Water availability is undoubtedly one of the most important environmental factors affecting crop production. Drought causes a gradual deprivation of water in the soil from top to deep layers and can occur at diverse stages of plant development. Roots are the first organs that perceive water deficit in soil and their adaptive development contributes to drought adaptation. Domestication has contributed to a bottleneck in genetic diversity. Wild species or landraces represent a pool of genetic diversity that has not been exploited yet in breeding program. In this study, we used a collection of 230 two-row spring barley landraces to detect phenotypic variation in root system plasticity in response to drought and to identify new quantitative trait loci (QTL) involved in root system architecture under diverse growth conditions. For this purpose, young seedlings grown for 21 days in pouches under control and osmotic-stress conditions were phenotyped and genotyped using the barley 50k iSelect SNP array, and genome-wide association studies (GWAS) were conducted using three different GWAS methods (MLM GAPIT, FarmCPU, and BLINK) to detect genotype/phenotype associations. In total, 276 significant marker-trait associations (MTAs; p-value (FDR)< 0.05) were identified for root (14 and 12 traits under osmotic-stress and control conditions, respectively) and for three shoot traits under both conditions. In total, 52 QTL (multi-trait or identified by at least two different GWAS approaches) were investigated to identify genes representing promising candidates with a role in root development and adaptation to drought stress.

• Keywords
• GWAS, QTL, barley landraces, candidate gene, osmotic stress, root system architecture,
• Publication type
• Journal Article MeSH

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

• MeSH
• Centaurium growth & development   metabolism   MeSH
• Cyclopentanes analysis   metabolism   MeSH
• Cytokinins analysis   metabolism   MeSH
• Plant Roots growth & development   metabolism   MeSH
• Abscisic Acid analysis   metabolism   MeSH
• Salicylic Acid metabolism   MeSH
• Indoleacetic Acids analysis   metabolism   MeSH
• Oxylipins analysis   metabolism   MeSH
• Plant Growth Regulators analysis   metabolism   MeSH
• Salt Stress * MeSH
• In Vitro Techniques MeSH
• Plant Shoots growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclopentanes MeSH
• Cytokinins MeSH
• indoleacetic acid MeSH Browser
• jasmonic acid MeSH Browser
• Abscisic Acid MeSH
• Salicylic Acid MeSH
• Indoleacetic Acids MeSH
• Oxylipins MeSH
• Plant Growth Regulators MeSH

Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6-position. They are involved in many physiological processes. While the metabolism of trans-zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis-zeatin, dihydrozeatin, and aromatic cytokinins, which have been comparatively neglected. To help explain this duality, we present a novel hypothesis metaphorically comparing various cytokinin forms, enzymes of CK metabolism, and their signalling and transporter functions to the comics superheroes Hulk and Deadpool. Hulk is a powerful but short-lived creation, whilst Deadpool presents a more subtle and enduring force. With this dual framework in mind, this review compares different cytokinin metabolites, and their biosynthesis, translocation, and sensing to illustrate the different mechanisms behind the two CK strategies. This is put together and applied to a plant developmental scale and, beyond plants, to interactions with organisms of other kingdoms, to highlight where future study can benefit the understanding of plant fitness and productivity.

• Keywords
• Hulk/Deadpool, aromatic cytokinins, cis-zeatin, cytokinin biosynthesis, cytokinin oxidase/dehydrogenase, cytokinin signalling, cytokinin transport, cytokinins, isopentenyl transferase,
• MeSH
• Arabidopsis metabolism   MeSH
• Models, Biological MeSH
• Biological Transport MeSH
• Biological Assay MeSH
• Cytokinins metabolism   MeSH
• Plant Physiological Phenomena * MeSH
• Glycosylation MeSH
• Hydrolysis MeSH
• Kinetics MeSH
• Kinetin metabolism   MeSH
• Oxidoreductases metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Plants metabolism   MeSH
• Signal Transduction * MeSH
• Protein Binding MeSH
• Zeatin analogs & derivatives   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• cytokinin oxidase MeSH Browser
• Cytokinins MeSH
• dihydrozeatin MeSH Browser
• Kinetin MeSH
• Oxidoreductases MeSH
• Plant Growth Regulators MeSH
• Zeatin MeSH

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.

• Keywords
• Arabidopsis thaliana, Drought stress, Phytohormones, Proteomics, Split-root systems,
• Publication type
• Journal Article MeSH

Cytokinin is a phytohormone involved in the regulation of diverse developmental and physiological processes in plants. Its potential in biotechnology and for development of higher-yield and more resilient plants has been recognized, yet the molecular mechanisms behind its action are far from understood. In this report, the roots of barley seedlings were explored as a new source to reveal as yet unknown cytokinin-responsive proteins for crop improvement. Here we found significant differences reproducibly observed for 178 proteins, for which some of the revealed cytokinin-responsive pathways were confirmed in metabolome analysis, including alterations phenylpropanoid pathway, amino acid biosynthesis and ROS metabolism. Bioinformatics analysis indicated a significant overlap between cytokinin response and response to abiotic stress. This was confirmed by comparing proteome and metabolome profiles in response to drought, salinity or a period of temperature stress. The results illustrate complex abiotic stress response in the early development of model crop plant and confirm an extensive crosstalk between plant hormone cytokinin and response to temperature stimuli, water availability or salinity stress.

