Cytokinin Levels Research Articles

Interactions Between Exogenous Cytokinin and Nitrogen Application Regulate Tiller Bud Growth via Sucrose and Nitrogen Allocation in Winter Wheat

In this study, we assessed the effects of exogenous cytokinin and nitrogen levels on sucrose and nitrogen allocation and tiller bud growth. Results showed that low nitrogen (1.25 mmol L−1, LN) inhibited tiller bud growth and decreased tiller occurrence rate, compared with high nitrogen (7.5 mmol L−1, HN). SPAD readings and leaf photosynthesis rate was decreased by LN, which resulted in the decrease of sucrose content in the tiller bud. Furthermore, LN promoted the primary and lateral root growth, which may compete with the tiller bud for sucrose and nitrogen. Application of 6-BA (30 mg L−1) increased tiller bud length and the occurrence rate. Compared with LN, leaf photosynthesis and root activity was improved by application of 6-BA under LN condition (LN + 6-BA), which caused the increase of sucrose and nitrogen accumulation in tiller bud. Moreover, LN + 6-BA significantly increased endogenous zeatin (Zt) contents and decreased abscisic acid (ABA) levels in the tiller node. Correlation analysis indicated that tiller bud growth was positively correlated with sucrose and nitrogen accumulation. The latter two were positively correlated with endogenous Zt but negatively correlated with ABA and auxin (IAA):Zt, gibberellin (GA3):Zt, and ABA:Zt. Application of 6-BA with reduced nitrogen fertilizer (240 kg N ha−1, N1 + 6-BA) increased both grain yield and the partial factor productivity of applied N. Therefore, an appropriate combination of exogenous cytokinin with reduced N application could regulate tiller growth to enhance yield by endogenous hormones balance and sucrose and nitrogen allocation in the plant.

Overexpression of MsGH3.5 inhibits shoot and root development through the auxin and cytokinin pathways in apple plants.

Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole-3-acetic acid (IAA)-amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA-amino acid conjugates, and MsGH3.5-overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type-B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.

Mutational analysis indicates that abnormalities in rhizobial infection and subsequent plant cell and bacteroid differentiation in pea (Pisum sativum) nodules coincide with abnormal cytokinin responses and localization.

Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation.

EARLY BUD BREAK 1 triggers bud break in peach trees by regulating hormone metabolism, the cell cycle, and cell wall modifications.

In a previous study we identified EARLY BUD BREAK 1 (EBB1), an ERF transcription factor, in peach (Prunus persica var. nectarina cultivar Zhongyou 4); however, little is known of how PpEBB1 may regulate bud break. To verify the function of PpEBB1 in bud break, PpEBB1 was transiently transformed into peach buds, resulting in early bud break. Bud break occurred earlier in PpEBB1-oe poplar (Populus trichocarpa) obtained by heterologous transformation than in wild type (WT), consistent with the peach bud results, indicating that PpEBB1 can promote bud break. To explore how PpEBB1 affects bud break, differentially expressed genes (DEGs) between WT and PpEBB1-oe poplar plants were identified by RNA-sequencing. The expression of DEGs associated with hormone metabolism, cell cycle, and cell wall modifications changed substantially according to qRT-PCR. Auxin, ABA, and total trans-zeatin-type cytokinin levels were higher in the PpEBB1-oe plants than in WT plants, while the total N6-(Δ 2-isopentenyl)-adenine-type cytokinins was lower. Yeast two-hybrid and bimolecular fluorescence complementation assays verified that a cell wall modification-related protein (PpEXBL1) interacted with PpEBB1 suggesting that PpEBB1 could interact with these cell wall modification proteins directly. Overall, our study proposed a multifaceted explanation for how PpEBB1 regulates bud break and showed that PpEBB1 promotes bud break by regulating hormone metabolism, the cell cycle, and cell wall modifications.

Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

Heat Acclimation and Inhibition of Cytokinin Degradation Positively Affect Heat Stress Tolerance of Arabidopsis.

