Cytokinin Synthesis Research Articles

Mitigation of salinity stress in plants using plant growth promoting bacteria

Salinity is one of the major constraints prevailing in environment that affects not only plant growth but also agriculture productivity and soil fertility. Salinity stress causes nutritional and hormonal imbalance, ion toxicity, oxidative and osmotic stress and increased susceptibility of plant to diseases. Increasing use of chemical based fertilizers creates demand for environmental friendly and ecological compatible alternative in agriculture. Plant growth promoting bacteria (PGPB) have tremendous ability to mitigate salinity stress and ameliorate plant growth, playing vital role in food security by increasing agriculture crop productivity. PGPB inoculation under salinity promotes plant growth by several traits including 1-aminocyclopropane 1 carboxylate (ACC) deaminase activity, synthesis of plant hormones (such as indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), cytokinin) and exopolysaccharides. PGPB alleviates salinity stress in plants by providing nutrients, maintaining high potassium and sodium ratio, increasing accumulation of osmolytes, enhanced photosynthesis and activity of antioxidant enzymes. The present review highlights and discusses current knowledge on effect of salinity stress on plant growth, PGPB mechanisms resulting in mitigation of salinity stress and increasing plant growth. It assesses morphological, biochemical, physiological and molecular changes induced in plants suffering from salinity when supplemented with PGPB.

Partial and full root-zone drought stresses account for differentiate root-sourced signal and yield formation in primitive wheat

BackgroundPartial and full root-zone drought stresses are two widely used methods to induce soil drying in plant container-culture experiments. Two methods might lead to different observational results in plant water relation, such as non-hydraulic root-sourced signal (nHRS). We compared partial and full stress methods to induce nHRS in two diploids (MO1 and MO4) and two tetraploids (DM 22 and DM 31) wheat varieties under pot-culture conditions. Partial root-zone stress (PS) was performed using split-root alternative water supply method (one half wetting and the other drying) to induce the continuous operation of nHRS, and full root-zone stress (FS) was exposed to whole soil block to induce periodic operation of nHRS since jointing stage.ResultsWe tested the two drought methods whether it influenced the nHRS mediated signalling and yield formation in primitive wheat species. Results showed that partial root-zone stress caused more increase in abscisic acid (ABA) production and decline in stomatal closure than full root-zone stress method. The incline in ABA was closely related to triggering reactive oxygen species (ROS) generation, and reducing cytokinin synthesis which, thereby, led to crosstalk with other signalling molecules. Furthermore, PS up-regulated the antioxidant defense system and proline content. Water use efficiency and harvest index was significantly increased in PS, suggesting that PS was more likely to simulate the occurrence of nHRS by increasing the adaptive strategies of plants and closer to natural status of soil drying than FS.ConclusionThese findings lead us to conclude that partial root-zone stress method is more feasible method to induce nHRS which has great capacity to reduce water consumption and enhance plant adaptation to constantly changing environment. These observations also suggest that different root-zone planting methods can be considered to improve the plant phenotypic plasticity and tolerance in water-limited rainfed environments.

Plug Cell Volume, Growing Media Quality and Benzyl Aminopurine (BAP) Spray Effects for Nursery Growth of Impatiens walleriana

Plant propagators must take two technological critical decisions: the plug-cell size and the growing medium, both of which have been mentioned as abiotic stress sources for bedding pot plants. However, only a few recent reports on bedding pot plants have simultaneously included limiting and non-limiting plug cell volumes and growing medium during nursery. The aim of this work was to assess the nursery performance of Impatiens walleriana seedlings grown in four plug cell volumes and four growing media with significant differences in both physical and chemical properties. Plants were sprayed or not with an early and single benzyl aminopurine (BAP) dose, aiming to understand how they interact on determining biomass accumulation at the pot transplant stage. The hypothesis tested was that, both plug cell volume and growing medium, must be seen as additive abiotic stress sources, which can be partially overridden by exogenous cytokinin supply. The main result was that, in I. walleriana seedlings, the abiotic stress imposed by the growing medium quality during nursery had a higher effect on biomass accumulation (on both fresh and dry base), leaf area expansion and photo assimilates partitioning than plug cell volume and constitute an interactive process associated with cytokinin synthesis. From a grower´s point of view, one expensive option to avoid root restriction is to use high quality growing media and increase the plug cell volume. In contrast, a single 100 mg L-1 BAP spray can partially override the root restriction symptoms related to abiotic stresses. The novelty of this work is related to the fact that growing media quality would be a more limited factor than plug cell volume for I. walleriana seedlings during nursery.

