Substrate recognition and transport mechanism of the PIN-FORMED auxin exporters.

Auxins are pivotal plant hormones that regulate plant growth and transmembrane polar auxin transport (PAT) direct patterns of development. The PIN-FORMED (PIN) family of membrane transporters mediate auxin export from the plant cell and play crucial roles in PAT. Here we describe the recently solved structures of PIN transporters, PIN1, PIN3, and PIN8, and also their mechanisms of substrate recognition and transport of auxin. We compare structures of PINs in both inward- and outward-facing conformations, as well as PINs with different binding configurations for auxin. By this comparative analysis, a model emerges for an elevator transport mechanism. Central structural elements necessary for function are identified, and we show that these are shared with other distantly related protein families.

Distinct guard cell-specific remodeling of chromatin accessibility during abscisic acid- and CO2-dependent stomatal regulation.

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2 (carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type-specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell-specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO2 had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance.

Dynamic interactions between SPX proteins, the ubiquitination machinery, and signalling molecules for stress adaptation at a whole-plant level.

The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF-CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.

KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana.

SignificanceKarrikins are chemicals in smoke that stimulate regrowth of many plants after fire. However, karrikin responses are not limited to species from fire-prone environments and can affect growth after germination. Putatively, this is because karrikins mimic an unknown signal in plants, KAI2 ligand (KL). Karrikins likely require modification in plants to become bioactive. We identify a gene, KUF1, that appears to negatively regulate biosynthesis of KL and metabolism of a specific karrikin. KUF1 expression increases in response to karrikin or KL signaling, thus forming a negative feedback loop that limits further activation of the signaling pathway. This discovery will advance understanding of how karrikins are perceived and how smoke-activated germination evolved. It will also aid identification of the elusive KL.

A carlactonoic acid methyltransferase that contributes to the inhibition of shoot branching in Arabidopsis.

SignificanceStrigolactones (SLs) are a group of apocarotenoid hormones, which regulates shoot branching and other diverse developmental processes in plants. The major bioactive form(s) of SLs as endogenous hormones has not yet been clarified. Here, we identify an Arabidopsis methyltransferase, CLAMT, responsible for the conversion of an inactive precursor to a biologically active SL that can interact with the SL receptor in vitro. Reverse genetic analysis showed that this enzyme plays an essential role in inhibiting shoot branching. This mutant also contributed to specifying the SL-related metabolites that could move from root to shoot in grafting experiments. Our work has identified a key enzyme necessary for the production of the bioactive form(s) of SLs.

Modeling reveals posttranscriptional regulation of GA metabolism enzymes in response to drought and cold.

The hormone gibberellin (GA) controls plant growth and regulates growth responses to environmental stress. In monocotyledonous leaves, GA controls growth by regulating division-zone size. We used a systems approach to investigate the establishment of the GA distribution in the maize leaf growth zone to understand how drought and cold alter leaf growth. By developing and parameterizing a multiscale computational model that includes cell movement, growth-induced dilution, and metabolic activities, we revealed that the GA distribution is predominantly determined by variations in GA metabolism. Considering wild-type and UBI::GA20-OX-1 leaves, the model predicted the peak in GA concentration, which has been shown to determine division-zone size. Drought and cold modified enzyme transcript levels, although the model revealed that this did not explain the observed GA distributions. Instead, the model predicted that GA distributions are also mediated by posttranscriptional modifications increasing the activity of GA 20-oxidase in drought and of GA 2-oxidase in cold, which we confirmed by enzyme activity measurements. This work provides a mechanistic understanding of the role of GA metabolism in plant growth regulation.

An in-frame deletion mutation in the degron tail of auxin coreceptor IAA2 confers resistance to the herbicide 2,4-D in Sisymbrium orientale.

The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides such as 2,4-D mimic the effects of IAA by inducing strong auxinic-signaling responses in plants. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and -susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing nine amino acids in the degron tail (DT) of the auxin coreceptor Aux/IAA2 (SoIAA2). The deletion allele cosegregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D-resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This mechanism of synthetic auxin herbicide resistance assigns an in planta function to the DT region of this Aux/IAA coreceptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin-resistant crops using gene-editing.

