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Abstract Background Mantle cell lymphoma (MCL) is a chronically relapsing malignancy with deregulated cell cycle progression. We analyzed efficacy, mode of action, and predictive markers of susceptibility to palbociclib, an approved CDK 4/6 inhibitor, and its combination with venetoclax, a BCL2 inhibitor. Methods A panel of nine MCL cell lines were used for in vitro experiments. Four patient derived xenografts (PDX) obtained from patients with chemotherapy and ibrutinib-refractory MCL were used for in vivo proof-of-concept studies. Changes of the mitochondrial membrane potential, energy-metabolic pathways, AKT activity, and pro-apoptotic priming of MCL cells were evaluated by JC-1 staining, Seahorse XF analyser, genetically encoded fluorescent AKT reporter, and BH3 profiling, respectively. MCL clones with gene knockout or transgenic (over)expression of CDKN2A, MYC, CDK4, and RB1 were used to estimate impact of these aberrations on sensitivity to palbociclib, and venetoclax. Results Co-targeting MCL cells with palbociclib and venetoclax induced cytotoxic synergy in vitro and in vivo. Molecular mechanisms responsible for the observed synthetic lethality comprised palbociclib-mediated downregulation of anti-apoptotic MCL1, increased levels of proapoptotic BIM bound on both BCL2, and BCL-XL and increased pro-apoptotic priming of MCL cells mediated by BCL2-independent mechanisms, predominantly palbociclib-triggered metabolic and mitochondrial stress. Loss of RB1 resulted in palbociclib resistance, while deletion of CDKN2A or overexpression of CDK4, and MYC genes did not change sensitivity to palbociclib. Conclusions Our data strongly support investigation of the chemotherapy-free palbociclib and venetoclax combination as an innovative treatment strategy for post-ibrutinib MCL patients without RB1 deletion.

Abstract Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive deterioration of upper and lower motor neurons. A definitive diagnostic test or biomarker for ALS is currently unavailable, leading to a diagnostic delay following the onset of initial symptoms. Our study focused on cerebrospinal fluid (CSF) concentrations of clusterin, tau protein, phosphorylated tau protein, and beta-amyloid1–42 in ALS patients and a control group. Methods Our study involved 54 ALS patients and 58 control subjects. Among the ALS patients, 14 presented with bulbar-onset ALS, and 40 with limb-onset ALS. We quantified biomarker levels using enzyme-linked immunosorbent assay (ELISA) and compared the results using the Mann–Whitney U-test. Results Significant elevations in neurodegenerative markers, including tau protein (p

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 μM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7–1.3 μM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 μM) compared to 11b–f (GI50 = 6.4–11.5 μM). Molecular docking studies explained the structure–activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Abstract Background Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. Results A high-throughput sample preparation method for liquid chromatography–tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. Conclusion The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

The transition from seed to seedling represents a critical developmental step in the life cycle of higher plants, dramatically affecting plant ontogenesis and stress tolerance. The release from dormancy to acquiring germination ability is defined by a balance of phytohormones, with the substantial contribution of abscisic acid (ABA), which inhibits germination. We studied the embryonic axis of Pisum sativum L. before and after radicle protrusion. Our previous work compared RNA sequencing-based transcriptomics in the embryonic axis isolated before and after radicle protrusion. The current study aims to analyze ABA-dependent gene regulation during the transition of the embryonic axis from the germination to post-germination stages. First, we determined the levels of abscisates (ABA, phaseic acid, dihydrophaseic acid, and neo-phaseic acid) using ultra-high-performance liquid chromatography–tandem mass spectrometry. Second, we made a detailed annotation of ABA-associated genes using RNA sequencing-based transcriptome profiling. Finally, we analyzed the DNA methylation patterns in the promoters of the PsABI3, PsABI4, and PsABI5 genes. We showed that changes in the abscisate profile are characterized by the accumulation of ABA catabolites, and the ABA-related gene profile is accompanied by the upregulation of genes controlling seedling development and the downregulation of genes controlling water deprivation. The expression of ABI3, ABI4, and ABI5, which encode crucial transcription factors during late maturation, was downregulated by more than 20-fold, and their promoters exhibited high levels of methylation already at the late germination stage. Thus, although ABA remains important, other regulators seems to be involved in the transition from seed to seedling.