Physiological and biochemical responses of two spring wheat genotypes to non-hydraulic root-to-shoot signalling of partial and full root-zone drought stress

Non-hydraulic root-sourced signal (nHRS) is so far affirmed to be a unique positive early-warning response to drying soil, however its physiological and agronomic implications are still unclear. We designed two contrast methods to induce nHRS in two wheat (Triticum aestivum L.) genotypes released in different decades under pot-culture conditions. Partial root-zone stress (PS) was performed using the method of split-root alternative water supply (one half wetting and the other drying) to induce the continuous operation of nHRS, and full root-zone stress (FS) was subjected to whole root system to periodic operation of nHRS. nHRS-mediated signalling increased abscisic acid (ABA) production and triggered ROS (reactive oxygen species) generation, which, thereby, led to up-regulation of antioxidant defense system. Cytokinin synthesis reduced during drought stress while proline and malodialdehyde (MDA) content were increased. Regardless of drought treatment methods and wheat genotype, a significant decrease in grain yield, root biomass and above-ground biomass (p < 0.05) was observed, without significant changes in root-to-shoot ratio. Harvest index was increased, proposing that more energy was allocated to reproductive organs during the action of nHRS. Moreover, higher water use efficiency was witnessed in PS. The data suggest that nHRS triggered ABA accumulation, thereby closing stomata, and reducing water use and also decreases the production of ROS and improves the antioxidant defence enzymes, thus enhancing drought tolerance. This survey of different-decade genotypes suggests that advances in grain yield and drought tolerance would be made by targeted selection for a wheat genetic resource.

Impatiens ‘New Guinea’ (Impatiens hawkeri Bull) Hormonal Effects during the Post-transplant Biomass Accumulation

Pot ornamental plant productivity is related to the environmental growth facilities but negatively affected by the pot root restriction syndrome so during nursery as the post-transplant stage. The physiological mechanism involved included both the synthesis and translocation of auxins and cytokinins. However, clear sink-source and dose-response relationships of exogenous plant regulators such as indole acetic acid (AIA) and benzyl amino purine (BAP) and environment on biomass accumulation in most ornamental foliage plants, including New Guinea Impatiens (Impatiens hawkeri) are lacking. The aim of this work was to analyze the effects of an exogenously shoot-applied auxin and a cytokinin, separately or successively, on the post-transplant biomass accumulation of I. hawkeri through the anatomical, morphological and physiological changes observed. Two experiments were performed. The first experiment included the response to only BAP-sprayed plants (0, 5, 50, or 100 mg L-1). For the second experiment, rooting cuttings of I. hawkeri were sprayed with different concentrations of IAA (0, 5, 50, or 100 mg L-1) followed by different BAP concentrations (0, 5, 50, or 100 mg L-1) one week later to run-off at sunset. Results showed that (a) a single BAP or AIA dose increased increase post-transplant biomass accumulation through a higher leaf area expansion and photo assimilate production, (b) as a result of both AIA and BAP spray, the higher NAR the higher post-transplant biomass accumulation, (c) leaf anatomical changes (leaf thickness, intercellular spaces) let a higher carbon dioxide diffusion and fixation with a correlative increase in photo assimilates, (d) a higher root system would be related to a higher cytokinin synthesis. In summary, similarities between responses to either hormone, together with the lack of any IAA - BAP interaction, provide two independent routes for commercial growers to increase the productivity of I. hawkeri ornamental plants by using early foliar sprays.

Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes.

Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Molecular role of cytokinin in bud activation and outgrowth in apple branching based on transcriptomic analysis

Axillary bud activation and outgrowth were dependent on local cytokinin, and that bud activation preceded the activation of cell cycle and cell growth genes in apple branching. Cytokinin is often applied to apple trees to produce more shoot branches in apple seedlings. The molecular response of apple to the application of cytokinin, and the relationship between bud activation and cell cycle in apple branching, however, are poorly understood. In this study, RNA sequencing was used to characterize differential expression genes in axillary buds of 1-year grafted "Fuji" apple at 4 and 96h after cytokinin application. And comparative gene expression analyses were performed in buds of decapitated shoots and buds of the treatment of biosynthetic inhibitor of cytokinin (Lovastatin) on decapitated shoots. Results indicated that decapitation and cytokinin increased ZR content in buds and internodes at 4-8h, and induced bud elongation at 96h after treatment, relative to buds in shoots receiving the Lovastatin treatment. RNA-seq analysis indicated that differential expression genes in auxin and cytokinin signal transduction were significantly enriched at 4h, and DNA replication was enriched at 96h. Cytokinin-responsive type-A response regulator, auxin polar transport, and axillary meristem-related genes were up-regulated at 4h in the cytokinin and decapitation treatments, while qRT-PCR analysis showed that cell cycle and cell growth genes were up-regulated after 8h. Collectively, the data indicated that bud activation and outgrowth might be dependent on local cytokinin synthesis in axillary buds or stems, and that bud activation preceded the activation of cell cycle genes during the outgrowth of ABs in apple shoots.