Resolving binding pathways and solvation thermodynamics of plant hormone receptors.

Plant hormones are small molecules that regulate plant growth, development, and responses to biotic and abiotic stresses. They are specifically recognized by the binding site of their receptors. In this work, we resolved the binding pathways for eight classes of phytohormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) to their canonical receptors using extensive molecular dynamics simulations. Furthermore, we investigated the role of water displacement and reorganization at the binding site of the plant receptors through inhomogeneous solvation theory. Our findings predict that displacement of water molecules by phytohormones contributes to free energy of binding via entropy gain and is associated with significant free energy barriers for most systems analyzed. Also, our results indicate that displacement of unfavorable water molecules in the binding site can be exploited in rational agrochemical design. Overall, this study uncovers the mechanism of ligand binding and the role of water molecules in plant hormone perception, which creates new avenues for agrochemical design to target plant growth and development.

MicroRNAs balance growth and salt stress responses in sweet sorghum.

Salt stress is one of the major causes of reduced crop production, limiting agricultural development globally. Plants have evolved with complex systems to maintain the balance between growth and stress responses, where signaling pathways such as hormone signaling play key roles. Recent studies revealed that hormones are modulated by microRNAs (miRNAs). Previously, two sweet sorghum (Sorghum bicolor) inbred lines with different salt tolerance were identified: the salt-tolerant M-81E and the salt-sensitive Roma. The levels of endogenous hormones in M-81E and Roma varied differently under salt stress, showing a different balance between growth and stress responses. miRNA and degradome sequencing showed that the expression of many upstream transcription factors regulating signal transduction and hormone-responsive genes was directly induced by differentially expressed miRNAs, whose levels were very different between the two sweet sorghum lines. Furthermore, the effects of representative miRNAs on salt tolerance in sorghum were verified through a transformation system mediated by Agrobacterium rhizogenes. Also, miR-6225-5p reduced the level of Ca2+ in the miR-6225-5p-overexpressing line by inhibiting the expression of the Ca2+ uptake gene SbGLR3.1 in the root epidermis and affected salt tolerance in sorghum. This study provides evidence for miRNA-mediated growth and stress responses in sweet sorghum.

Transcriptome disclosure of hormones inducing stigma exsertion in Nicotiana tabacum by corolla shortening.

BACKGROUND: Stigma exsertion is an essential agricultural trait that can promote cross-pollination to improve hybrid seed production efficiency. However, the molecular mechanism controlling stigma exsertion remains unknown. RESULTS: In this study, the Nicotiana tabacum cv. K326 and its two homonuclear-heteroplasmic lines, MSK326 (male-sterile) and MSK326SE (male-sterile and stigma exserted), were used to investigate the mechanism of tobacco stigma exsertion. A comparison of the flowers between the three lines showed that the stigma exsertion of MSK326SE was mainly due to corolla shortening. Therefore, the corollas of the three lines were sampled and presented for RNA-seq analysis, which found 338 candidate genes that may cause corolla shortening. These genes were equally expressed in K326 and MSK326, but differentially expressed in MSK326SE. Among these 338 genes, 15 were involved in hormone synthesis or signal transduction pathways. Consistently, the content of auxin, dihydrozeatin, gibberellin, and jasmonic acid was significantly decreased in the MSK326SE corolla, whereas abscisic acid levels were significantly increased. Additionally, seven genes involved in cell division, cell cycle, or cell expansion were identified. Protein-protein interaction network analysis identified 45 nodes and 79 protein interactions, and the largest module contained 20 nodes and 52 protein interactions, mainly involved in the hormone signal transduction and pathogen defensive pathways. Furthermore, a putative hub gene coding a serine/threonine-protein kinase was identified for the network. CONCLUSIONS: Our results suggest that hormones may play a key role in regulating tobacco stigma exsertion induced by corolla shortening.

Title: Bona Fide Plant Steroid Receptors Are Innovated in Seed Plants and Angiosperms through Successive Whole Genome Duplication Events.