In order to pinpoint phytohormone changes associated with enhanced heat stress tolerance, the complex phytohormone profiles [cytokinins, auxin, abscisic acid (ABA), jasmonic acid (JA), salicylic acid and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)] were compared in Arabidopsis thaliana after direct heat shock (45°C, 3 h) and in heat-stressed pre-acclimated plants (1 h at 37°C followed by 2 h at optimal temperature 20°C). Organ-specific responses were followed in shoot apices, leaves, and roots immediately after heat shock and after 24-h recovery at 20°C. The stress strength was evaluated via membrane ion leakage and the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) and antioxidant enzymes [superoxide dismutases, guaiacol peroxidases (POD), catalases, ascorbate peroxidases (APX)]. Heat acclimation diminished negative effects of heat stress, especially in apices and roots, no significant differences being observed in leaves. Low NOX1-3 activities indicated diminished production of reactive oxygen species. Higher activity of APX, POD1, and the occurrence of POD3-4 reflected acclimation-stimulated readiness of the antioxidant system. Acclimation diminished heat shock-induced changes of ABA, JA, cytokinin, and auxin levels in apices. Excess of ABA catabolites suggested an early stress response. The strong up-regulation of ABA and ACC in roots indicated defense boost in roots of acclimated plants compared to the non-acclimated ones. To evaluate the possibility to enhance stress tolerance by cytokinin pool modulation, INCYDE-F, an inhibitor of cytokinin oxidase/dehydrogenase, was applied. As cytokinin effects on stress tolerance may depend on timing of their regulation, INCYDE was applied at several time-points. In combination with acclimation, INCYDE treatment had a slight positive effect on heat stress tolerance, mainly when applied after 2-h period of the optimal temperature. INCYDE increased contents of cytokinins trans-zeatin and cis-zeatin in roots and auxin in all tissues after heat shock. INCYDE also helped to suppress the content of ABA in leaves, and ethylene in apices and roots. INCYDE application to non-acclimated plants (applied before or after heat shock) strengthened negative stress effects, probably by delaying of the repair processes. In conclusion, pre-treatment with moderately elevated temperature enhanced heat stress tolerance and accelerated recovery after stress. Inhibition of cytokinin degradation by INCYDE slightly improved recovery of acclimated plants.

Arbuscular mycorrhizal fungi promote the growth of plants in the mining associated clay

It is urgent to restore the ecological function in open-pit mining areas on grassland in Eastern China. The open-pit mines have abundant of mining associated clay, which is desirable for using as a soil source for ecological restoration. The mining associated clay in Hulunbuir district, Inner Mongolia was selected and mixed with a sandy soil at a ratio of 1:1 (S_C soil). Also, effects of arbuscular mycorrhizal fungi (AMF) inoculation on soil functions were studied. The aboveground and underground biomass of maize in S_C soil was 1.49 and 2.41 times higher than that of clay soil, respectively. In the topsoil and S_C soil, the growth hormone (IAA) and cytokinin (CTK) levels of maize were higher than that of clay, while abscission acid (ABA) levels were lower. The inoculation with AMF could significantly improve the biomass of maize and enhance the stress resistance of plants. Through structural equation model (SEM) analyses, it was found that the soil type and AMF inoculation had the most direct impact on maize growth and biomass content. These finds extend our knowledge regarding a low-cost method for physical and biological improvement of mining associated clay, and to provide theoretical support for large-scale application in the future.

Overexpressing AtCKX1 in Potato Plants grown In Vitro: The Effects on Cytokinin Composition and Tuberization

Over the last years, cytokinin deficiency has been studied in a variety of plant species, using transgenic expression of cytokinin oxidase/dehydrogenase genes (CKX). In tobacco and Arabidopsis thaliana, overexpression of A. thaliana CKX genes caused changes in plant morphology known as the "cytokinin deficiency syndrome", including stunted shoot growth, decreased apical dominance, and enhanced root growth. For a valid comparison with the previously obtained in vitro grown AtCKX2-transgenic potato lines, we report here on the construction of transgenic lines of the cultivar Desiree constitutively expressing the gene AtCKX1 under control of the CaMV 35S promoter. Three lines expressing the transgene in both shoot and root tissues were used for analysis. Analyses of CKX activity of tissue extracts, cytokinin levels, morphology, and tuberization parameters revealed similarities as well as differences from the AtCKX2-transgenic plants, including lower levels of CKX activity, and more pronounced morphological effects of cytokinin deficiency. One of the three AtCKX1-overexpressing potato lines was able to start forming tubers before 30 days of growth in long day conditions (16 h/8 h photoperiod) as has been previously observed in some AtCKX2 overexpressors. The extension of the experimental period to 120 days of growth in vitro showed that the other two AtCKX1-overexpressing lines showed impaired tuberization in terms of the number of tubers per plant, and tuber size. We conclude that cytokinin deficiency has distinct effects on tuber induction and tuber initiation/growth, possibly mediated by distinct signaling mechanisms.