Abstract Members of the Viola genus play important roles in traditional Asian herbal medicine. This study investigates the ability of Viola odorata L. extracts to inhibit Na+,K+-ATPase, an essential animal enzyme responsible for membrane potential maintenance. The root extract of V. odorata strongly inhibited Na+,K+-ATPase, while leaf and seeds extracts were basically inactive. A UHPLC-QTOF-MS/MS metabolomic approach was used to identify the chemical principle of the root extract’s activity, resulting in the detection of 35,292 features. Candidate active compounds were selected by correlating feature area with inhibitory activity in 14 isolated fractions. This yielded a set of 15 candidate compounds, of which 14 were preliminarily identified as procyanidins. Commercially available procyanidins (B1, B2, B3 and C1) were therefore purchased and their ability to inhibit Na+,K+-ATPase was investigated. Dimeric procyanidins B1, B2 and B3 were found to be inactive, but the trimeric procyanidin C1 strongly inhibited Na+,K+-ATPase with an IC50 of 4.5 µM. This newly discovered inhibitor was docked into crystal structures mimicking the Na3E1∼P·ADP and K2E2·Pi states to identify potential interaction sites within Na+,K+-ATPase. Possible binding mechanisms and the principle responsible for the observed root extract activity are discussed.

Increasing crop productivity under optimal conditions and mitigating yield losses under stressful conditions is a major challenge in contemporary agriculture. We have recently identified an effective anti-senescence compound (MTU, [1-(2-methoxyethyl)-3-(1,2,3-thiadiazol-5yl)urea]) in in vitro studies. Here, we show that MTU delayed both age- and stress-induced senescence of wheat plants (Triticum aestivum L.) by enhancing the abundance of PSI supercomplex with LHCa antennae (PSI-LHCa) and promoting the cyclic electron flow (CEF) around PSI. We suppose that this rarely-observed phenomenon blocks the disintegration of photosynthetic apparatus and maintains its activity as was reflected by the faster growth rate of wheat in optimal conditions and under drought and heat stress. Our multiyear field trial analysis further shows that the treatment with 0.4 g ha-1 of MTU enhanced average grain yields of field-grown wheat and barley (Hordeum vulgare L.) by 5-8%. Interestingly, the analysis of gene expression and hormone profiling confirms that MTU acts without the involvement of cytokinins or other phytohormones. Moreover, MTU appears to be the only chemical reported to date to affect PSI stability and activity. Our results indicate a central role of PSI and CEF in the onset of senescence with implications in yield management at least for cereal species.

Dormancy and heteromorphism are innate seed properties that control germination timing through adaptation to the prevailing environment. The degree of variation in dormancy depth within a seed population differs considerably depending on the genotype and maternal environment. Dormancy is therefore a key trait of annual weeds to time seedling emergence across seasons. Seed heteromorphism, the production of distinct seed morphs (in color, mass or other morphological characteristics) on the same individual plant, is considered to be a bet-hedging strategy in unpredictable environments. Heteromorphic species evolved independently in several plant families and the distinct seed morphs provide an additional degree of variation. Here we conducted a comparative morphological and molecular analysis of the dimorphic seeds (black and brown) of the Amaranthaceae weed Chenopodium album. Freshly harvested black and brown seeds differed in their dormancy and germination responses to ambient temperature. The black seed morph of seedlot #1 was dormant and 2/3rd of the seed population had non-deep physiological dormancy which was released by after-ripening (AR) or gibberellin (GA) treatment. The deeper dormancy of the remaining 1/3rd non-germinating seeds required in addition ethylene and nitrate for its release. The black seeds of seedlot #2 and the brown seed morphs of both seedlots were non-dormant with 2/3rd of the seeds germinating in the fresh mature state. The dimorphic seeds and seedlots differed in testa (outer seed coat) thickness in that thick testas of black seeds of seedlot #1 conferred coat-imposed dormancy. The dimorphic seeds and seedlots differed in their abscisic acid (ABA) and GA contents in the dry state and during imbibition in that GA biosynthesis was highest in brown seeds and ABA degradation was faster in seedlot #2. Chenopodium genes for GA and ABA metabolism were identified and their distinct transcript expression patterns were quantified in dry and imbibed C. album seeds. Phylogenetic analyses of the Amaranthaceae sequences revealed a high proportion of expanded gene families within the Chenopodium genus. The identified hormonal, molecular and morphological mechanisms and dormancy variation of the dimorphic seeds of C. album and other Amaranthaceae are compared and discussed as adaptations to variable and stressful environments.

Dinoflagellate inhabitants of the reef-building corals exchange nutrients and signals with host cells, which often benefit the growth of both partners. Phytohormones serve as central hubs for signal integration between symbiotic microbes and their hosts, allowing appropriate modulation of plant growth and defense in response to various stresses. However, the presence and function of phytohormones in photosynthetic dinoflagellates and their function in the holobionts remain elusive. We hypothesized that endosymbiotic dinoflagellates may produce and employ phytohormones for stress responses. Using the endosymbiont of reef corals Breviolum minutum as model, this study aims to exam whether the alga employ analogous signaling systems by an integrated multiomics approach. We show that key gibberellin (GA) biosynthetic genes are widely present in the genomes of the selected dinoflagellate algae. The non-13-hydroxylation pathway is the predominant route for GA biosynthesis and the multifunctional GA dioxygenase in B. minutum has distinct substrate preference from high plants. GA biosynthesis is modulated by the investigated bleaching-stimulating stresses at both transcriptional and metabolic levels and the exogenously applied GAs improve the thermal tolerance of the dinoflagellate. Our results demonstrate the innate ability of a selected Symbiodiniaceae to produce the important phytohormone and the active involvement of GAs in the coordination and the integration of the stress response.