Whole genome duplication (WGD) events are widespread in plants and animals, thus their long-term evolutionary contribution has long been speculated, yet a specific contribution is difficult to verify. Here, we show that ɛ-WGD and ζ-WGD contribute to the origin and evolution of bona fide brassinosteroid (BR) signaling through the innovation of active BR biosynthetic enzymes and active BR receptors from their respective ancestors. We found that BR receptors BRI1 (BR Insensitive 1) and BRL1/3 (BRI1-likes 1/3) derived by ɛ-WGD and ζ-WGD, which occurred in the common ancestor of angiosperms and seed plants, respectively, while orphan BR receptor BRL2 first appeared in stomatophytes. Additionally, CYP85A enzymes synthesizing the bioactive BRs derived from a common ancestor of seed plants while its sister enzymes CYP90 synthesizing BR precursors presented in all land plants, implying possible ligand-receptor coevolution. Consistently, the island domains (IDs) responsible for BR perception in BR receptors were most divergent among different receptor branches, supporting ligand-driven evolution. As a result, BRI1 was the most diversified BR receptor in angiosperms. Importantly, relative to the BR biosynthetic DET2 gene presented in all land plants, BRL2, BRL1/3 and BRI1 had high expression in vascular plants ferns, gymnosperms and angiosperms, respectively. Notably, BRI1 is the most diversified BR receptor with the most abundant expression in angiosperms, suggesting potential positive selection. Therefore, WGDs initiate a neofunctionalization process diverged by ligand-perception and transcriptional expression, which might optimize both BR biosynthetic enzymes and BR receptors, likely contributing to the evolution of land plants, especially seed plants and angiosperms.

Comparative transcriptome revealed the molecular responses of Aconitum carmichaelii Debx. to downy mildew at different stages of disease development.

BACKGROUND: Aconitum carmichaelii Debx. has been widely used as a traditional medicinal herb for a long history in China. It is highly susceptible to various dangerous diseases during the cultivation process. Downy mildew is the most serious leaf disease of A. carmichaelii, affecting plant growth and ultimately leading to a reduction in yield. To better understand the response mechanism of A. carmichaelii leaves subjected to downy mildew, the contents of endogenous plant hormones as well as transcriptome sequencing were analyzed at five different infected stages. RESULTS: The content of 3-indoleacetic acid, abscisic acid, salicylic acid and jasmonic acid has changed significantly in A. carmichaelii leaves with the development of downy mildew, and related synthetic genes such as 9-cis-epoxycarotenoid dioxygenase and phenylalanine ammonia lyase were also significant for disease responses. The transcriptomic data indicated that the differentially expressed genes were primarily associated with plant hormone signal transduction, plant-pathogen interaction, the mitogen-activated protein kinase signaling pathway in plants, and phenylpropanoid biosynthesis. Many of these genes also showed potential functions for resisting downy mildew. Through weighted gene co-expression network analysis, the hub genes and genes that have high connectivity to them were identified, which could participate in plant immune responses. CONCLUSIONS: In this study, we elucidated the response and potential genes of A. carmichaelii to downy mildew, and observed the changes of endogenous hormones content at different infection stages, so as to contribute to the further screening and identification of genes involved in the defense of downy mildew.

Identification and functional analysis of long non-coding RNA (lncRNA) and metabolites response to mowing in hulless barley (Hordeum vulgare L. var. nudum hook. f.).

