A contribution to cell growth and morphogenesis has also been reported for HERCULES1 (Guo et al., 2009) and CURVY (Gachomo et al., 2014), but details regarding their functions remain limited.     

Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis

The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log₂ fold change of ±0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.     

The cloning and characterization of a poplar stomatal density gene

EPIDERMAL PATTERNING FACTOR1 (EPF1) is a well characterized negative regulator of cell division in Arabidopsis thaliana (AtEPF1) where the primary region of localization is the leaf. However, little data have been reported on the role of EPF1 in other plant species. In this study, the EPF1 gene from Arabidopsis and the newly identified poplar ortholog from Populus trichocarpa (PtaEPF1) were overexpressed in a hybrid poplar genotype. We attempted to identify the physiological role of PtaEPF1. Gene overexpression experiments were performed to determine if and how stomatal density (SD) numbers were affected. The poplar 717-1B4 (P. tremula × P. alba) genotype was used in the study. Results presented here suggest that overexpression of PtaEPF1 and AtEPF1 in poplar led to significantly altered SD and also affected transgenic water stress tolerance. Overexpression of AtEPF1 in 717-1B4 led to the most dramatic decrease in SD while overexpression of PtaEPF1 in 717-1B4 significantly increased SD in several transgenic lines, an indication that EPF1 may have additional functions in poplar. Also, abnormalities in leaf morphology were discovered that indicated overexpression of AtEPF1 or PtaEPF1 in poplar triggered aberrant phenotypes not seen in other published Arabidopsis studies, an indication of additional pathway involvement.     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

A MORN-domain protein regulates growth and seed production and enhances freezing tolerance in Arabidopsis

AtRGP (AT4G17080, Arabidopsis thaliana reduction in growth and productivity) contains two N-terminal transmembrane helices and seven membrane occupation and recognition nexus motifs at its C-terminus, and associates with phosphatidylinositol phosphate kinase. To elucidate the function of AtRGP, we employed mutant plants to analyze gene expression, plant phenotypes, protein localization, structure and function of the chloroplast, and freezing tolerance. Overexpression of AtRGP increased growth rate, hypocotyl elongation, leaf size, seed production, photosynthetic rate, and freezing tolerance, and promoted chloroplast organization and stacking of grana. By contrast, Atrgp null mutants exhibited a smaller plant size, reduced seed production, photosynthetic rate, and freezing tolerance, and displayed abnormal chloroplast organization with insufficient stacking of grana. Considering these data, we postulate that AtRGP may bind transiently to the chloroplast envelope and interact with other proteins under certain conditions, thereby regulating cellular processes involved in growth and abiotic stress responses.     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

Plant PA signaling via diacylglycerol kinase

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential ³²P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGK?, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.