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

Tangled history of a multigene family: The evolution of ISOPENTENYLTRANSFERASE genes.

ISOPENTENYLTRANSFERASE (IPT) genes play important roles in the initial steps of cytokinin synthesis, exist in plant and pathogenic bacteria, and form a multigene family in plants. Protein domain searches revealed that bacteria and plant IPT proteins were to assigned to different protein domains families in the Pfam database, namely Pfam IPT (IPTPfam) and Pfam IPPT (IPPTPfam) families, both are closely related in the P-loop NTPase clan. To understand the origin and evolution of the genes, a species matrix was assembled across the tree of life and intensively in plant lineages. The IPTPfam domain was only found in few bacteria lineages, whereas IPPTPfam is common except in Archaea and Mycoplasma bacteria. The bacterial IPPTPfam domain miaA genes were shown as ancestral of eukaryotic IPPTPfam domain genes. Plant IPTs diversified into class I, class II tRNA-IPTs, and Adenosine-phosphate IPTs; the class I tRNA-IPTs appeared to represent direct successors of miaA genes were found in all plant genomes, whereas class II tRNA-IPTs originated from eukaryotic genes, and were found in prasinophyte algae and in euphyllophytes. Adenosine-phosphate IPTs were only found in angiosperms. Gene duplications resulted in gene redundancies with ubiquitous expression or diversification in expression. In conclusion, it is shown that IPT genes have a complex history prior to the protein family split, and might have experienced losses or HGTs, and gene duplications that are to be likely correlated with the rise in morphological complexity involved in fine tuning cytokinin production.

Interactions between cytokinin and nitrogen contribute to grain mass in wheat cultivars by regulating the flag leaf senescence process

Premature senescence after anthesis reduces crop yields. Delaying leaf senescence could maintain photosynthetic activity for a longer period and lead to a higher photosynthetic rate. Recent studies have provided some insights into the interaction between cytokinin and nitrogen (N) in the regulation of plant development. In the present study, foliar application of exogenous 6-benzylaminopurine (6-BA) and lovastatin, an inhibitor of cytokinin synthesis, was combined with three N rates [0 kg ha−1 (low nitrogen, LN), 240 kg ha−1 (normal nitrogen, NN), and 360 kg ha−1 (high nitrogen, HN)] in two wheat cultivars, Wennong 6 (with a staygreen phenotype) and Jimai 20 (with a non-staygreen phenotype). Flag leaf senescence was assessed using a Gompertz growth curve. Grain mass, dry matter accumulation and distribution, total N of flag leaf, and concentrations of zeatin riboside (ZR) and abscisic acid (ABA) were also used to evaluate the functional characteristics of flag leaves. Grain mass was negatively correlated with initial senescence rate (r0) and duration of rapid chlorophyll loss (Chlloss), whereas it was positively correlated with maximum senescence rate (rmax), average senescence rate (raver), persistence phase (Chlper), total duration of flag leaf (Chltotal) and inflection point cumulative temperature (M). Compared to Jimai 20, Wennong 6 had larger raver, Chlper, and Chltotal. The concentration of ZR was highest under the 6-BA × NN treatment, followed by the 6-BA × HN and 6-BA × LN treatments. However, the concentration of ABA showed the opposite trend. It was concluded that the staygreen phenotype Wennong 6 was associated with greater grain mass and altered cytokinin metabolism and could be classified as a functional staygreen type. Foliar application of 6-BA interacting with N at the NN level (240 kg ha−1) may be a beneficial strategy for improving grain yield of wheat by regulating endogenous hormones and the flag leaf senescence process. Increasing endogenous cytokinin promoted the transport of dry matter to grain.

WUSCHEL-RELATED HOMEOBOX4 acts as a key regulator in early leaf development in rice.