BACKGROUND: Hulless barley (Hordeum vulgare L. var. nudum Hook. f.) is a significant cereal crop and a substantial source of forage for livestock. Long non-coding RNAs (lncRNAs) and metabolites play crucial roles in the nutrient accumulation and regeneration of hulless barley plants following mowing. The study aimed to identify differentially expressed lncRNAs and metabolites in hulless barley plants by analyzing transcriptomic and metabolomic datasets at 2 h, 24 h, and 72 h following mowing. RESULTS: The study revealed that 190, 90, and 438 lncRNA genes were differentially expressed at the 2 h, 24 h, and 72 h time points compared to the non-mowing control. We identified 14 lncRNA genes-11 downregulated and 3 upregulated-showing consistently significant differential expression across all time points after mowing. These differentially expressed lncRNAs target genes involved in critical processes such as cytokinin signaling, cell wall degradation, storage protein accumulation, and biomass increase. In addition, we identified ten differentially expressed metabolites targeting diverse metabolic pathways, including plant hormones, alkaloids, and flavonoids, before and after mowing at various time points. Endogenous hormone analysis revealed that cytokinin most likely played a crucial role in the regeneration of hulless barley after mowing. CONCLUSIONS: This study created a comprehensive dataset of lncRNAs, metabolites, and hormones in hulless barley after mowing, revealing valuable insights into the functional characteristics of lncRNAs, metabolites, and hormones in regulating plant regeneration. The results indicated that cytokinin plays a significant role in facilitating the regeneration process of hulless barley after mowing. This comprehensive dataset is an invaluable resource for better understanding the complex mechanisms that underlie plant regeneration, with significant implications for crop improvement.

Transcriptomic analyses provide molecular insight into the cold stress response of cold-tolerant alfalfa.

BACKGROUND: Daye No.3 is a novel cultivar of alfalfa (Medicago sativa L.) that is well suited for cultivation in high-altitude regions such as the Qinghai‒Tibet Plateau owing to its high yield and notable cold resistance. However, the limited availability of transcriptomic information has hindered our investigation into the potential mechanisms of cold tolerance in this cultivar. Consequently, we conducted de novo transcriptome assembly to overcome this limitation. Subsequently, we compared the patterns of gene expression in Daye No. 3 during cold acclimatization and exposure to cold stress at various time points. RESULTS: A total of 15 alfalfa samples were included in the transcriptome assembly, resulting in 141.97 Gb of clean bases. A total of 441 DEGs were induced by cold acclimation, while 4525, 5016, and 8056 DEGs were identified at 12 h, 24 h, and 36 h after prolonged cold stress at 4 °C, respectively. The consistency between the RT‒qPCR and transcriptome data confirmed the accuracy and reliability of the transcriptomic data. KEGG enrichment analysis revealed that many genes related to photosynthesis were enriched under cold stress. STEM analysis demonstrated that genes involved in nitrogen metabolism and the TCA cycle were consistently upregulated under cold stress, while genes associated with photosynthesis, particularly antenna protein genes, were downregulated. PPI network analysis revealed that ubiquitination-related ribosomal proteins act as hub genes in response to cold stress. Additionally, the plant hormone signaling pathway was activated under cold stress, suggesting its vital role in the cold stress response of alfalfa. CONCLUSIONS: Ubiquitination-related ribosomal proteins induced by cold acclimation play a crucial role in early cold signal transduction. As hub genes, these ubiquitination-related ribosomal proteins regulate a multitude of downstream genes in response to cold stress. The upregulation of genes related to nitrogen metabolism and the TCA cycle and the activation of the plant hormone signaling pathway contribute to the enhanced cold tolerance of alfalfa.

Overexpression of MtIPT gene enhanced drought tolerance and delayed leaf senescence of creeping bentgrass (Agrostis stolonifera L.).

BACKGROUND: Isopentenyltransferases (IPT) serve as crucial rate-limiting enzyme in cytokinin synthesis, playing a vital role in plant growth, development, and resistance to abiotic stress. RESULTS: Compared to the wild type, transgenic creeping bentgrass exhibited a slower growth rate, heightened drought tolerance, and improved shade tolerance attributed to delayed leaf senescence. Additionally, transgenic plants showed significant increases in antioxidant enzyme levels, chlorophyll content, and soluble sugars. Importantly, this study uncovered that overexpression of the MtIPT gene not only significantly enhanced cytokinin and auxin content but also influenced brassinosteroid level. RNA-seq analysis revealed that differentially expressed genes (DEGs) between transgenic and wild type plants were closely associated with plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis. And numerous DEGs related to growth, development, and stress tolerance were identified, including cytokinin signal transduction genes (CRE1, B-ARR), antioxidase-related genes (APX2, PEX11, PER1), Photosynthesis-related genes (ATPF1A, PSBQ, PETF), flavonoid synthesis genes (F3H, C12RT1, DFR), wax synthesis gene (MAH1), senescence-associated gene (SAG20), among others. CONCLUSION: These findings suggest that the MtIPT gene acts as a negative regulator of plant growth and development, while also playing a crucial role in the plant's response to abiotic stress.

