Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid.

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

Influence of Exogenous 24-Epicasterone on the Hormonal Status of Soybean Plants.

Brassinosteroids (BRs) are key phytohormones involved in the regulation of major processes of cell metabolism that guide plant growth. In the past decades, new evidence has made it clear that BRs also play a key role in the orchestration of plant responses to many abiotic and biotic stresses. In the present work, we analyzed the impact of foliar treatment with 24-epicastasterone (ECS) on the endogenous content of major phytohormones (auxins, salicylic acid, jasmonic acid, and abscisic acid) and their intermediates in soybean leaves 7 days following the treatment. Changes in the endogenous content of phytohormones have been identified and quantified by LC/MS. The obtained results point to a clear role of ECS in the upregulation of auxin content (indole-3-acetic acid, IAA) and downregulation of salicylic, jasmonic, and abscisic acid levels. These data confirm that under optimal conditions, ECS in tested concentrations of 0.25 µM and 1 µM might promote growth in soybeans by inducing auxin contents. Benzoic acid (a precursor of salicylic acid (SA)), but not SA itself, has also been highly accumulated under ECS treatment, which indicates an activation of the adaptation strategies of cell metabolism to possible environmental challenges.

The PROSCOOP10 Gene Encodes Two Extracellular Hydroxylated Peptides and Impacts Flowering Time in Arabidopsis.

The Arabidopsis PROSCOOP genes belong to a family predicted to encode secreted pro-peptides, which undergo maturation steps to produce peptides named SCOOP. Some of them are involved in defence signalling through their perception by a receptor complex including MIK2, BAK1 and BKK1. Here, we focused on the PROSCOOP10 gene, which is highly and constitutively expressed in aerial organs. The MS/MS analyses of leaf apoplastic fluids allowed the identification of two distinct peptides (named SCOOP10#1 and SCOOP10#2) covering two different regions of PROSCOOP10. They both possess the canonical S-X-S family motif and have hydroxylated prolines. This identification in apoplastic fluids confirms the biological reality of SCOOP peptides for the first time. NMR and molecular dynamics studies showed that the SCOOP10 peptides, although largely unstructured in solution, tend to assume a hairpin-like fold, exposing the two serine residues previously identified as essential for the peptide activity. Furthermore, PROSCOOP10 mutations led to an early-flowering phenotype and increased expression of the floral integrators SOC1 and LEAFY, consistent with the de-regulated transcription of PROSCOOP10 in several other mutants displaying early- or late-flowering phenotypes. These results suggest a role for PROSCOOP10 in flowering time, highlighting the functional diversity within the PROSCOOP family.

DIACYLGLYCEROL KINASE 5 participates in flagellin-induced signaling in Arabidopsis.

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant-microbe interactions is discussed.

An Arabidopsis mutant deficient in phosphatidylinositol-4-phosphate kinases ß1 and ß2 displays altered auxin-related responses in roots.

Phosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kß1 and ß2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kß1ß2 double mutant was insensitive to exogenous auxin concerning inhibition of root length and cell elongation; it also responded more slowly to gravistimulation. The pi4kß1ß2 root transcriptome displayed some similarities to a wild type plant response to auxin. Yet, not all the genes displayed such a constitutive auxin-like response. Besides, most assessed genes did not respond to exogenous auxin. This is consistent with data with the transcriptional reporter DR5-GUS. The content of bioactive auxin in the pi4kß1ß2 roots was similar to that in wild-type ones. Yet, an enhanced auxin-conjugating activity was detected and the auxin level reporter DII-VENUS did not respond to exogenous auxin in pi4kß1ß2 mutant. The mutant exhibited altered subcellular trafficking behavior including the trapping of PIN-FORMED 2 protein in rapidly moving vesicles. Bigger and less fragmented vacuoles were observed in pi4kß1ß2 roots when compared to the wild type. Furthermore, the actin filament web of the pi4kß1ß2 double mutant was less dense than in wild-type seedling roots, and less prone to rebuilding after treatment with latrunculin B. A mechanistic model is proposed in which an altered PI4K activity leads to actin filament disorganization, changes in vesicle trafficking, and altered auxin homeostasis and response resulting in a pleiotropic root phenotypes.

Phosphatidic Acid in Plant Hormonal Signaling: From Target Proteins to Membrane Conformations.

Cells sense a variety of extracellular signals balancing their metabolism and physiology according to changing growth conditions. Plasma membranes are the outermost informational barriers that render cells sensitive to regulatory inputs. Membranes are composed of different types of lipids that play not only structural but also informational roles. Hormones and other regulators are sensed by specific receptors leading to the activation of lipid metabolizing enzymes. These enzymes generate lipid second messengers. Among them, phosphatidic acid (PA) is a well-known intracellular messenger that regulates various cellular processes. This lipid affects the functional properties of cell membranes and binds to specific target proteins leading to either genomic (affecting transcriptome) or non-genomic responses. The subsequent biochemical, cellular and physiological reactions regulate plant growth, development and stress tolerance. In the present review, we focus on primary (genome-independent) signaling events triggered by rapid PA accumulation in plant cells and describe the functional role of PA in mediating response to hormones and hormone-like regulators. The contributions of individual lipid signaling enzymes to the formation of PA by specific stimuli are also discussed. We provide an overview of the current state of knowledge and future perspectives needed to decipher the mode of action of PA in the regulation of cell functions.