SEEDSTICK Controls Arabidopsis Fruit Size by Regulating Cytokinin Levels and FRUITFULL

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.

Flower-Specific Overproduction of Cytokinins Altered Flower Development and Sex Expression in the Perennial Woody Plant Jatropha curcas L.

Jatropha curcas L. is monoecious with a low female-to-male ratio, which is one of the factors restricting its seed yield. Because the phytohormone cytokinins play an essential role in flower development, particularly pistil development, in this study, we elevated the cytokinin levels in J. curcas flowers through transgenic expression of a cytokinin biosynthetic gene (AtIPT4) from Arabidopsis under the control of a J. curcas orthologue of TOMATO MADS BOX GENE 6 (JcTM6) promoter that is predominantly active in flowers. As expected, the levels of six cytokinin species in the inflorescences were elevated, and flower development was modified without any alterations in vegetative growth. In the transgenic J. curcas plants, the flower number per inflorescence was significantly increased, and most flowers were pistil-predominantly bisexual, i.e., the flowers had a huge pistil surrounded with small stamens. Unfortunately, both the male and the bisexual flowers of transgenic J. curcas were infertile, which might have resulted from the continuously high expression of the transgene during flower development. However, the number and position of floral organs in the transgenic flowers were well defined, which suggested that the determinacy of the floral meristem was not affected. These results suggest that fine-tuning the endogenous cytokinins can increase the flower number and the female-to-male ratio in J. curcas.

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

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

The <i>rolC</i> gene of agrobacteria: towards the understanding of its functions

Agrobacterium rhizogenesConn is a soil bacterium, which can transform plants by inserting a plasmid fragment into the plant genome. This fragment contains a “root locus”: four genes that cause root overgrowth of the transformed plant, the so-called “hairy root syndrome”. The most studied gene of the root locus isrolC. For more than 30 years of research on this gene, data have been obtained on its expression, protein localization and putative functions of the protein as well as on its effect on plant morphology and biochemistry. TherolCtransformation leads to multiple morphological effects, most common among which are dwarfism, bushiness, and a change in the shape of the leaf blade. Such specific plant reactions are associated with changes in hormone balance under the influence ofrolC. The levels of auxins, cytokinins, and abscisic acid do change in transformed plants, but no regularities have been revealed. Also, the signaling pathways ofrolCaffecting the hormonal system of plants are not established. Morphogenetic effects can occur in varying degrees depending on the promoter under which therolCworks. A constitutive promoter usually leads to a more pronounced effect when compared to a gene that operates under a native promoter. Secondary plant metabolism is also affected byrolC. The synthesis of various metabolites is amplified in transformants, and, in contrast to morphological effects, this biochemical effect does not depend on the promoter. Some secondary metabolites are associated with the plant defense system; thus,rolCis able to indirectly influence this aspect of plant physiology. This review summarizes the results of therolCgene studies in plants. The authors formulate the main hypotheses regarding the mechanisms of the gene in order to promote our understanding of its function in plants.

Cytokinin dynamics in xylem sap and leaves of fruiting and de-fruited sunflower hybrids of contrasting post-anthesis canopy senescence patterns during flowering and grain-filling

To determine whether xylem sap and leaf cytokinin levels were associated with live root length density (LRLD) and leaf senescence, these variables were measured in intact (control) plants and those from which florets were removed, in hybrids of contrasting post-anthesis senescence patterns. Field experiments were carried out during two consecutive seasons using sunflower hybrids (Paraiso75, stay-green [SG]; and Paraiso65, fast dry down [FDD]) of contrasting canopy senescence patterns. Crop carbon partitioning was altered by removing all florets (-F treatment) before anthesis. At crop level, the dynamics of LRLD, xylem sap flow rate, cytokinins in the xylem sap, and green leaf area index (GLAI) were measured, while at leaf level, dynamics of total chlorophyll content, trans-Zeatin content, and net photosynthesis were recorded for leaf positions 17, 20, 22, 24 and 27. In both hybrids, the –F treatment had higher LRLD, cytokinins in the xylem sap, and leaf trans-Zeatin concentration at anthesis and during the grain filling period than control plants. All measured variables decreased earlier in the control treatment than the -F treatment (in both hybrids), and in the FDD hybrid than the SG hybrid. The dynamics of xylem sap cytokinins were related to the dynamics of LRLD and that of leaf trans-Zeatin levels, contributing to contrasting canopy senescence patterns and underlying the responses to defruiting in both hybrids.