A delayed response in phytohormone signaling and production contributes to pine susceptibility to Fusarium circinatum.

BACKGROUND: Fusarium circinatum is the causal agent of pine pitch canker disease, which affects Pinus species worldwide, causing significant economic and ecological losses. In Spain, two Pinus species are most affected by the pathogen; Pinus radiata is highly susceptible, while Pinus pinaster has shown moderate resistance. In F. circinatum-Pinus interactions, phytohormones are known to play a crucial role in plant defense. By comparing species with different degrees of susceptibility, we aimed to elucidate the fundamental mechanisms underlying resistance to the pathogen. For this purpose, we used an integrative approach by combining gene expression and metabolomic phytohormone analyses at 5 and 10 days post inoculation. RESULTS: Gene expression and metabolite phytohormone contents suggested that the moderate resistance of P. pinaster to F. circinatum is determined by the induction of phytohormone signaling and hormone rearrangement beginning at 5 dpi, when symptoms are still not visible. Jasmonic acid was the hormone that showed the greatest increase by 5 dpi, together with the active gibberellic acid 4 and the cytokinin dehydrozeatin; there was also an increase in abscisic acid and salicylic acid by 10 dpi. In contrast, P. radiata hormonal changes were delayed until 10 dpi, when symptoms were already visible; however, this increase was not as high as that in P. pinaster. Indeed, in P. radiata, no differences in jasmonic acid or salicylic acid production were found. Gene expression analysis supported the hormonal data, since the activation of genes related to phytohormone synthesis was observed earlier in P. pinaster than in the susceptible P. radiata. CONCLUSIONS: We determine that the moderate resistance of P. pinaster to F. circinatum is in part a result of early and strong activation of plant phytohormone-based defense responses before symptoms become visible. We suggest that jasmonic acid signaling and production are strongly associated with F. circinatum resistance. In contrast, P. radiata susceptibility was attributed to a delayed response to the fungus at the moment when symptoms were visible. Our results contribute to a better understanding of the phytohormone-based defense mechanism involved in the Pinus-F. circinatum interactions and provide insight into the development of new strategies for disease mitigation.

Unraveling the molecular mechanisms governing axillary meristem initiation in plants.

MAIN CONCLUSION: Axillary meristems (AMs) located in the leaf axils determine the number of shoots or tillers eventually formed, thus contributing significantly to the plant architecture and crop yields. The study of AM initiation is unavoidable and beneficial for crop productivity. Shoot branching is an undoubted determinant of plant architecture and thus greatly impacts crop yield due to the panicle-bearing traits of tillers. The emergence of the AM is essential for the incipient bud formation, and then the bud is dormant or outgrowth immediately to form a branch or tiller. While numerous reviews have focused on plant branching and tillering development networks, fewer specifically address AM initiation and its regulatory mechanisms. This review synthesizes the significant advancements in the genetic and hormonal factors governing AM initiation, with a primary focus on studies conducted in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.). In particular, by elaborating on critical genes like LATERAL SUPPRESSOR (LAS), which specifically regulates AM initiation and the networks in which they are involved, we attempt to unify the cascades through which they are positioned. We concentrate on clarifying the precise mutual regulation between shoot apical meristem (SAM) and AM-related factors. Additionally, we examine challenges in elucidating AM formation mechanisms alongside opportunities provided by emerging omics approaches to identify AM-specific genes. By expanding our comprehension of the genetic and hormonal regulation of AM development, we can develop strategies to optimize crop production and address global food challenges effectively.

Light at the end of the tunnel: integrating signaling pathways in the coordination of lateral root development.