Abstract Background Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination of parasitic plants induced by host strigolactones leads to serious agricultural problems worldwide. More insight in these signaling molecules is hampered by their extremely low concentrations in complex soil and plant tissue matrices, as well as their instability. So far, the combination of tailored isolation—that would replace current unspecific, time-consuming and labour-intensive processing of large samples—and a highly sensitive method for the simultaneous profiling of a broad spectrum of strigolactones has not been reported. Results Depending on the sample matrix, two different strategies for the rapid extraction of the seven structurally similar strigolactones and highly efficient single-step pre-concentration on polymeric RP SPE sorbent were developed and validated. Compared to conventional methods, controlled temperature during the extraction and the addition of an organic modifier (acetonitrile, acetone) to the extraction solvent helped to tailor strigolactone isolation from low initial amounts of root tissue (150 mg fresh weight, FW) and root exudate (20 ml), which improved both strigolactone stability and sample purity. We have designed an efficient UHPLC separation with sensitive MS/MS detection for simultaneous analysis of seven natural strigolactones including their biosynthetic precursors—carlactone and carlactonoic acid. In combination with the optimized UHPLC–MS/MS method, attomolar detection limits were achieved. The new method allowed successful profiling of seven strigolactones in small exudate and root tissue samples of four different agriculturally important plant species—sorghum, rice, pea and tomato. Conclusion The established method provides efficient strigolactone extraction with aqueous mixtures of less nucleophilic organic solvents from small root tissue and root exudate samples, in combination with rapid single-step pre-concentration. This method improves strigolactone stability and eliminates the co-extraction and signal of matrix-associated contaminants during the final UHPLC–MS/MS analysis with an electrospray interface, which dramatically increases the overall sensitivity of the analysis. We show that the method can be applied to a variety of plant species.

Cytokinin receptors predominantly localize to the endoplasmic reticulum. Here, Kubiasová et al. use a cytokinin fluoroprobe to show that ER-localized cytokinin receptors can enter the secretory pathway, reach the plasma membrane and undergo vesicular recycling, suggesting multiple sites of cytokinin perception.

Plasmodiophora brassicae is an obligate biotrophic pathogen causing clubroot disease in cruciferous plants. Infected plant organs are subject to profound morphological changes, the roots form characteristic galls, and the leaves are chlorotic and abscise. The process of gall formation is governed by timely changes in the levels of endogenous plant hormones that occur throughout the entire life cycle of the clubroot pathogen. The homeostasis of two plant hormones, cytokinin and auxin, appears to be crucial for club development. To investigate the role of cytokinin and auxin in gall formation, we used metabolomic and transcriptomic profiling of Arabidopsis thaliana infected with clubroot, focusing on the late stages of the disease, where symptoms were more pronounced. Loss-of-function mutants of three cytokinin receptors, AHK2, AHK3, and CRE1/AHK4, were employed to further study the homeostasis of cytokinin in response to disease progression; ahk double mutants developed characteristic symptoms of the disease, albeit with varying intensity. The most susceptible to clubroot disease was the ahk3 ahk4 double mutant, as revealed by measuring its photosynthetic performance. Quantification of phytohormone levels and pharmacological treatment with the cytokinin antagonist PI-55 showed significant changes in the levels of endogenous cytokinin and auxin, which was manifested by both enhanced and reduced development of disease symptoms in different genotypes.

The understanding of neurodegenerative diseases, traditionally considered to be well-defined entities with distinguishable clinical phenotypes, has undergone a major shift over the last 20 years. The diagnosis of neurodegenerative diseases primarily requires functional brain imaging techniques or invasive tests such as lumbar puncture to assess cerebrospinal fluid. A new biological approach and research efforts, especially in vivo, have focused on biomarkers indicating underlying proteinopathy in cerebrospinal fluid and blood serum. However, due to the complexity and heterogeneity of neurodegenerative processes within the central nervous system and the large number of overlapping clinical diagnoses, identifying individual proteinopathies is relatively difficult and often not entirely accurate. For this reason, there is an urgent need to develop laboratory methods for identifying specific biomarkers, understand the molecular basis of neurodegenerative disorders and classify the quantifiable and readily available tools that can accelerate efforts to translate the knowledge into disease-modifying therapies that can improve and simplify the areas of differential diagnosis, as well as monitor the disease course with the aim of estimating the prognosis or evaluating the effects of treatment. The aim of this review is to summarize the current knowledge about clinically relevant biomarkers in different neurodegenerative diseases.

