Mass spectrometric exploration of phytohormone profiles and signaling networks

Phytohormones have crucial roles in plant growth, development, and acclimation to environmental stress; however, measuring phytohormone levels and unraveling their complex signaling networks and interactions remains challenging. Mass spectrometry (MS) has revolutionized the study of complex biological systems, enabling the comprehensive identification and quantification of phytohormones and their related targets. Here, we review recent advances in MS technologies and highlight studies that have used MS to discover and analyze phytohormone-mediated molecular events. In particular, we focus on the application of MS for profiling phytohormones, elucidating phosphorylation signaling, and mapping protein interactions in plants.     

Mining for treatment-specific and general changes in target compounds and metabolic fingerprints in response to herbivory and phytohormones in Plantago lanceolata

Induction studies focusing on target metabolites may not reveal metabolic changes occurring in plants after various challenges. By contrast, metabolic fingerprinting can be a powerful tool to find patterns that are either treatment-specific or general and was therefore used to depict plant responses after various challenges. Plants of Plantago lanceolata were challenged by mechanical damage, specialist herbivores (aphids or sawfly larvae), generalist herbivores (Lepidopteran caterpillars) or phytohormones (jasmonic or salicylic acid). After 3 d of treatment, local and systemic leaves were analyzed for characteristic target metabolites (iridoid glucosides and verbascoside) by gas chromatography coupled with mass spectrometry (GC-MS) and for metabolic fingerprints by liquid chromatography coupled with time of flight mass spectrometry (LC-TOF-MS). Whereas only marginal changes in target metabolite concentrations were found, metabolic fingerprints were substantially affected especially by generalist and phytohormone treatments. By contrast, mechanical damage and specialist herbivory caused fewer changes. Responses to generalists partly overlapped with the changes caused by jasmonic acid, but many additional peaks were up-regulated. Furthermore, many peaks were co-induced by jasmonic and salicylic acid. The surprisingly high co-induction of peaks by both phytohormones suggests that the signaling pathways regulate a set of common targets. Furthermore, only metabolic fingerprinting could reveal that herbivores induce additional species-specific pathways beyond these phytohormone responses.     

Metabolic and miRNA profiling of TMV infected plants reveals biphasic temporal changes

Plant viral infections induce changes including gene expression and metabolic components. Identification of metabolites and microRNAs (miRNAs) differing in abundance along infection may provide a broad view of the pathways involved in signaling and defense that orchestrate and execute the response in plant-pathogen interactions. We used a systemic approach by applying both liquid and gas chromatography coupled to mass spectrometry to determine the relative level of metabolites across the viral infection, together with a miRs profiling using a micro-array based procedure. Systemic changes in metabolites were characterized by a biphasic response after infection. The first phase, detected at one dpi, evidenced the action of a systemic signal since no virus was detected systemically. Several of the metabolites increased at this stage were hormone-related. miRs profiling after infection also revealed a biphasic alteration, showing miRs alteration at 5 dpi where no virus was detected systemically and a late phase correlating with virus accumulation. Correlation analyses revealed a massive increase in the density of correlation networks after infection indicating a complex reprogramming of the regulatory pathways, either in response to the plant defense mechanism or to the virus infection itself. Our data propose the involvement of a systemic signaling on early miRs alteration.     

Plant apocarotenoids: from retrograde signaling to interspecific communication

Carotenoids are isoprenoid compounds synthesized by all photosynthetic and some non-photosynthetic organisms. They are essential for photosynthesis and contribute to many other aspects of a plant's life. The oxidative breakdown of carotenoids gives rise to the formation of a diverse family of essential metabolites called apocarotenoids. This metabolic process either takes place spontaneously through reactive oxygen species or is catalyzed by enzymes generally belonging to the CAROTENOID CLEAVAGE DIOXYGENASE family. Apocarotenoids include the phytohormones abscisic acid and strigolactones (SLs), signaling molecules and growth regulators. Abscisic acid and SLs are vital in regulating plant growth, development and stress response. SLs are also an essential component in plants’ rhizospheric communication with symbionts and parasites. Other apocarotenoid small molecules, such as blumenols, mycorradicins, zaxinone, anchorene, β-cyclocitral, β-cyclogeranic acid, β-ionone and loliolide, are involved in plant growth and development, and/or contribute to different processes, including arbuscular mycorrhiza symbiosis, abiotic stress response, plant–plant and plant–herbivore interactions and plastid retrograde signaling. There are also indications for the presence of structurally unidentified linear cis-carotene-derived apocarotenoids, which are presumed to modulate plastid biogenesis and leaf morphology, among other developmental processes. Here, we provide an overview on the biology of old, recently discovered and supposed plant apocarotenoid signaling molecules, describing their biosynthesis, developmental and physiological functions, and role as a messenger in plant communication.     