Rice (Oryza sativa) has long and narrow leaves with parallel veins, similar to other grasses. Relative to Arabidopsis thaliana which has oval-shaped leaves, our understanding of the mechanism of leaf development is insufficient in grasses. In this study, we show that OsWOX4, a member of the WUSCHEL-RELATED HOMEOBOX gene family, plays important roles in early leaf development in rice. Inducible downregulation of OsWOX4 resulted in severe defects in leaf development, such as an arrest of vascular differentiation, a partial defect in the early cell proliferation required for midrib formation, and a failure to maintain cellular activity in general parenchyma cells. In situ analysis showed that knockdown of OsWOX4 reduced the expression of two LONELY GUY genes, which function in the synthesis of active cytokinin, in developing vascular bundles. Consistent with this, cytokinin levels were downregulated by OsWOX4 knockdown. Transcriptome analysis further showed that OsWOX4 regulates multiple genes, including those responsible for cell cycle progression and hormone action, consistent with the effects of OsWOX4 downregulation on leaf phenotypes. Collectively, these results suggest that OsWOX4 acts as a key regulator at an early stage of leaf development. Our previous work revealed that OsWOX4 is involved in the maintenance of shoot apical meristem in rice, whereas AtWOX4 is specifically associated with the maintenance of vascular stem cells in Arabidopsis. Thus, the function of the two orthologous genes seems to be diversified between rice and Arabidopsis.

Proteasomal regulation of hormone signaling in Mycobacterium tuberculosis

Tuberculosis (TB) kills about 1.5 million people globally every year, making it the leading causes of death by an infectious agent. The lack of an effective TB vaccine or the development of immunity after TB infection suggests antimicrobial drugs may be our best chance for controlling this disease. The Mycobacterium tuberculosis (Mtb) proteasome is essential to cause lethal infections in animals and, therefore, it is currently being targeted for tuberculosis chemotherapy.My lab is working to understand how a bacterial proteasome allows Mtb to persist in animals by characterizing substrates of the Mtb proteasome. Dozens if not hundreds of proteins that are degraded by the bacterial proteasome must first be post‐translationally modified with the small protein Pup (prokaryotic ubiquitin‐like protein). We reported the identification of a pupylated protein called Lonely Guy (Log) that catalyzes the synthesis of cytokinins, which have previously only been characterized as plant hormones. Log levels are maintained at very low levels in Mtb by the Pup‐proteasome system, suggesting the presence of cytokinins in Mtb requires tight regulation. In my talk, I will discuss exciting new data that addresses the function of Log and cytokinins in Mtb physiology.Support or Funding InformationThis work was supported by NIH grant R01HL092774 awarded to K.H.D. K.H.D. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Trainees were supported by NIH grant T32 AT007180.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Auxin-cytokinin interactions in the regulation of correlative inhibition in two-branched pea seedlings.

A model system of 10-12 day-old, two-branched (2-B) pea (Pisum sativum L. cv. Adagumsky) seedlings was used to study the roles of endogenous auxin indole-3-acetic acid (IAA) and cytokinins (CKs) in the interaction between the shoots. The IAA export activity (IEA) from shoots was 2-fold higher in one-branched (1-B) plants with one shoot removed than in the 2-B plants, while tZ-type cytokinin contents in xylem sap were 4-fold greater in the 1-B plants than in 2-B plants. Exogenous 6-benzylaminopurine introduced into the vascular stream of one shoot enhanced its IEA. Therefore, xylem cytokinin appears to control both growth and IEA in branches. In the hypocotyls of 1-B and 2-B plants, IAA contents were equal in both cases, while the levels of tZ-type cytokinins were different. These data do not agree with the well-supported role of auxin in down-regulating CK content. The observed paradox may be explained by assuming that a steady-state IAA level in the hypocotyls is feedback regulated via xylem cytokinin, which controls the delivery of IAA from the shoots. As a result, the level of IAA in the hypocotyl is most likely maintained at a threshold below which a decrease in auxin content can switch on CK synthesis that will increase xylem cytokinin levels, thereby stabilizing the level of IAA in the hypocotyl. Therefore, our results suggest that correlative inhibition in the 2-B pea system is a function of an IAA/CK feedback loop, in which cytokinin essentially acts as a second messenger for IAA.

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

Identification and expression analysis of the IPT and CKX gene families during axillary bud outgrowth in apple (Malus domestica Borkh.)

Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT, MdCKX, and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX, and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10, however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth.

The Agrobacterium Phenotypic Plasticity (Plast) Genes.

The transfer of T-DNA sequences from Agrobacterium to plant cells is a well-understood process of natural genetic engineering. The expression of T-DNA genes in plants leads to tumors, hairy roots, or transgenic plants. The transformed cells multiply and synthesize small molecules, called opines, used by Agrobacteria for their growth. Several T-DNA genes stimulate or influence plant growth. Among these, iaaH and iaaM encode proteins involved in auxin synthesis, whereas ipt encodes a protein involved in cytokinin synthesis. Growth can also be induced or modified by other T-DNA genes, collectively called plast genes (for phenotypic plasticity). The plast genes are defined by their common ancestry and are mostly found on T-DNAs. They can influence plant growth in different ways, but the molecular basis of their morphogenetic activity remains largely unclear. Only some plast genes, such as 6b, rolB, rolC, and orf13, have been studied in detail. Plast genes have a significant potential for applied research and may be used to modify the growth of crop plants. In this review, I summarize the most important findings and models from 30years of plast gene research and propose some outlooks for the future.

Chemoenzymatic synthesis of cytokinins from nucleosides: ribose as a blocking group

Nucleoside phosphorylases are involved in the salvage pathways of nucleoside biosynthesis and catalyze the reversible reaction of a nucleobase with α-d-ribose-1-phosphate to yield a corresponding nucleoside and an inorganic phosphate. The equilibrium of these reactions is shifted towards nucleosides, especially in the case of purines. Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is widely used in labs and industry for the synthesis of nucleosides of practical importance. Bacterial PNPs have relatively broad substrate specificity utilizing a wide range of purines with different substituents to form the corresponding nucleosides. To shift the reaction in the opposite direction we have used arsenolysis instead of phosphorolysis. This reaction is irreversible due to the hydrolysis of the resulting α-d-ribose-1-arsenate. As a result, heterocyclic bases are formed in quantitative yields and can be easily isolated. We have developed a novel method for the preparation of cytokinins based on the enzymatic cleavage of the N-glycosidic bond of N6-substituted adenosines in the presence of PNP and Na2HAsO4. According to the HPLC analysis the conversion proceeds in quantitative yields. In the proposed strategy the ribose residue acts as a protective group. No contamination of the final products with AsO43- has been detected via HPLC-HRMS; simple analytical arsenate detection via ESI-MS has been proposed.

Alternate bearing in fruit tree crops: past, present and future

Alternate bearing, i.e., year-to-year alternation between large and small yields of fruit tree crops worldwide, is often caused by abiotic stress such as late frost at flowering. Economic losses for apple due to alternate bearing were calculated at between ½ and 5 mil US$ year-1 depending on growing region. We recorded the number of flower clusters over three years (autumn 2009 to May 2011) and yield of 2,086 trees of alternate-susceptible 'Elstar' apple on M.9 trees with and without hail nets (for light deprivation) resulting in a data set of 16,000 points. We calculated 'biennial bearing index' (BBI), the ratio of differences in tree yields to cumulative tree yield, and graphed to data to show the tree-to-tree alternation. A Pearson correlation between yield in year x and year x+1 and flower clusters in year x, gave an intersect of 91-106 flower clusters, whereas the mean yield in year x and year x+1, resulted in a range of 72-133 flower clusters, or 9.6 kg tree-1, as a thinning target for sustainable cultivation of 'Elstar' in a pedestrian orchard at 50°N or larger yields with taller trees at a lower latitude. The interaction between the crop levels on the spur and on the whole tree showed that if the tree crop level was low, the tree provided adequate resources for flower bud development in spurs carrying 1 or 2 fruit. Conversely, to obtain good return bloom on normal-to-heavily cropping trees required having good numbers of non-fruiting (also called “resting”) spurs. A scheme for the underlying regulatory mechanisms was developed, which includes light deprivation and subsequent lack of flower initiation, polar basipetal GA7 transport, cytokinin synthesis in the root and level in the xylem and phloem. Countermeasures include early chemical or mechanical thinning of young trees before reaching full crop load and early in terms of the flowering stage.

Wounding Triggers Callus Formation via Dynamic Hormonal and Transcriptional Changes.

Wounding is a primary trigger of organ regeneration, but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study, we combined transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis (Arabidopsis thaliana). Our time course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes, starting from rapid stress responses followed by the activation of metabolic processes and protein synthesis and subsequent activation of cell cycle regulators. Gene ontology analyses further uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signaling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin is critical for wound-induced callus formation. We further demonstrate that type-B ARABIDOPSIS RESPONSE REGULATOR-mediated cytokinin signaling regulates the expression of CYCLIN D3;1 (CYCD3;1) and that mutations in CYCD3;1 and its homologs CYCD3;2 and 3 cause defects in callus formation. In addition to these hormone-mediated changes, our transcriptome data uncovered that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 and PLETHORA3 (PLT3), PLT5, and PLT7 in callus generation. All together, these results provide novel mechanistic insights into how wounding reactivates cell proliferation during callus formation.