Genome-wide transcriptomic analysis during rhizome development of ginger (Zingiber officinale Roscoe.) reveals hormone and transcriptional regulation involved in cellulose production

Ginger is a popular vegetable crop primarily consumed in Chongqing and Sichuan, China. The rhizome becomes tough and fibrous rapidly with the tissue maturation, resulting in loss of edible quality. To characterize biochemical and molecular mechanisms of rhizome texture modification, we investigated the changes of the cell wall components, hormones and transcriptome profiles during rhizome development. With rhizomes maturation, the contents of cellulose and hemicellulose increased gradually, and no obvious change was observed in lignin accumulation. The levels of ABA and cytokinin exhibited a gradual decline during maturation of ginger rhizome, which were negatively correlated with cellulose content, while GA3 displayed an increasing trend and a positive correlation with cellulose production. Transcriptomic analysis identified candidate genes involved in cellulose biosynthesis including cellulose synthase subunit A (CesA) genes, hormone metabolism and signaling genes, and NAC-MYB-based transcription factors. Further analysis demonstrated ZoCESA7, ZoNAC16 and ZoMYB37/39/41, homologs of AtCESA7, AtVND6/7 and AtMYB46/83, respectively, positively correlated with cellulose contents, whereas ZoNAC3/9/13 and ZoMYB7/11/18, homologs of AtVND6/7 and AtMYB4, respectively, negatively correlated with cellulose levels, indicating their involvements in cellulose synthesis during rhizome development. These findings provide new insight into molecular events of cellulose accumulation mainly mediated by hormones and transcription factors in ginger rhizomes.

ИОННЫЙ ГОМЕОСТАЗ У РАСТЕНИЙ ПШЕНИЦЫ, ИНОКУЛИРОВАННЫХ ПРОДУЦИРУЮЩИМИ ФИТОГОРМОНЫ БАКТЕРИЯМИ НА ФОНЕ ЗАСОЛЕНИЯ

Сytokinins content, concentration of sodium and potassium ions and K+/Na+ratio in wheat plants (Triticum durum Desf., Bashkirskaya 27) were evaluated under salinity during treatment with hormone-producing bacteria Pseudomonas mandelii IB-Ki14 (auxin-producer) or Bacillus subtilis IB-22 (cytokinin-producer). An increased level of cytokinins was observed in the roots on 6th day and then in the shoots (on 11th day) both in the absence of salt and under 100 mM NaCl in the presence of B. subtilis IB-22. The introduction of P. mandelii IB-Ki14 into the rhizosphere did not lead to the accumulation of cytokinins under salinity. The presence of NaCl in the soil led to the expected increase in the concentration of sodium ions both in the roots and in the shoots, and inoculation did not significantly change their values. Salinity decreased the content of potassium ions in the roots by 25% in plants uninoculated with bacteria and in plants treated with P. mandelii IB-Ki14, as a result of which the ratio of K+/Na+ ions decreased by 4 times compared with the control, indicating a disruption of the ionic homeostasis. In plants inoculated with B. subtilis IB-22, no decrease in the concentration of potassium ions was found as compared to the control, and the ratio of K+/Na+ ions in the roots under salinity decreased to a lesser extent than in other variants of the experiment. This indicates the ability of cytokinin-producing bacteria to stabilize ionic balance and reduce the consequences of the negative effects of salinity on photosynthesis, protein synthesis, and growth. Our data indicate that this property of bacteria is associated with their ability to synthesize cytokinins and increase cytokinin content in plants under salinity.

Physiological response of North China red elder container seedlings to inoculation with plant growth-promoting rhizobacteria under drought stress.

