The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.

• Keywords
• Hormonomics, Internal standard, Liquid chromatography, Mass spectrometry, Matrix effect, Metabolomics, Omics, Plant hormone, Solid phase extraction,
• Publication type
• Journal Article MeSH
• Review MeSH

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

• MeSH
• Sugars metabolism   MeSH
• Cytokinins * metabolism   MeSH
• Hordeum * genetics   metabolism   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Sucrose metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sugars MeSH
• Cytokinins * MeSH
• Plant Proteins MeSH
• Sucrose MeSH

Cytokinins (CKs) regulate numerous plant developmental processes, including photosynthesis and leaf senescence. Isopentenyltransferase (IPT) is a rate-limiting enzyme in the CK-biosynthesis pathway. We overexpressed ipt under tissue-specific promoters to study the long-range effect of CK on the functioning of tomato source leaves. Photosynthetic activity over time provided the measure for leaf aging. Significantly delayed leaf senescence was observed in plants expressing ipt under a root-specific promoter, but not in those expressing the gene under a source leaf-specific promoter. The root-derived influence on leaf aging was further confirmed by grafting experiments. CK concentration in source leaves of both transgenic lines increased significantly, with different proportions of its various derivatives. On the other hand, root CK concentration was only slightly elevated. Nevertheless, the significant change in the proportion of CK derivatives in the root indicated that CK biosynthesis and metabolism were altered. Partial leaf defoliation upregulates photosynthetic rate in the remaining leaf; however, overexpression of ipt in either tissues eliminated this response. Interestingly, stem girdling also eliminated the photosynthetic response. Taken together, our findings suggest that leaf senescence is regulated by a CK-mediated root-shoot communication network. We propose that CK-mediated signal is translocated to the leaf via the xylem where it alters CK biosynthesis, resulting in delayed senescence.

• Keywords
• Solanum lycopersicum, cytokinin, isopentenyltransferase, photosynthesis, shoot-to-root ratio,
• Publication type
• Journal Article MeSH

Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels.

• Keywords
• CKX, CPPU, INCYDE, IPT, TD-K, barley, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, thidiazuron, wheat, yield,
• Publication type
• Journal Article MeSH

Modifying the cytokinin content in plants is a means of improving plant productivity. Here, we report the development and biological activity of compound TD-K (1-(furan-2-ylmethyl)-3-(1,2,3-thiadiazol-5-yl)urea)which is related to thidiazuron. TD-K-which exhibited extremely high antisenescence activity in the wheat leaf bioassay-and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine)-a plant growth regulator reported to inhibit cytokinin oxidase/dehydrogenase (CKX), an enzyme involved in the degradation of the plant hormone cytokinin-were selected for investigation of their effects on the model plant Rapid Cycling Brassica rapa (RCBr). We monitored the expression of BrCKX and isopentenyl transferase (BrIPT), which codes for the key cytokinin biosynthesis enzyme, in developing leaves following INCYDE and TD-K application. Growth room experiments revealed that INCYDE increased RCBr seed yield per plant, but only when applied multiple times and when grown in 5 mM KNO3. Expression in control leaves showed transient, high levels of expression of BrCKX and BrIPT at true leaf appearance. Following INCYDE application, there was a rapid and strong upregulation of BrCKX3, and a transient downregulation of BrIPT1 and BrIPT3. Interestingly, the upregulation of BrCKX3 persisted in a milder form throughout the course of the experiment (16 days). TD-K also upregulated BrCKX3. However, in contrast to INCYDE, this effect disappeared after two days. These results suggest that both compounds (CKX inhibitor and cytokinin TD-K) influenced cytokinin homeostasis in RCBr leaves, but with different mechanisms.

• Keywords
• CKX, INCYDE, IPT, TD-K, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, thidiazuron,
• 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

Inositol trisphosphate 5/6 kinases (ITPK) constitute a small group of enzymes participating in the sequential phosphorylation of inositol phosphate to inositol hexakisphosphate (IP6), which is a major storage form of phosphate in cereal grains. The development of lines with reduced IP6 content could enhance phosphate and mineral bioavailability. Moreover, plant ITPKs participate in abiotic stress signaling. To elucidate the role of HvITPK1 in IP6 synthesis and stress signaling, a barley itpk1 mutant was created using programmable nuclease Cas9. Homozygous single bp insertion and deletion mutant lines were obtained. The mutants contained altered levels of phosphate in the mature grains, ranging from 65% to 174% of the wild type (WT) content. Homozygous mutant lines were tested for their response to salinity during germination. Interestingly, insertion mutant lines revealed a higher tolerance to salinity stress than deletion mutants. Mature embryos of an insertion mutant itpk1-2 and deletion mutant itpk1-33 were cultivated in vitro on MS medium supplemented with NaCl at 50, 100, and 200 mM. While both mutants grew less well than WT on no or low salt concentrations, the itpk1-2 mutant was affected less than the WT and itpk33 when grown on the highest NaCl concentration. The expression of all ITPKs was induced in roots in response to salt stress. In shoots, the differential effect of high salt on IPTK expression in the two iptk1 mutants was consistent with their different sensitivities to salt stress. The results extend the evidence for the involvement of ITPK genes in phosphate storage and abiotic stress signaling.

• Keywords
• CRISPR, abiotic stress, barley mutant, phosphate, phytic acid, salinity,
• Publication type
• Journal Article MeSH

Photosynthetic activity is affected by exogenous and endogenous inputs, including source-sink balance. Reducing the source to sink ratio by partial defoliation or heavy shading resulted in significant elevation of the photosynthetic rate in the remaining leaf of tomato plants within 3 d. The remaining leaf turned deep green, and its area increased by almost 3-fold within 7 d. Analyses of photosynthetic activity established up-regulation due to increased carbon fixation activity in the remaining leaf, rather than due to altered water balance. Moreover, senescence of the remaining leaf was significantly inhibited. As expected, carbohydrate concentration was lower in the remaining leaf than in the control leaves; however, expression of genes involved in sucrose export was significantly lower. These results suggest that the accumulated fixed carbohydrates were primarily devoted to increasing the size of the remaining leaf. Detailed analyses of the cytokinin content indicated that partial defoliation alters cytokinin biosynthesis in the roots, resulting in a higher concentration of trans-zeatin riboside, the major xylem-translocated molecule, and a higher concentration of total cytokinin in the remaining leaf. Together, our findings suggest that trans-zeatin riboside acts as a signal molecule that traffics from the root to the remaining leaf to alter gene expression and elevate photosynthetic activity.

• Keywords
• Solanum lycopersicum, Cytokinin, leaf development, photosynthesis, source–sink relationship, tomato, xylem,
• MeSH
• Cytokinins physiology   MeSH
• Photosynthesis * MeSH
• Plant Roots physiology   MeSH
• Plant Leaves metabolism   MeSH
• Signal Transduction * MeSH
• Solanum lycopersicum physiology   MeSH
• Plant Shoots physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH

Plant phenotyping platforms offer automated, fast scoring of traits that simplify the selection of varieties that are more competitive under stress conditions. However, indoor phenotyping methods are frequently based on the analysis of plant growth in individual pots. We present a reproducible indoor phenotyping method for screening young barley populations under water stress conditions and after subsequent rewatering. The method is based on a simple read-out of data using RGB imaging, projected canopy height, as a useful feature for indirectly following the kinetics of growth and water loss in a population of barley. A total of 47 variables including 15 traits and 32 biochemical metabolites measured (morphometric parameters, chlorophyll fluorescence imaging, quantification of stress-related metabolites; amino acids and polyamines, and enzymatic activities) were used to validate the method. The study allowed the identification of metabolites related to water stress response and recovery. Specifically, we found that cadaverine (Cad), 1,3-aminopropane (DAP), tryptamine (Tryp), and tyramine (Tyra) were the major contributors to the water stress response, whereas Cad, DAP, and Tyra, but not Tryp, remained at higher levels in the stressed plants even after rewatering. In this work, we designed, optimized and validated a non-invasive image-based method for automated screening of potential water stress tolerance genotypes in barley populations. We demonstrated the applicability of the method using transgenic barley lines with different sensitivity to drought stress showing that combining canopy height and the metabolite profile we can discriminate tolerant from sensitive genotypes. We showed that the projected canopy height a sensitive trait that truly reflects other invasively studied morphological, physiological, and metabolic traits and that our presented methodological setup can be easily applicable for large-scale screenings in low-cost systems equipped with a simple RGB camera. We believe that our approach will contribute to accelerate the study and understanding of the plant water stress response and recovery capacity in crops, such as barley.

• Keywords
• Hordeum vulgare, amino acids, antioxidative enzymes, blue (RGB) imaging, canopy height, fluorescence, green, indoor phenotyping, polyamines, red,
• Publication type
• Journal Article MeSH