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development.

Abstract Background Mal de Río Cuarto virus (MRCV) infects several monocotyledonous species including maize and wheat. Infected plants show shortened internodes, partial sterility, increased tillering and reduced root length. To better understand the molecular basis of the plant-virus interactions leading to these symptoms, we combined RNA sequencing with metabolite and hormone measurements. Results More than 3000 differentially accumulated transcripts (DATs) were detected in MRCV-infected wheat plants at 21 days post inoculation compared to mock-inoculated plants. Infected plants exhibited decreased levels of TaSWEET13 transcripts, which are involved in sucrose phloem loading. Soluble sugars, starch, trehalose 6-phosphate (Tre6P), and organic and amino acids were all higher in MRCV-infected plants. In addition, several transcripts related to plant hormone metabolism, transport and signalling were increased upon MRCV infection. Transcripts coding for GA20ox, D14, MAX2 and SMAX1-like proteins involved in gibberellin biosynthesis and strigolactone signalling, were reduced. Transcripts involved in jasmonic acid, ethylene and brassinosteroid biosynthesis, perception and signalling and in auxin transport were also altered. Hormone measurements showed that jasmonic acid, brassinosteroids, abscisic acid and indole-3-acetic acid were significantly higher in infected leaves. Conclusions Our results indicate that MRCV causes a profound hormonal imbalance that, together with alterations in sugar partitioning, could account for the symptoms observed in MRCV-infected plants.

Shoot branching from axillary buds (AXBs) is regulated by a network of inhibitory and promotive forces, which includes hormones. In perennials, the dwarfed stature of the embryonic shoot inside AXBs is indicative of gibberellin (GA) deficiency, suggesting that AXB activation and outgrowth require GA. Nonetheless, the role of GA in branching has remained obscure. We here carried out comprehensive GA transcript and metabolite analyses in hybrid aspen, a perennial branching model. The results indicate that GA has an inhibitory as well as promotive role in branching. The latter is executed in two phases. While the expression level of GA2ox is high in quiescent AXBs, decapitation rapidly downregulated it, implying increased GA signaling. In the second phase, GA3ox2-mediated de novo GA-biosynthesis is initiated between 12 and 24 h, prior to AXB elongation. Metabolite analyzes showed that GA1/4 levels were typically high in proliferating apices and low in the developmentally inactive, quiescent AXBs, whereas the reverse was true for GA3/6. To investigate if AXBs are differently affected by GA3, GA4, and GR24, an analog of the branch-inhibitor hormone strigolactone, they were fed into AXBs of single-node cuttings. GA3 and GA4 had similar effects on GA and SL pathway genes, but crucially GA3 induced AXB abscission whereas GA4 promoted outgrowth. Both GA3 and GA4 strongly upregulated GA2ox genes, which deactivate GA1/4 but not GA3/6. Thus, the observed production of GA3/6 in quiescent AXBs targets GA1/4 for GA2ox-mediated deactivation. AXB quiescence can therefore be maintained by GA3/6, in combination with strigolactone. Our discovery of the distinct tasks of GA3 and GA4 in AXB activation might explain why the role of GA in branching has been difficult to decipher. Together, the results support a novel paradigm in which GA3/6 maintains high levels of GA2ox expression and low levels of GA4 in quiescent AXBs, whereas activation and outgrowth require increased GA1/4 signaling through the rapid reduction of GA deactivation and subsequent GA biosynthesis.

Abstract Background Cytokinin is a negative regulator of root growth, and a reduction of the cytokinin content or signalling causes the formation a larger root system in model plants, improves their growth under drought and nutrient limitation and causes increased accumulation of elements in the shoot. Roots are an important but understudied target of plant breeding. Here we have therefore explored whether root enhancement by lowering the cytokinin content can also be achieved in oilseed rape (Brassica napus L.) plants. Results Transgenic plants overexpressing the CKX2 gene of Arabidopsis thaliana encoding a cytokinin-degrading cytokinin oxidase/dehydrogenase showed higher CKX activity and a strongly reduced cytokinin content. Cytokinin deficiency led to the formation of a larger root system under different growth conditions, which was mainly due to an increased number of lateral and adventitious roots. In contrast, shoot growth was comparable to wild type, which caused an enhanced root-to-shoot ratio. Transgenic plants accumulated in their leaves higher concentrations of macro- and microelements including P, Ca, Mg, S, Zn, Cu, Mo and Mn. They formed more chlorophyll under Mg- and S-deficiency and accumulated a larger amount of Cd and Zn from contaminated medium and soil. Conclusions These findings demonstrate the usefulness of ectopic CKX gene expression to achieve root enhancement in oilseed rape and underpin the functional relevance of a larger root system. Furthermore, the lack of major developmental consequences on shoot growth in cytokinin-deficient oilseed rape indicates species-specific differences of CKX gene and/or cytokinin action.

The total phenolic content, anthocyanins, phenolic acids, antioxidant capacity and α-amylase inhibitory activity of black (Aydin Siyahi), purple (Kadife Kemer) and white (Trabzon Kadife) eggplants grown in Turkey were subjected to a comparative investigation. The black cultivar exhibited the highest total phenolic (17,193 and 6552 mg gallic acid equivalent/kg fw), flavonoid (3019 and 1160 quercetin equivalent/kg fw) and anthocyanin (1686 and 6167 g delphinidin-3-O-glucoside equivalent/kg fw) contents in crude extracts of the peel and pulp. The majority of the caffeic acid was identified in the ester (2830 mg/kg fw) and ester-bound (2594 mg/kg fw) forms in the peel of ‘Kadife Kemer’ and in the glycoside form (611.9 mg/kg fw) in ‘Aydin Siyahi’, as well as in the pulp of these two eggplants. ‘Kadife Kemer’ (purple eggplant) contained the majority of the chlorogenic acid in free form (27.55 mg/kg fw), compared to ‘Aydin Siyahi’ in the ester (7.82 mg/kg fw), glycoside (294.1 mg/kg dw) and ester-bound (2.41 mg/kg fw) forms. The eggplant cultivars (peel and pulp, mg/kg fw) exhibited a relatively high delphinidin-3-O-rutinoside concentration in the peel of ‘Aydin Siyahi’ (avg. 1162), followed by ‘Kadife Kemer’ (avg. 336.6), and ‘Trabzon Kadife’ (avg. 215.1). The crude phenolic extracts of the eggplants exhibited the highest antioxidant capacity values (peel and pulp, µmoL Trolox equivalent/kg fw) of 2,2-diphenyl-1-picrylhydrazyl (DPPH, 8156 and 2335) and oxygen radical absorbance capacity (ORAC, 37,887 and 17,648). The overall results indicate that black and purple eggplants are the cultivars with greater potential benefits in terms of their phenolics and antioxidant values than the white eggplant.

Acclimation to salt stress in plants is regulated by complex signaling pathways involving endogenous phytohormones. The signaling role of salicylic acid (SA) in regulating crosstalk between endogenous plant growth regulators’ levels was investigated in barley (Hordeum vulgare L. ‘Ince’; 2n = 14) leaves and roots under salt stress. Salinity (150 and 300 mM NaCl) markedly reduced leaf relative water content (RWC), growth parameters, and leaf water potential (LWP), but increased proline levels in both vegetative organs. Exogenous SA treatment did not significantly affect salt-induced negative effects on RWC, LWP, and growth parameters but increased the leaf proline content of plants under 150 mM salt stress by 23.1%, suggesting that SA enhances the accumulation of proline, which acts as a compatible solute that helps preserve the leaf’s water status under salt stress. Changes in endogenous phytohormone levels were also investigated to identify agents that may be involved in responses to increased salinity and exogenous SA. Salt stress strongly affected endogenous cytokinin (CK) levels in both vegetative organs, increasing the concentrations of CK free bases, ribosides, and nucleotides. Indole-3-acetic acid (IAA, auxin) levels were largely unaffected by salinity alone, especially in barley leaves, but SA strongly increased IAA levels in leaves at high salt concentration and suppressed salinity-induced reductions in IAA levels in roots. Salt stress also significantly increased abscisic acid (ABA) and ethylene levels; the magnitude of this increase was reduced by treatment with exogenous SA. Both salinity and SA treatment reduced jasmonic acid (JA) levels at 300 mM NaCl but had little effect at 150 mM NaCl, especially in leaves. These results indicate that under high salinity, SA has antagonistic effects on levels of ABA, JA, ethylene, and most CKs, as well as basic morphological and physiological parameters, but has a synergistic effect on IAA, which was well exhibited by principal component analysis (PCA).

Herein, we present a new strategy for the preparation of a broad range of brassinosteroid biosynthetic precursors/metabolites differing by the ring A fragment. The protocol is based on the use of readily available phytohormones of this class bearing a 2α,3α-diol moiety (epibrassinolide or epicastasterone) as starting materials. The required functionalities (Δ2-, 2α,3α- and 2β,3β-epoxy-, 2α,3β-, 2β,3α-, and 2β,3β-dihydroxy-, 3-keto-, 3α- and 3β-hydroxy-, 2α-hydroxy-3-keto-) were synthesized from 2α,3α-diols in a few simple steps (Corey–Winter reaction, epoxidation, oxidation, hydride reduction, etc.).

Summary: Upon fertilization, the ovary increases in size and undergoes a complex developmental process to become a fruit. We show that cytokinins (CKs), which are required to determine ovary size before fertilization, have to be degraded to facilitate fruit growth. The expression of CKX7, which encodes a cytosolic CK-degrading enzyme, is directly positively regulated post-fertilization by the MADS-box transcription factor STK. Similar to stk, two ckx7 mutants possess shorter fruits than wild type. Quantification of CKs reveals that stk and ckx7 mutants have high CK levels, which negatively control cell expansion during fruit development, compromising fruit growth. Overexpression of CKX7 partially complements the stk fruit phenotype, confirming a role for CK degradation in fruit development. Finally, we show that STK is required for the expression of FUL, which is essential for valve elongation. Overall, we provide insights into the link between CKs and molecular pathways that control fruit growth. : Di Marzo et al. establish that defects in Arabidopsis fruit size are accompanied by alterations in the metabolism of the plant hormone cytokinin, which is critical for cell elongation. SEEDSTICK influences fruit elongation by directly regulating cytokinin degradation via CKX7 and indirectly modulating the expression of the MADS-box gene FRUITFULL. Keywords: Arabidopsis, cell elongation, CKX7, cytokinins, FRUITFULL, fruit size, MADS-box, SEEDSTICK

Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms.

Soil salinity is severely affecting crop productivity in many countries, particularly in the Mediterranean area. To evaluate early plant responses to increased salinity and characterize tolerance markers, three important Brassica crops – Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata) and kale (B. oleracea var. acephala) were subjected to short-term (24 h) salt stress by exposing them to NaCl at concentrations of 50, 100, or 200 mM. Physiological (root growth, photosynthetic performance parameters, and Na+/K+ ratio) and biochemical parameters (proline content and lipid peroxidation as indicated by malondialdehyde, MDA, levels) in the plants’ roots and leaves were then measured. Photosynthetic parameters such as the total performance index PItotal (describing the overall efficiency of PSI, PSII and the intersystem electron transport chain) appeared to be the most salinity-sensitive parameter and informative stress marker. This parameter was decreased more strongly in Chinese cabbage than in white cabbage and kale. It indicated that salinity reduced the capacity of the photosynthetic system for efficient energy conversion, particularly in Chinese cabbage. In parallel with the photosynthetic impairments, the Na+/K+ ratio was highest in Chinese cabbage leaves and lowest in kale leaves while kale root is able to keep high Na+/K+ ratio without a significant increase in MDA. Thus Na+/K+ ratio, high in root and low in leaves accompanying with low MDA level is an informative marker of salinity tolerance. The crops’ tolerance was positively correlated with levels of the stress hormone abscisic acid (ABA) and negatively correlated with levels of jasmonic acid (JA), and jasmonoyl-L-isoleucine (JA-Ile). Furthermore, salinity induced contrasting changes in levels of the growth-promoting hormones brassinosteroids (BRs). The crop’s tolerance was positively correlated with levels of BR precursor typhasterol while negatively with the active BR brassinolide. Principal Component Analysis revealed correlations in observed changes in phytohormones, biochemical, and physiological parameters. Overall, the results show that kale is the most tolerant of the three species and Chinese cabbage the most sensitive to salt stress, and provide holistic indications of the spectrum of tolerance mechanisms involved.

Neuroactive steroids are a family of all steroid-based compounds, of both natural and synthetic origin, which can affect the nervous system functions. Their biosynthesis occurs directly in the nervous system (so-called neurosteroids) or in peripheral endocrine tissues (hormonal steroids). Steroid hormone levels may fluctuate due to physiological changes during life and various pathological conditions affecting individuals. A deeper understanding of neuroactive steroids’ production, in addition to reliable monitoring of their levels in various biological matrices, may be useful in the prevention, diagnosis, monitoring, and treatment of some neurodegenerative and psychiatric diseases. The aim of this review is to highlight the most relevant methods currently available for analysis of neuroactive steroids, with an emphasis on immunoanalytical methods and gas, or liquid chromatography combined with mass spectrometry.

Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson’s disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N6-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.

Six new (1, 2, 6, 8, 13, and 20) and twenty previously isolated (3–5, 7, 9–12, 14–19, and 21–26) steroids featuring thirteen different carbocycle motifs were isolated from the organic extract of the soft coral Sinularia polydactyla collected from the Hurghada reef in the Red Sea. The structures and the relative configurations of the isolated natural products have been determined based on extensive analysis of their NMR and MS data. The cytotoxic, anti-inflammatory, anti-angiogenic, and neuroprotective activity of compounds 3–7, 9–12, 14–20, and 22–26, as well as their effect on androgen receptor-regulated transcription was evaluated in vitro in human tumor and non-cancerous cells. Steroids 22 and 23 showed significant cytotoxicity in the low micromolar range against the HeLa and MCF7 cancer cell lines, while migration of endothelial cells was inhibited by compounds 11, 12, 22, and 23 at 20 µM. The results of the androgen receptor (AR) reporter assay showed that compound 11 exhibited the strongest inhibition of AR at 10 µM, while it is noteworthy that steroids 10, 16, and 20 displayed increased inhibition of AR with decreasing concentrations. Additionally, compounds 11 and 23 showed neuroprotective activity on neuron-like SH-SY5Y cells.