• Keywords
• Hordeum vulgare, ROS, abiotic stress, metabolome, phenylpropanoid biosynthesis, proteome, root, zeatin,
• Publication type
• Journal Article MeSH

Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. In planta, endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley.

• Keywords
• CRISPR-Cas9, barley, cytokinin, silencing, yield,
• Publication type
• Journal Article MeSH

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to drought and examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs.

• Keywords
• Brassica crops, drought, phytohormones, recovery, tolerance,
• MeSH
• Brassica classification   genetics   metabolism   MeSH
• Stress, Physiological * MeSH
• Droughts * MeSH
• Gene Expression Regulation, Plant * MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Plant Growth Regulators MeSH
• Plant Proteins MeSH

Cytokinin is a multifaceted plant hormone that plays major roles not only in diverse plant growth and development processes, but also stress responses. We summarize knowledge of the roles of its metabolism, transport, and signalling in responses to changes in levels of both macronutrients (nitrogen, phosphorus, potassium, sulphur) and micronutrients (boron, iron, silicon, selenium). We comment on cytokinin's effects on plants' xenobiotic resistance, and its interactions with light, temperature, drought, and salinity signals. Further, we have compiled a list of abiotic stress-related genes and demonstrate that their expression patterns overlap with those of cytokinin metabolism and signalling genes.

• Keywords
• abiotic stress, cytokinin, drought, nutrient, stress tolerance, temperature,
• MeSH
• Acclimatization MeSH
• Circadian Clocks MeSH
• Cytokinins metabolism   MeSH
• Stress, Physiological * MeSH
• Plant Physiological Phenomena * MeSH
• Droughts MeSH
• Gene Expression Regulation, Plant MeSH
• Plants genetics   metabolism   MeSH
• Salinity MeSH
• Signal Transduction * MeSH
• Light MeSH
• Temperature MeSH
• Plant Development MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH

Our phenotyping and hormonal study has characterized the role of cytokinins (CK) in the drought and recovery responses of Arabidopsis thaliana. CK down-regulation was achieved by overexpression of the gene for CK deactivating enzyme cytokinin oxidase/dehydrogenase (CKX): constitutive (35S:CKX) or at the stress onset using a dexamethasone-inducible pOp/LhGR promoter (DEX:CKX). The 35S:CKX plants exhibited slow ontogenesis and higher expression levels of stress-associated genes, e.g., AtP5CS1, already at well-watered conditions. CK down-regulation resulted during drought in higher stress tolerance (indicated by relatively low up-regulation of the expression of drought stress marker gene AtRD29B) accompanied with lower leaf water loss. Nevertheless, these plants exhibited slow and delayed recovery after re-watering. CK levels were increased at the stress onset by stimulation of the expression of CK biosynthetic gene isopentenyl transferase (ipt) (DEX:IPT) or by application of exogenous CK meta-topolin. After water withdrawal, long-term CK elevation resulted in higher water loss in comparison with CKX transformants as well as with plants overexpressing ipt driven by senescence-inducible SAG12 promoter (SAG:IPT), which gradually enhanced CKs during the stress progression. In all cases, CK up-regulation resulted in fast and more vigorous recovery. All drought-stressed plants exhibited growth suppression associated with elevation of abscisic acid and decrease of auxins and active CKs (with the exception of SAG:IPT plants). Apart from the ipt overexpressers, also increase of jasmonic and salicylic acid was found.

• Keywords
• abscisic acid, auxin, cytokinin, cytokinin oxidase/dehydrogenase, drought stress, isopentenyl transferase, phytohormone,
• Publication type
• Journal Article MeSH

Plant growth and development are critically influenced by unpredictable abiotic factors. To survive fluctuating changes in their environments, plants have had to develop robust adaptive mechanisms. The dynamic and complementary actions of the auxin and cytokinin pathways regulate a plethora of developmental processes, and their ability to crosstalk makes them ideal candidates for mediating stress-adaptation responses. Other crucial signaling molecules responsible for the tremendous plasticity observed in plant morphology and in response to abiotic stress are reactive oxygen species (ROS). Proper temporal and spatial distribution of ROS and hormone gradients is crucial for plant survival in response to unfavorable environments. In this regard, the convergence of ROS with phytohormone pathways acts as an integrator of external and developmental signals into systemic responses organized to adapt plants to their environments. Auxin and cytokinin signaling pathways have been studied extensively. Nevertheless, we do not yet understand the impact on plant stress tolerance of the sophisticated crosstalk between the two hormones. Here, we review current knowledge on the function of auxin and cytokinin in redirecting growth induced by abiotic stress in order to deduce their potential points of crosstalk.

• Keywords
• ROS, abiotic stress, adaptation, auxin, crosstalk, cytokinin, growth,
• MeSH
• Biological Transport MeSH
• Cytokinins metabolism   MeSH
• Adaptation, Physiological MeSH
• Stress, Physiological * MeSH
• Plant Physiological Phenomena * MeSH
• Gene Regulatory Networks MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Plants genetics   metabolism   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH