Cytokinins regulate spatially-specific ethylene production to control root growth in Arabidopsis.

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatial-specific regulation of ethylene biosynthesis as a key aspect of hormonal control over root growth.

Epigenetics and plant hormone dynamics - a functional and methodological perspective.

Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

iReenCAM: automated imaging system for kinetic analysis of photosynthetic pigment biosynthesis at high spatiotemporal resolution during early deetiolation.

Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type Arabidopsis and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.

Light Quality and Intensity Modulate Cold Acclimation in Arabidopsis.

Plant survival in temperate zones requires efficient cold acclimation, which is strongly affected by light and temperature signal crosstalk, which converge in modulation of hormonal responses. Cold under low light conditions affected Arabidopsis responses predominantly in apices, possibly because energy supplies were too limited for requirements of these meristematic tissues, despite a relatively high steady-state quantum yield. Comparing cold responses at optimal light intensity and low light, we found activation of similar defence mechanisms-apart from CBF1-3 and CRF3-4 pathways, also transient stimulation of cytokinin type-A response regulators, accompanied by fast transient increase of trans-zeatin in roots. Upregulated expression of components of strigolactone (and karrikin) signalling pathway indicated involvement of these phytohormones in cold responses. Impaired response of phyA, phyB, cry1 and cry2 mutants reflected participation of these photoreceptors in acquiring freezing tolerance (especially cryptochrome CRY1 at optimal light intensity and phytochrome PHYA at low light). Efficient cold acclimation at optimal light was associated with upregulation of trans-zeatin in leaves and roots, while at low light, cytokinin (except cis-zeatin) content remained diminished. Cold stresses induced elevation of jasmonic acid and salicylic acid (in roots). Low light at optimal conditions resulted in strong suppression of cytokinins, jasmonic and salicylic acid.

Mixed Models as a Tool for Comparing Groups of Time Series in Plant Sciences.

Plants adapt to continual changes in environmental conditions throughout their life spans. High-throughput phenotyping methods have been developed to noninvasively monitor the physiological responses to abiotic/biotic stresses on a scale spanning a long time, covering most of the vegetative and reproductive stages. However, some of the physiological events comprise almost immediate and very fast responses towards the changing environment which might be overlooked in long-term observations. Additionally, there are certain technical difficulties and restrictions in analyzing phenotyping data, especially when dealing with repeated measurements. In this study, a method for comparing means at different time points using generalized linear mixed models combined with classical time series models is presented. As an example, we use multiple chlorophyll time series measurements from different genotypes. The use of additional time series models as random effects is essential as the residuals of the initial mixed model may contain autocorrelations that bias the result. The nature of mixed models offers a viable solution as these can incorporate time series models for residuals as random effects. The results from analyzing chlorophyll content time series show that the autocorrelation is successfully eliminated from the residuals and incorporated into the final model. This allows the use of statistical inference.

Signal Integration in Plant Abiotic Stress Responses via Multistep Phosphorelay Signaling.

Plants growing in any particular geographical location are exposed to variable and diverse environmental conditions throughout their lifespan. The multifactorial environmental pressure resulted into evolution of plant adaptation and survival strategies requiring ability to integrate multiple signals that combine to yield specific responses. These adaptive responses enable plants to maintain their growth and development while acquiring tolerance to a variety of environmental conditions. An essential signaling cascade that incorporates a wide range of exogenous as well as endogenous stimuli is multistep phosphorelay (MSP). MSP mediates the signaling of essential plant hormones that balance growth, development, and environmental adaptation. Nevertheless, the mechanisms by which specific signals are recognized by a commonly-occurring pathway are not yet clearly understood. Here we summarize our knowledge on the latest model of multistep phosphorelay signaling in plants and the molecular mechanisms underlying the integration of multiple inputs including both hormonal (cytokinins, ethylene and abscisic acid) and environmental (light and temperature) signals into a common pathway. We provide an overview of abiotic stress responses mediated via MSP signaling that are both hormone-dependent and independent. We highlight the mutual interactions of key players such as sensor kinases of various substrate specificities including their downstream targets. These constitute a tightly interconnected signaling network, enabling timely adaptation by the plant to an ever-changing environment. Finally, we propose possible future directions in stress-oriented research on MSP signaling and highlight its potential importance for targeted crop breeding.

Split-root systems: detailed methodology, alternative applications, and implications at leaf proteome level.

BACKGROUND: Split-root systems (SRS) have many applications in plant sciences, but their implementation, depending on the experimental design, can be difficult and time-consuming. Additionally, the system is not exempt from limitations, since the time required for the establishment of the SRS imposes a limit to how early in plant development experiments can be performed. Here, we optimized and explained in detail a method for establishing a SRS in young Arabidopsis thaliana seedlings, both in vitro and in soil. RESULTS: We found that the partial de-rooting minimized the recovery time compared to total de-rooting, thus allowing the establishment of the split-root system in younger plants. Analysis of changes in the Arabidopsis leaf proteome following the de-rooting procedure highlighted the distinct metabolic alterations that totally and partially de-rooted plants undergo during the healing process. This system was also validated for its use in drought experiments, as it offers a way to apply water-soluble compounds to plants subjected to drought stress. By growing plants in a split-root system with both halves being water-deprived, it is possible to apply the required compound to one half of the root system, which can be cut from the main plant once the compound has been absorbed, thus minimizing rehydration and maintaining drought conditions. CONCLUSIONS: Partial de-rooting is the suggested method for obtaining split-root systems in small plants like Arabidopsis thaliana, as growth parameters, survival rate, and proteomic analysis suggest that is a less stressful procedure than total de-rooting, leading to a final rosette area much closer to that of uncut plants. Additionally, we provide evidence that split root-systems can be used in drought experiments where water-soluble compounds are applied with minimal effects of rehydration.

Steady-State Levels of Cytokinins and Their Derivatives May Serve as a Unique Classifier of Arabidopsis Ecotypes.

We determined steady-state (basal) endogenous levels of three plant hormones (abscisic acid, cytokinins and indole-3-acetic acid) in a collection of thirty different ecotypes of Arabidopsis that represent a broad genetic variability within this species. Hormone contents were analysed separately in plant shoots and roots after 21 days of cultivation on agar plates in a climate-controlled chamber. Using advanced statistical and machine learning methods, we tested if basal hormonal levels can be considered a unique ecotype-specific classifier. We also explored possible relationships between hormone levels and the prevalent environmental conditions in the site of origin for each ecotype. We found significant variations in basal hormonal levels and their ratios in both root and shoot among the ecotypes. We showed the prominent position of cytokinins (CK) among the other hormones. We found the content of CK and CK metabolites to be a reliable ecotype-specific identifier. Correlation with the mean temperature at the site of origin and the large variation in basal hormonal levels suggest that the high variability may potentially be in response to environmental factors. This study provides a starting point for ecotype-specific genetic maps of the CK metabolic and signalling network to explore its contribution to the adaptation of plants to local environmental conditions.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

Stimulation of ipt overexpression as a tool to elucidate the role of cytokinins in high temperature responses of Arabidopsis thaliana.

Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS - targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.

Plant responses to ambient temperature fluctuations and water-limiting conditions: A proteome-wide perspective.

BACKGROUND: Every year, environmental stresses such as limited water and nutrient availability, salinity, and temperature fluctuations inflict significant losses on crop yields across the globe. Recently, developments in analytical techniques, e.g. mass spectrometry, have led to great advances towards understanding how plants respond to environmental stresses. These processes are mediated by many molecular pathways and, at least partially, via proteome-environment interactions. SCOPE OF REVIEW: This review focuses on the current state of knowledge about interactions between the plant proteome and the environment, with a special focus on drought and temperature responses of plant proteome dynamics, and subcellular and organ-specific compartmentalization, in Arabidopsis thaliana and crop species. MAJOR CONCLUSIONS: Correct plant development under non-optimal conditions requires complex self-protection mechanisms, many of them common to different abiotic stresses. Proteome analyses of plant responses to temperature and drought stresses have revealed an intriguing interplay of modifications, mainly affecting the photosynthetic machinery, carbohydrate metabolism, and ROS activation and scavenging. Imbalances between transcript-level and protein-level regulation observed during adaptation to abiotic stresses suggest that many of the regulatory processes are controlled at translational and post-translational levels; proteomics is thus essential in revealing important regulatory networks. GENERAL SIGNIFICANCE: Because information from proteomic data extends far beyond what can be deduced from transcriptome analysis, the results of proteome studies have substantially deepened our understanding of stress adaptation in plants; this is clearly a prerequisite for designing strategies to improve the yield and quality of crops grown under unfavorable conditions brought about by ongoing climatic change. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Roles of proteome dynamics and cytokinin signaling in root to hypocotyl ratio changes induced by shading roots of Arabidopsis seedlings.

In nature, root systems of most terrestrial plants are protected from light exposure by growing in a dark soil environment. Hence, in vitro cultivation in transparent Petri dishes leads to physiological perturbations, but the mechanisms underlying root-mediated light perception and responses have not been fully elucidated. Thus, we compared Arabidopsis thaliana seedling development in transparent and darkened Petri dishes at low light intensity (20 µmol m(-2) s(-1)), allowing us to follow (inter alia) hypocotyl elongation, which is an excellent process for studying interactions of signals involved in the regulation of growth and developmental responses. To obtain insights into molecular events underlying differences in seedling growth under these two conditions, we employed liquid chromatography-mass spectrometry (LC-MS) shotgun proteomics (available via the PRIDE deposit PXD001612). In total, we quantified the relative abundances of peptides representing 1,209 proteins detected in all sample replicates of LC-MS analyses. Comparison of MS spectra after manual validation revealed 48 differentially expressed proteins. Functional classification, analysis of available gene expression data and literature searches revealed alterations associated with root illumination (inter alia) in autotrophic CO2 fixation, C compound and carbohydrate metabolism, and nitrogen metabolism. The results also indicate a previously unreported role for cytokinin plant hormones in the escape-tropism response to root illumination. We complemented these results with reverse transcription followed by quantitative PCR (RT-qPCR), chlorophyll fluorescence and detailed cytokinin signaling analyses, detecting in the latter a significant increase in the activity of the cytokinin two-component signaling cascade in roots and implicating the cytokinin receptor AHK3 as the major mediator of root to hypocotyl signaling in responses to root illumination.

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis.

Targeting of the heat stress (HS, 40°C) to shoots, roots or whole plants substantially affects Arabidopsis physiological responses. Effective stress targeting was proved by determination of the expression of HS markers, HsfA2 and HSA32, which were quickly stimulated in the targeted organ(s), but remained low in non-stressed tissues for at least 2h. When shoots or whole plants were subjected to HS, a transient decrease in abscisic acid, accompanied by a small increase in active cytokinin levels, was observed in leaves, consistent with stimulation of transpiration, the main cooling mechanism in leaves. HS application targeted to part of plant resulted in a rapid stimulation of expression of components of cytokinin signaling pathway (especially of receptor genes) in the non-exposed tissues, which indicated fast inter-organ communication. Under all HS treatments, shoot apices responded by transient elevation of active cytokinin contents and stimulation of transcription of genes involved in photosynthesis and carbohydrate metabolism. Duration of this stimulation was negatively correlated with stress strength. The impact of targeted HS on the expression of 63 selected genes, including those coding regulatory 14-3-3 proteins, was compared. Stimulation of GRF9 (GRF14µ) in stressed organs after 2-6h may be associated with plant stress adaptation.

Advances in purification and separation of posttranslationally modified proteins.

Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene - transcript - protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

High cytokinin levels induce a hypersensitive-like response in tobacco.

BACKGROUND AND AIMS: Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens. METHODS: The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange. KEY RESULTS: Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure. CONCLUSIONS: Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.