Light Regulation of Gibberellins Metabolism in Seedling Development

Light affects many aspects of plant development, including seed germination, stem elongation, and floral initiation. How photoreceptors control photomorphogenic processes is not yet fully understood. Because phytohormones are chemical regulators of plant development, it may not be surprising that light affects, directly or indirectly, cellular levels and signaling processes of various phytohormones, such as auxin, gibberellins （GA）, cytokinin, ethylene, abscisic acid （ABA）, and brassinosteroids （BR）. Among those phytohormones, light regulation of GA metabolism has probably attracted more attention among photoblologists and it is arguably the most extensively studied plant hormone at present with respect to its role in photomorphogenesis. It has become Increasingly clear that phytochromes and cryptochromes are the major photoreceptors mediating light regulation of GA homeostasis. This short article attempts to examine some recent developments in our understanding of how light and photoreceptors regulate GA blosynthesis and catabolism during seedling development. It is not our intention to carry out a comprehensive review of the field, and readers are referred to recent review articles for a more complete view of this area of study （Kamiya and Garcia-Martinez 1999; Hedden and Phillips 2000; Garcla-Martinez and GI12001; Olszewski et al. 2002; Halliday and Fankhauser 2003; Sun and Gubler 2004）.     

The role of phytohormone signaling in ozone-induced cell death in plants

Ozone is the main photochemical oxidant that causes leaf damage in many plant species, and can thereby significantly decrease the productivity of crops and forests. When ozone is incorporated into plants, it produces reactive oxygen species (ROS), such as superoxide radicals and hydrogen peroxide. These ROS induce the synthesis of several plant hormones, such as ethylene, salicylic acid, and jasmonic acid. These phytohormones are required for plant growth, development, and defense responses, and regulate the extent of leaf injury in ozone-fumigated plants. Recently, responses to ozone have been studied using genetically modified plants and mutants with altered hormone levels or signaling pathways. These researches have clarified the roles of phytohormones and the complexity of their signaling pathways. The present paper reviews the biosynthesis of the phytohormones ethylene, salicylic acid, and jasmonic acid, their roles in plant responses to ozone, and multiple interactions between these phytohormones in ozone-exposed plants.Key words: cross-talk, ethylene, jasmonic acid, ozone, phytohormones, programmed cell death, salicylic acid, signaling pathways     

Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive method was developed for simultaneous quantification of multiple classes of phytohormones and some related metabolites in crude plant extracts without purification or derivatization. High-performance liquid chromatography and electrospray ionization–tandem mass spectrometry with multiple reaction monitoring were used to quantify auxins, cytokinins, abscisic acid, gibberellins, jasmonates, salicylates, and a number of related metabolites in crude plant extracts. The technology was applied to analyze biotic and abiotic stress-induced changes of phytohormones in Arabidopsis tissues, starting with 50–100 mg fresh tissue. Biotic and/or abiotic stresses were shown to differentially affect levels of salicylic acid, jasmonic acid, indole-3-acetic acid, and benzoic acid, in comparison to their methyl esters. Compared with previous methods, sample preparation time and amount of sample required for analysis of phytohormones are reduced, and more classes of hormones are quantitatively profiled. Structurally diverse compounds from complicated biological matrices are determined with high selectivity and sensitivity.     

Current understanding of the interplays between host hormones and plant viral infections

Phytohormones mediate plant development and responses to stresses caused by biotic agents or abiotic factors. The functions of phytohormones in responses to viral infection have been intensively studied, and the emerging picture of complex mechanisms provides insights into the roles that phytohormones play in defense regulation as a whole. These hormone signaling pathways are not simple linear or isolated cascades, but exhibit crosstalk with each other. Here, we summarized the current understanding of recent advances for the classical defense hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) and also the roles of abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinins (CKs), and brassinosteroids (BRs) in modulating plant–virus interactions.     

Electrostatic fasteners hold the T cell receptor-CD3 complex together

Assembly of the T cell receptor includes the formation of trimers stabilized by electrostatic interactions inside the membrane (Call et al., 2003). Such interactions can strongly stabilize subunit associations while permitting conformation changes during signaling.     

Light participates in the auxin‐dependent regulation of plant growth

Light is the energy source for plant photosynthesis and influences plant growth and development. Through multiple photoreceptors, plant interprets light signals through various downstream phytohormones such as auxin. Recently, Chen et al. (2020) uncover a new layer of regulation in IPyA pathway of auxin biosynthesis by light. Here we highlight recent studies about how light controls plant growth through regulating auxin biosynthesis and signaling.     