Root system architecture (RSA) encompasses a range of physical root attributes, including the lateral roots (LRs), root hairs and adventitious roots, in addition to the primary or main root. This overall structure is a crucial trait for efficient water and mineral capture alongside providing anchorage to the plant in the soil and is vital for plant productivity and fitness. RSA dynamics are dependent upon various environmental cues such as light, soil pH, water, mineral nutrition and the belowground microbiome. Among these factors, light signaling through HY5 significantly influences the flexibility of RSA by controlling different signaling pathways that converge at photoreceptors-mediated signaling, also present in the 'hidden half'. Furthermore, several phytohormones also drive the formation and emergence of LRs and are critical to harmonize intra and extracellular stimuli in this regard. This review endeavors to elucidate the impact of these interactions on RSA, with particular emphasis on LR development and to enhance our understanding of the fundamental mechanisms governing the light-regulation of LR growth and physiology.

Enhancement of in situ detection and imaging of phytohormones in plant tissues by MALDI-MSI using 2,4-dihydroxy-5-nitrobenzoic acid as a novel matrix.

Phytohormones possess unique chemical structures, and their physiological effects are regulated through intricate interactions or crosstalk among multiple phytohormones. MALDI-MSI enables the simultaneous detection and imaging of multiple hormones. However, its application for tracing phytohormones is currently restricted by low abundance of hormone in plant and suboptimal matrix selection. 2,4-Dihydroxy-5-nitrobenzoic acid (DHNBA) was reported as a new MALDI matrix for the enhanced detection and imaging of multiple phytohormones in plant tissues. DHNBA demonstrates remarkable sensitivity improvement when compared to the commonly used matrix, 2,5-dihydroxybenzoic acid (DHB), in the detection of isoprenoid cytokinins (trans-zeatin (tZ), dihy-drozeatin (DHZ), meta-topolin (mT), and N6-(Δ2-isopentenyl) adenine (iP)), jasmonic acid (JA), abscisic acid (ABA), and 1-aminocyclo-propane-1-carboxylic acid (ACC) standards. The distinctive properties of DHNBA (i.e. robust UV absorption, uniform matrix deposition, negligible background interference, and high ionization efficiency of phytohormones) make it as an ideal matrix for enhanced detection and imaging of phytohormones, including tZ, DHZ, ABA, indole-3-acetic acid (IAA), and ACC, by MALDI-MSI in various plant tissues, for example germinating seeds, primary/lateral roots, and nodules. Employing DHNBA significantly enhances our capability to concurrently track complex phytohormone biosynthesis pathways while providing precise differentiation of the specific roles played by individual phytohormones within the same category. This will propel forward the comprehensive exploration of phytohormonal functions in plant science.

Leaf ontogeny modulates epinasty through shifts in hormone dynamics during waterlogging in tomato.

Waterlogging leads to hypoxic conditions in the root zone that subsequently cause systemic adaptive responses in the shoot, including leaf epinasty. Waterlogging-induced epinasty in tomato has long been ascribed to the coordinated action of ethylene and auxins. However, other hormonal signals have largely been neglected, despite evidence of their importance in leaf posture control. To cover a large group of growth regulators, we performed a tissue-specific and time-dependent hormonomics analysis. This revealed that multiple hormones are differentially affected throughout a 48 h waterlogging treatment, and that leaf age determines hormone homeostasis and modulates their changes during waterlogging. In addition, we distinguished early hormonal signals that contribute to fast responses to oxygen deprivation from those that potentially sustain the waterlogging response. We found that abscisic acid (ABA) levels peak in petioles within the first 12 h of the treatment, while its precursors only increase much later, suggesting that ABA transport is altered. At the same time, cytokinins (CKs) and their derivatives drastically decline during waterlogging in leaves of all ages. This drop in CKs possibly releases the inhibition of ethylene- and auxin-mediated cell elongation to establish epinastic bending. Auxins themselves rise substantially in the petiole of mature leaves, but mostly after 48 h of root hypoxia. Based on our hormone profiling, we propose that ethylene and ABA might act synergistically as an early signal to induce epinasty, while the balance of indole-3-acetic acid and CKs in the petiole ultimately regulates differential growth.