Plant steroid alcohols, plant sterols, are essential components of cell membranes that perform many functions. Their most prominent function is maintaining membrane semipermeability and regulating its fluidity through their specific interaction with phospholipids and membrane proteins. This work is focused on the study of the interaction of two groups of plant sterols, brassinosteroids (BRs) and phytoecdysteroids (PE). Steroid substances belonging to both groups are important signaling molecules essential for plant growth and development, but while the first group has all the known attributes of plant hormones, the second lacks hormonal function in plants. The aim of this preliminary study was to determine at what concentration level and to what extent substances of this type are able to interact with each other, and thus influence the early growth and development of a plant. It was found that exogenously applied PE 20-hydroxyecdysone (20E) significantly reduced the level of endogenous BRs in four-day-old garden cress (Lepidium sativum) seedlings. On the other hand, exogenously applied BRs, 24-epibrassinolide (epiBL), caused the opposite effect. Endogenous 20E was further detected at the picogram level in garden cress seedlings. Thus, this is the first report indicating that this plant species is PE-positive. The level of endogenous 20E in garden cress seedlings can be decreased by exogenous epiBL, but only at a relatively high concentration of 1·10−6 M in a culture medium. The image analysis of garden cress seedlings revealed that the length of shoot is affected neither by exogenous BRs nor PE, whereas the root length varies depending on the type and concentration of steroid applied.

Cytokinins and their sugar or non-sugar conjugates are very active growth-promoting factors in plants, although they occur at very low concentrations. These compounds have been identified in numerous plant species. This review predominantly focuses on 9-substituted adenine-based cytokinin conjugates, both artificial and endogenous, sugar and non-sugar, and their roles in plants. Acquired information about their biological activities, interconversions, and metabolism improves understanding of their mechanisms of action and functions in planta. Although a number of 9-substituted cytokinins occur endogenously, many have also been prepared in laboratories to facilitate the clarification of their physiological roles and the determination of their biological properties. Here, we chart advances in knowledge of 9-substituted cytokinin conjugates from their discovery to current understanding and reciprocal interactions between biological properties and associated structural motifs. Current organic chemistry enables preparation of derivatives with better biological properties, such as improved anti-senescence, strong cell division stimulation, shoot forming, or more persistent stress tolerance compared to endogenous or canonical cytokinins. Many artificial cytokinin conjugates stimulate higher mass production than naturally occurring cytokinins, improve rooting, or simply have high stability or bioavailability. Thus, knowledge of the biosynthesis, metabolism, and activity of 9-substituted cytokinins in various plant species extends the scope for exploiting both natural and artificially prepared cytokinins in plant biotechnology, tissue culture, and agriculture.

Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.

Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (Triticum aestivum L.) and Arabidopsis thaliana. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound. In vivo, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.

ABSTRACT It was recently reported that the human-exclusive pathogen Mycobacterium tuberculosis secretes cytokinins, which had only been known as plant hormones. While cytokinins are well-established, adenine-based signaling molecules in plants, they have never been shown to participate in signal transduction in other kingdoms of life. M. tuberculosis is not known to interact with plants. Therefore, we tested the hypothesis that cytokinins trigger transcriptional changes within this bacterial species. Here, we show cytokinins induced the strong expression of the M. tuberculosis gene Rv0077c. We found that Rv0077c expression is repressed by a TetR-like transcriptional repressor, Rv0078. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis. While acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice, consistent with an unaltered mycolic acid profile of Rv0077c-expressing cells. Collectively, these findings show cytokinins signal transcriptional changes that can affect M. tuberculosis acid-fastness and that cytokinin signaling is no longer limited to the kingdom Plantae. IMPORTANCE Cytokinins have only previously been known as plant hormones. The discovery that they can be used as signaling molecules outside of plants broadens the repertoire of small molecules that can potentially affect gene expression in all domains of life.

Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, 11 [15N]-labelled C6-purine derivatives were prepared, among them 5 aromatic (4, 5, 6, 7, 8) and 3 isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine) and 6-chloro-9H-[15N4]-purine (6-chloropurine) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC–MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.

Salinity is a major abiotic stress negatively affecting plant growth and consequently crop production. The effects of short-term salt stress were evaluated on seedlings of three globally important Brassica crops—Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (Brassica oleracea var. capitata), and kale (Brassica oleracea var. acephala)—with particular focus on phenolic acids. The physiological and biochemical stress parameters in the seedlings and the levels of three main groups of metabolites (total glucosinolates, carotenoids, and phenolics) and individual phenolic acids were determined. The salt treatments caused a dose-dependent reduction in root growth and biomass and an increase in stress parameters (Na+/K+ ratio, reactive oxygen species (ROS) and glutathione (GSH)) in all seedlings but most prominently in Chinese cabbage. Based on PCA, specific metabolites grouped close to the more tolerant species, white cabbage and kale. The highest levels of phenolic acids, particularly hydroxycinnamic acids, were determined in the more tolerant kale and white cabbage. A reduction in caffeic, salicylic, and 4-coumaric acid was found in Chinese cabbage and kale, and an increase in ferulic acid levels was found in kale upon salinity treatments. Phenolic acids are species-specific among Brassicaceae, and some may participate in stress tolerance. Salt-tolerant varieties have higher levels of some phenolic acids and suffer less from metabolic stress disorders under salinity stress.

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

Verticillium longisporum is a vascular pathogen that infects the Brassicaceae host plants Arabidopsis thaliana and Brassica napus. The soilborne fungus enters the plant via the roots and colonizes the xylem of roots, stems, and leaves. During late stages of infections, Verticillium spp. spread into senescing tissue and switch from biotrophic to a necrotrophic life style. Typical symptoms of V. longisporum-induced disease are stunted growth and leaf chlorosis. Expression analyses of the senescence marker genes SENESCENCE-ASSOCIATED GENE12, SENESCENCE-ASSOCIATED GENE13, and WRKY53 revealed that the observed chlorosis is a consequence of premature senescence triggered by Verticillium infection. Our analyses show that, concomitant with the development of chlorosis, levels of trans-zeatin decrease in infected plants. Potentially, induction of cytokinin oxidase/dehydrogenase expression by Verticillium infection contributes to the observed decreases in cytokinin levels. Stabilization of Arabidopsis cytokinin levels by both pharmacological and genetic approaches inhibits Verticillium proliferation and coincides with reduced disease symptom development. In summary, our results indicate that V. longisporum triggers premature plant senescence for efficient host plant colonization.

Despite numerous studies, the role of hormones in the induction of shoot apical meristem leading to reproductive development, especially regarding thermoperiodic plants, is still not fully understood. The key problem is separating the effects of the low temperature required for vernalization from those responsible for low temperature stress. An earlier experiment demonstrated the correlation between an increase of cytokinin level in the apical parts of winter rapeseed and the transition time into their reproductive phase during vernalization, i.e., low temperature treatment. From data obtained from the presented experiments, this study aims to contribute to the understanding the role of cytokinins in the induction of flowering based on the grafting of vegetative apical parts of winter rapeseed (scion) on the reproductive (stock) winter and spring genotypes. On the basis of analyses carried out using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in combination with microscopic observation of changes at the apical meristem, it was indicated that the increase in the amount of trans-zeatin and trans- and cis-zeatin-O-glucoside derivatives appeared in the early stages of apex floral differentiation. During further development, the content of all investigated cytokinins passed through the maximum level followed by their decrease. The final level in reproductive apices was found to be higher than that in vegetative ones.

The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica–induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica–mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica–induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.

The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.

Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

Current inhibitors of angiogenesis comprise either therapeutic antibodies (e.g. bevacicumab binding to VEGF-A) or small molecular inhibitors of receptor tyrosin kinases like e.g. sunitinib, which inhibits PDGFR and VEGFR. We have recently identified cyclin-dependent kinase 5 (Cdk5) as novel alternative and pharmacologically accessible target in the context of angiogenesis. In the present work we demonstrate that trisubstituted pyrazolo[4,3-d]pyrimidines constitute a novel class of compounds which potently inhibit angiogenesis. All seven tested compounds inhibited endothelial cell proliferation with IC(50) values between 1 and 18 µM. Interestingly, this seems not to be due to cytotoxicity, since none of them showed acute cytotoxic effects on endothelial cells at a concentration of 10 µM,. The three most potent compounds (LGR1404, LGR1406 and LGR1407) also inhibited cell migration (by 27, 51 and 31%, resp.), chemotaxis (by 50, 70 and 60% in accumulative distance, resp.), and tube formation (by 25, 60 and 30% of total tube length, resp.) at the non-toxic concentration of 10 µM. Furthermore, angiogenesis was reduced in vivo in the CAM assay by these three compounds. A kinase selectivity profiling revealed that the compounds prevalently inhibit Cdk2, Cdk5 and Cdk9. The phenotype of the migrating cells (reduced formation of lamellipodia, loss of Rac-1 translocation to the membrane) resembles the previously described effects of silencing of Cdk5 in endothelial cells. We conclude that especially LGR1406 and LGR1407 are highly attractive anti-angiogenic compounds, whose effects seem to largely depend on their Cdk5 inhibiting properties.

BackgroundWhen applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta.Methodology/principal findingsThe main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants.Conclusions/significanceTetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.