Application of metabolite profiling to the identification of traits in a population of tomato introgression lines

Naturally occurring variation in wild species can be used to increase the genetic diversity of cultivated crops and improve agronomic value. Populations of introgression lines carrying wild species alleles afford an opportunity to identify traits associated with the introgressed regions, and facilitate characterization of the biochemistry and genetics underlying these phenotypes. Understanding plant metabolic pathways and the interactions between genes, phenotype, and environment is fundamental to functional genomics. Successful analysis of the complex network of plant metabolism requires analytical methods able to record information on as many metabolites as possible. Metabolite profiling is used to provide a snapshot of the metabolome in samples which differ in a known factor such as genetic background. Differences between the metabolite profiles can identify those metabolites/metabolic pathways affected by the introgression and allow genetic maps for metabolic alterations to be established. A Time-of-Flight Mass Spectrometry method is presented, with associated data reduction, used for profiling aqueous metabolites fom tomato. Analysis of ripe fruits of two tomato species, Lycopersicon esculentum and L. pennellii, showed differences in the amounts of many metabolites, including organic acids and sugars. Six introgression lines, L. pennellii introgressions within L. esculentum, were also examined and showed that Principal Component Analysis can reveal subtle differences in metabolism of the introgressed lines when compared to their parents.     

Role of salicylic acid on physiological and biochemical mechanism of salinity stress tolerance in plants

Salinity stress is one of the major abiotic stresses affecting plant growth and productivity globally. In order to improve the yields of plants growing under salt stress bear remarkable importance to supply sustainable agriculture. Acclimation of plants to salinized condition depends upon activation of cascade of molecular network involved in stress sensing/perception, signal transduction, and the expression of specific stress-related genes and metabolites. Isolation of salt overly sensitive (SOS) genes by sos mutants shed us light on the relationship between ion homeostasis and salinity tolerance. Regulation of antioxidative system to maintain a balance between the overproduction of reactive oxygen species and their scavenging to keep them at signaling level for reinstating metabolic activity has been elucidated. However, osmotic adaptation and metabolic homeostasis under abiotic stress environment is required. Recently, role of phytohormones like Abscisic acid, Jasmonic acid, and Salicylic acid in the regulation of metabolic network under osmotic stress condition has emerged through crosstalk between chemical signaling pathways. Thus, abiotic stress signaling and metabolic balance is an important area with respect to increase crop yield under suboptimal conditions. This review focuses on recent developments on improvement in salinity tolerance aiming to contribute sustainable plant yield under saline conditions in the face of climate change.     

Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems

Metabolic profiling using gas chromatography-mass spectrometry technologies is a technique whose potential in the field of functional genomics is largely untapped. To demonstrate the general usefulness of this technique, we applied to diverse plant genotypes a recently developed profiling protocol that allows detection of a wide range of hydrophilic metabolites within a single chromatographic run. For this purpose, we chose four independent potato genotypes characterized by modifications in sucrose metabolism. Using data-mining tools, including hierarchical cluster analysis and principle component analysis, we were able to assign clusters to the individual plant systems and to determine relative distances between these clusters. Extraction analysis allowed identification of the most important components of these clusters. Furthermore, correlation analysis revealed close linkages between a broad spectrum of metabolites. In a second, complementary approach, we subjected wild-type potato tissue to environmental manipulations. The metabolic profiles from these experiments were compared with the data sets obtained for the transgenic systems, thus illustrating the potential of metabolic profiling in assessing how a genetic modification can be phenocopied by environmental conditions. In summary, these data demonstrate the use of metabolic profiling in conjunction with data-mining tools as a technique for the comprehensive characterization of a plant genotype.     

Integrating genomics and metabolomics for engineering plant metabolic pathways

Plant metabolites are characterized by an enormous chemical diversity, every plant having its own complex set of metabolites. This variety poses analytical challenges, both for profiling multiple metabolites in parallel and for the quantitative analysis of selected metabolites. We are only just starting to understand the roles of these metabolites, many of them being involved in adaptations to specific ecological niches and some finding beneficial use (e.g. as pharmaceuticals). Spectacular advances in plant metabolomics offer new possibilities, together with the aid of systems biology, to explore the extraordinary complexity of the plant biochemical capacity. State-of-the art genomics tools can be combined with metabolic profiling to identify key genes that could be engineered for the production of improved crop plants.     

The role of phytohormones ethylene and auxin in plant-nematode interactions

The phytohormones ethylene and auxin regulate many important processes in plants, including cell differentiation, cell expansion, and responses to abiotic stresses. These hormones also play important roles in many plant-pathogen interactions, including regulation of plant defense responses and symptom development. Sedentary plant-parasitic nematodes, which require the formation of a complex feeding site within the host root, are among the world’s most destructive plant pathogens. Nematode-induced feeding sites show dramatic changes in host cell morphology and gene expression. These changes are likely mediated, at least in part, by phytohormones. In the present review, current knowledge of the roles of ethylene and auxin will be explored in two main areas: the specific role of phytohormones in mediating feeding site development by plant-parasitic nematodes and the general role of phytohormones in affecting the ability of parasitic nematodes to cause disease. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 3–7. This article was presented in original.     

Kinome profiling reveals an interaction between jasmonate, salicylate and light control of hyponastic petiole growth in Arabidopsis thaliana

Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ～omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.