A ménage à trois: salicylic acid, growth inhibition, and immunity.

Salicylic acid (SA) is a plant hormone almost exclusively associated with the promotion of immunity. It is also known that SA has a negative impact on plant growth, yet only limited efforts have been dedicated to explain this facet of SA action. In this review, we focus on SA-related reduced growth and discuss whether it is a regulated process and if the role of SA in immunity imperatively comes with growth suppression. We highlight molecular targets of SA that interfere with growth and describe scenarios where SA can improve plant immunity without a growth penalty.

BODIPY Conjugate of Epibrassinolide as a Novel Biologically Active Probe for In Vivo Imaging.

Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

Correction: Pokotylo, I., et al. Deciphering the Binding of Salicylic Acid to Arabidopsis thaliana Chloroplastic GAPDH-A1. Int. J. Mol. Sci. 2020, 21, 4678.

The authors wish to make the following correction to their published paper [...].

Deciphering the Binding of Salicylic Acid to Arabidopsis thaliana Chloroplastic GAPDH-A1.

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.

Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant.

BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kß1ß2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kß1ß2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kß1 and PI4Kß2. METHODS: To achieve this we used the sid2pi4kß1ß2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kß1ß2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. KEY RESULTS: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kß1ß2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kß1ß2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kß1ß2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. CONCLUSIONS: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kß1ß2 triple mutant revealed the role of PI4Kß1/ß2 per se, thus showing the importance of these enzymes in plant defence responses.

Salicylic Acid Binding Proteins (SABPs): The Hidden Forefront of Salicylic Acid Signalling.

Salicylic acid (SA) is a phytohormone that plays important roles in many aspects of plant life, notably in plant defenses against pathogens. Key mechanisms of SA signal transduction pathways have now been uncovered. Even though details are still missing, we understand how SA production is regulated and which molecular machinery is implicated in the control of downstream transcriptional responses. The NPR1 pathway has been described to play the main role in SA transduction. However, the mode of SA perception is unclear. NPR1 protein has been shown to bind SA. Nevertheless, NPR1 action requires upstream regulatory events (such as a change in cell redox status). Besides, a number of SA-induced responses are independent from NPR1. This shows that there is more than one way for plants to perceive SA. Indeed, multiple SA-binding proteins of contrasting structures and functions have now been identified. Yet, all of these proteins can be considered as candidate SA receptors and might have a role in multinodal (decentralized) SA input. This phenomenon is unprecedented for other plant hormones and is a point of discussion of this review.

The SCOOP12 peptide regulates defense response and root elongation in Arabidopsis thaliana.

Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.

The phosphatidic acid paradox: Too many actions for one molecule class? Lessons from plants.

Phosphatidic acid (PA) is a simple phospholipid observed in most organisms. PA acts as a key metabolic intermediate and a second messenger that regulates many cell activities. In plants, PA is involved in numerous cell responses induced by hormones, stress inputs and developmental processes. Interestingly, PA production can be triggered by opposite stressors, such as cold and heat, or by hormones that are considered to be antagonistic, such as abscisic acid and salicylic acid. This property questions the specificity of the responses controlled by PA. Are there generic responses to PA, meaning that cell regulation triggered by PA would be always the same, even in opposite physiological situations? Alternatively, do the responses to PA differ according to the physiological context within the cells? If so, the mechanisms that regulate the divergence of PA-controlled reactions are poorly defined. This review summarizes the latest opinions on how PA signalling is directed in plant cells and examines the intrinsic properties of PA that enable its regulatory diversity. We propose a concept whereby PA regulatory messages are perceived as complex "signatures" that take into account their production site, the availability of target proteins and the relevant cellular environments.

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz.

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12-24 hr later in leaves. Relative levels of species with short-chained odd-numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long-chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd-numbered fatty acids in plant phosphoglycerolipids.

Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

Corrigendum: Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome.

[This corrects the article on p. 608 in vol. 5, PMID: 25426125.].

Molecular mechanisms of gravity perception and signal transduction in plants.

Gravity is one of the environmental cues that direct plant growth and development. Recent investigations of different gravity signalling pathways have added complexity to how we think gravity is perceived. Particular cells within specific organs or tissues perceive gravity stimulus. Many downstream signalling events transmit the perceived information into subcellular, biochemical, and genomic responses. They are rapid, non-genomic, regulatory, and cell-specific. The chain of events may pass by signalling lipids, the cytoskeleton, intracellular calcium levels, protein phosphorylation-dependent pathways, proteome changes, membrane transport, vacuolar biogenesis mechanisms, or nuclear events. These events culminate in changes in gene expression and auxin lateral redistribution in gravity response sites. The possible integration of these signalling events with amyloplast movements or with other perception mechanisms is discussed. Further investigation is needed to understand how plants coordinate mechanisms and signals to sense this important physical factor.