The issue of how to alleviate the negative effects imposed by water stress is an interesting problem. Plant growth-promoting rhizobacteria (PGPR) colonize the rhizosphere of plants and are known to promote the growth of crops. However, there are few studies characterizing the physiological response of plants to drought stress after PGPR inoculation. The aim of this study was to investigate the effectiveness of different PGPRs in arid environments and then investigated the effects of PGPR inoculation under drought stress on the physiological characteristics and growth of North China red elder (Sambucus williamsii) nursery container seedlings. The viable count of different PGPRs under drought stress varies widely, and the drought tolerance of Acinetobacter calcoaceticus X128 was significantly higher than that of other PGPRs. In comparison with non-inoculation, inoculation with X128 in an arid environment significantly increased stomatal conductance and mitigated the inhibition of net photosynthetic rate caused by drought stress; this mitigating effect of inoculation is enhanced as the level of drought stress increases. Relative to non-inoculated seedlings, cytokinin levels in the leaves increased by 91.17% under severe drought stress conditions in inoculated seedlings. However, X128 inoculation decreased this deficit to only 44.54%. Compared with non-inoculated seedlings, the relative water content of inoculated seedlings under severe drought stress increased by 15.06%, however the relative conductivity decreased by 12.48%. Consequently, X128 could increase dry matter accumulation of S. williamsii regardless of watering status, indicative of the greater benefits of PGPR on shoot growth than root. Therefore, inoculation of A. calcoaceticus X128 under drought conditions play a significant role for alleviating the negative effects imposed by water stress and promoting plant growth.

Galling impacts of the gall wasp Leptocybe invasa (Hymenoptera: Eulophidae) on Eucalyptus trees vary with plant genotype

Impacts of galling on the distributions of plant metabolites can vary greatly with plant genotype. In this study, two Eucalyptus genotypes with different resistance levels were chosen, and the levels of several primary and secondary metabolites, as well as phytohormones in galls and ungalled portions of galled leaves infested by the gall wasp Leptocybe invasa, and gall-free (control) leaves were compared. It was found that galls of both two plant genotypes accumulated higher concentrations of carbon, total phenolics, gibberellins (GA), and abscisic acid (ABA) but had lower chlorophyll content than ungalled portions. However, galls of highly susceptible genotype contained higher nitrogen (N), cytokinins (CK), GA, and chlorophyll content but lower C/N ratio, total phenolics, tannins, and ABA than less susceptible genotype. For both two genotypes, ABA in galls and ungalled portions increased compared with adjacent control leaves. CK and GA levels increased in galls but decreased in ungalled portions of highly susceptible genotype, compared with control leaves. For less susceptible genotype, CK levels increased in both galls and ungalled portions compared with control leaves, but higher levels of tannins, total phenolics, and GA were only detected in galls. Therefore, our study found insufficient evidence that the impact of galling on the distributions of these metabolites and phytohormones extended beyond the attacked leaves, because they varied greatly with plant genotype.

Characterization of five CHASE-containing histidine kinase receptors from Populus × canadensis cv. Robusta sensing isoprenoid and aromatic cytokinins.

Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays. Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins' biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.

Distinct redox signalling and nickel tolerance in Brassica juncea and Arabidopsis thaliana.

Despite of its essentiality, nickel (Ni) in excess is toxic for plants partly due to the overproduction of reactive oxygen species (ROS) and the consequent increase in oxidative stress signalling. However, in Ni-stressed plants little is known about the signal transduction of reactive nitrogen species (RNS) and protein tyrosine nitration as the protein-level consequence of increased RNS formation. Our experiments compared the nickel accumulation and tolerance, the redox signalling and the protein nitration in the agar-grown Arabidopsis thaliana and Brassica juncea exposed to Ni (50μM nickel chloride). Studying GUS-tagged Arabidopsis lines (ARR5::GUS, ACS8::GUS and DR5::GUS) revealed that Ni-increased lateral root (LR) emergence, and concomitantly reduced LR initiation were accompanied by elevated levels of auxin, cytokinin, and ethylene in the LRs or in upper root parts, whereas Ni-induced primary root shortening is related to decreased auxin, and increased cytokinin and ethylene levels. These suggest the Ni-induced disturbance of hormonal balance in the root system. Results of the comparative study showed that weaker Ni tolerance of A. thaliana was coupled with a Ni-induced increase in RNS, ROS, and hydrogen sulfide levels, as well as with an increase in redox signalling and consequent increment of protein nitration. However, in relative Ni tolerant B. juncea, redox signalling (except for peroxynitrite) was not modified, and Ni-induced intensification of protein tyrosine nitration was less pronounced. Data collectively show that the better Ni tolerance of Brassica juncea may be related to the capability of preventing the induction of redox signalling and consequently to the slighter increase in protein nitration.

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots.