Chao Zhang

Background: Phytohormones are low-abundance biomolecules essential for regulating plant growth and responses to various environmental factors. Direct identification and quantification of these compounds in heterogeneous tissues, especially after wounding or pathogen infection, remain challenging due to the presence of naturally occurring isomers and structurally similar molecules. Conventional liquid chromatography-mass spectrometry (LC–MS)-based metabolomics profiling enables the detection of major stress-related hormones but does not provide spatial or temporal resolution within infected tissues. Results: To enhance in situ separation and localisation of low-abundance metabolites, we applied ion mobility-assisted desorption electrospray ionisation mass spectrometry imaging (DESI‑IM‑MSI). This method was used to characterise key stress‑related phytohormones in Arabidopsis thaliana leaves inoculated with Pseudomonas syringae . DESI‑IM‑MSI revealed distinct drift ion distribution patterns reflecting the in situ abundance of targeted phytohormones and their metabolites (salicylic acid, jasmonic acid, auxin, abscisic acid and cytokinins), while minimising interference from background isomers. Importantly, changes in salicylic acid and its metabolites were consistent with parallel LC‑MS analysis, demonstrating the accuracy of the spatially resolved measurements. Conclusions: DESI‑IM‑MSI provides precise spatial and temporal insights into selected biomolecules, including structurally related isomers. The method represents a promising approach for generating comprehensive hormonomic and metabolomic maps during plant responses to pathogen attack, offering an alternative to conventional LC‑MS profiling.

Nitric oxide (NO) is a plant gasotransmitter that regulates plant growth and development by interacting with different regulatory pathways. Among these, brassinosteroids (BRs) have become candidate phytohormones that interact antagonistically and closely with NO to regulate plant growth. Seedling growth in NO-deficient mutants is enhanced and hyper-responds to BR treatments, while NO overaccumulator mutants show some developmental defects. Interestingly, this hyper-response was also observed in mutants that had both constitutively activated BR signalling and reduced NO levels. BR signalling mutants exhibited different responses to the repressive role of NO in seedlings. The phenotypes observed were attributed to the induction of BR-repressed biosynthetic genes and the repression of BR-induced growth-promoting genes detected in NO-overaccumulating plants, with opposite gene expression patterns detected in NO-deficient plants. Furthermore, the activation of BR signalling has an impact on NO accumulation, as BR-treated plants or mutants with activated BR signalling showed hyperaccumulation of NO. Finally, elevated endogenous levels of brassinolide, the most bioactive BR, were found in NO-deficient plants, which could explain the growth promotion observed in this mutant. These findings contribute to a better understanding of the growth-repressive role of NO during seedling development through its interaction with BR biosynthesis, signalling, and responses.

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defense versus growth’ responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1 (COI1)-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA-amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 (GH3) and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1 (ILR1)/ILR1-like (ILL) families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se.

Due to technological advances in mass spectrometry, significant progress has been achieved recently in plant hormone research. Nowadays, plant hormonomics is well established as a fully integrated scientific field focused on the analysis of phytohormones, mainly on their isolation, identification, and spatiotemporal quantification in plants. This review represents a comprehensive meta-study of the advances in the phytohormone analysis by mass spectrometry over the past decade. To address current trends and future perspectives, Web of Science data were systematically collected and key features such as mass spectrometry-based analyses were evaluated using multivariate data analysis methods. Our findings showed that plant hormonomics is currently divided into targeted and untargeted approaches. Both aim to miniaturize the sample, allowing high-resolution quantification to be covered in plant organs as well as subcellular compartments. Therefore, we can study plant hormone biosynthesis, metabolism, and signalling at a spatio-temporal resolution. Moreover, this trend has recently been accelerated by technological advances such as fluorescence-activated cell sorting or mass spectrometry imaging.

In situ separation and visualization of synthetic and naturally occurring isomers from heterogeneous plant tissues, especially when they share similar molecular structures, are a challenging task. In this study, we combined the ion mobility separation with desorption electrospray ionization mass spectrometry imaging (DESI-IM-MSI) to achieve a direct separation and visualization of two synthetic auxin derivatives, auxinole and its structural isomer 4pTb-MeIAA, as well as endogenous auxins from Arabidopsis samples. Distinct distribution of these synthetic isomers and endogenous auxins in Arabidopsis primary roots and hypocotyls was achieved in the same imaging analysis from both individually treated and cotreated samples. We also observed putative metabolites of synthetic auxin derivatives, i.e. auxinole amino acid conjugates and hydrolysed 4pTb-MeIAA product — 4pTb-IAA, based on their unique drifting ion intensity patterns. Furthermore, DESI-IM-MSI-revealed abundance of endogenous auxins and synthetic isomers was validated by liquid chromatography–mass spectrometry (LC-MS). Our results demonstrate that DESI-IM-MSI could be used as a robust technique for detecting endogenous and exogenous isomers and provide a spatiotemporal evaluation of hormonomics profiles in plants. Graphical abstract

Phytohormones (plant hormones) are a group of small signalling molecules that act as important endogenous regulators in plant development and stress responses. Previous research has identified the phytohormone species, jasmonates, auxins and abscisic acid, and their related compounds in stressed leaf extracts. However, in situ visualisations of endogenous phytohormones from intact plant tissues remain elusive without the usage of labels or reporters. Mass spectrometry imaging is a label-free analytical technique that has been successfully applied for the direct detection of plant proteins, lipids, carbohydrates and many other biomolecules. In this study, desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) was used for high throughput visualisation and evaluation of wound-induced phytohormones inside Arabidopsis thaliana leaves. The results showed higher levels of jasmonates, salicylic acid, abscisic acid and indole-3-acetic acid in their ion intensity maps established from wounded leaves compared to control leaves, which have been validated in the parallel liquid chromatography–mass spectrometry quantification, and the untainted distributions of the identified phytohormones in leaves were confirmed by mass spectrometry imaging of instant leaf imprinted thin-layer chromatography plate samples. Further statistical analysis has not only demonstrated a significant increase of jasmonic acid and its precursor compounds in wounded leaves/regions but also highlighted a potential correlation in different phytohormone species. Our results suggest that DESI-MSI can be used to in situ characterise multiple phytohormone compounds from intact leaves with 200 μm spatial resolution to provide insight into phytohormone distributions in wounded leaves, along with their correlated precursors and metabolites under mechanical stress.