Cytokinin Synthesis Research Articles

Identification of GhLOG gene family revealed that GhLOG3 is involved in regulating salinity tolerance in cotton (Gossypium hirsutum L.)

Cytokinin (CK) is an important plant hormone that promotes plant cell division and differentiation, and participates in salt response under osmotic stress. LOGs (LONELY GUY) are CK-activating enzymes involved in CK synthesis. The LOG gene family has not been comprehensively characterized in cotton. In this study we identified 151 LOG genes from nine plant species, including 28 LOG genes in Gossypium hirsutum. Phylogenetic analysis divided LOG genes into three groups. Exon/intron structures and protein motifs of GhLOG genes were highly conserved. Synteny analysis revealed that several gene loci were highly conserved between the A and D sub-genomes of G. hirsutum with purifying selection pressure during evolution. Expression profiles showed that most LOG genes were constitutively expressed in eight different tissues. Furthermore, LOG genes can be regulated by abiotic stresses and phytohormone treatments. Moreover, subcellular localization revealed that GhLOG3_At resides inside the cell membrane. Overexpression of GhLOG3 enhanced salt tolerance in Arabidopsis. Virus-induced gene silencing (VIGS) of GhLOG3_At in cotton enhanced sensitivity of plants to salt stress with increased H2O2 contents and decreased chlorophyll and proline (PRO) activity. Our results suggested that GhLOG3_At induces salt stress tolerance in cotton, and provides a basis for the use of CK synthesis genes to regulate cotton growth and stress resistance.

Moniliophthoraperniciosa, the causal agent of witches’ broom disease of cacao, interferes with cytokinin metabolism during infection of Micro‐Tom tomato and promotes symptom development

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

The Cytokinin Status of the Epidermis Regulates Aspects of Vegetative and Reproductive Development in Arabidopsis thaliana.

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.

Clade III cytokinin response factors share common roles in response to oxidative stress responses linked to cytokinin synthesis.

Cytokinin response factors (CRFs) are transcription factors that are involved in cytokinin (CK) response, as well as being linked to abiotic stress tolerance. In particular, oxidative stress responses are activated by Clade III CRF members, such as AtCRF6. Here we explored the relationships between Clade III CRFs and oxidative stress. Transcriptomic responses to oxidative stress were determined in two Clade III transcription factors, Arabidopsis AtCRF5 and tomato SlCRF5. AtCRF5 was required for regulated expression of >240 genes that are involved in oxidative stress response. Similarly, SlCRF5 was involved in the regulated expression of nearly 420 oxidative stress response genes. Similarities in gene regulation by these Clade III members in response to oxidative stress were observed between Arabidopsis and tomato, as indicated by Gene Ontology term enrichment. CK levels were also changed in response to oxidative stress in both species. These changes were regulated by Clade III CRFs. Taken together, these findings suggest that Clade III CRFs play a role in oxidative stress response as well as having roles in CK signaling.

Exogenous DA-6 Improves the Low Night Temperature Tolerance of Tomato Through Regulating Cytokinin.

Low night temperature (LNT) causes environmental stress and has a severe and negative impact on plant growth and productivity. Synthetic elicitors can regulate plant growth and induce defense mechanisms from this type of stress. Here, we evaluated the effect of the exogenous growth regulator diethyl aminoethyl hexanoate (DA-6) in tomato leaf response to LNT stress. Our results showed that exogenous DA-6 activates the expression of chlorophyll synthesis and photosystem-related genes, and results in higher photosynthetic activity and chlorophyll production. Furthermore, DA-6 can regulate the synthesis of endogenous cytokinin (CTK) and the expression of decomposition genes to stabilize chloroplast structure, reduce oxidative damage, and maintain the photochemical activity of tomato leaves under LNT stress. DA-6 maintains a high level of ABA content and induces the expression of CBF genes, indicating that DA-6 may participate in the cold response signaling pathway and induce the expression of downstream low temperature response genes and accumulation of compatible osmolytes. This study unravels a mode of action by which plant growth regulators can improve low temperature tolerance and provides important considerations for their application to alleviate the harmful effects of cold stress.

Seed Yield and Nitrogen Efficiency in Oilseed Rape After Ammonium Nitrate or Urea Fertilization.

In agricultural plant production, nitrate, ammonium, and urea are the major fertilized nitrogen forms, which differ in root uptake and downstream signaling processes in plants. Nitrate is known to stimulate cytokinin synthesis in roots, while for urea no hormonal effect has been described yet. Elevated cytokinin levels can delay plant senescence favoring prolonged nitrogen uptake. As the cultivation of winter oilseed rape provokes high nitrogen-balance surpluses, we tested the hypotheses whether nitrogen use efficiency increases under ammonium nitrate- relative to urea-based nutrition and whether this is subject to genotypic variation. In a 2-year field study, 15 oilseed rape lines were fertilized either with ammonium nitrate or with urease inhibitor-stabilized urea and analyzed for seed yield and nitrogen-related yield parameters. Despite a significant environmental impact on the performance of the individual lines, which did not allow revealing consistent impact of the genotype, ammonium nitrate-based nutrition tended to increase seed yield in average over all lines. To resolve whether the fertilizer N forms act on grain yield via phytohormones, we collected xylem exudates at three developmental stages and determined the translocation rates of cytokinins and N forms. Relative to urea, ammonium nitrate-based nutrition enhanced the translocation of nitrate or total nitrogen together with cytokinins, whereas in the urea treatment translocation rates were lower as long as urea remained stable in the soil solution. At later developmental stages, i.e., when urea became hydrolyzed, nitrogen and cytokinin translocation increased. In consequence, urea tended to increase nitrogen partitioning in the shoot toward generative organs. However, differences in overall nitrogen accumulation in shoots were not present at the end of the vegetation period, and neither nitrogen uptake nor utilization efficiency was consistently different between the two applied nitrogen forms.

РІВЕНЬ ГЛЮКОЗИ ТА ГАММА-ГЛУТАМІЛТРАНСПЕПТИДАЗНА АКТИВНІСТЬ У КЛІТИНАХ ГЕПАТОЦИТАРНОГО ПОХОДЖЕННЯ ЗА ДІЇ ЕКСТРАКТІВ ТА ЦИТОКІНІНОВИХ ФРАКЦІЙ ЛІКАРСЬКИХ ГРИБІВ

Mushroom extracts show the multifunctional activity and have a wide range of applications for the treatment of various diseases, including cancer. However, the full composition of the compounds that produce macromycetes that exhibit antitumor properties has not yet been established. Impaired glucose metabolism and activation of gamma-glutamyltranspeptidase (GGT) in tumor cells may be a key marker of biochemical anaplasia in neoplasms. The aim of the study was to investigate the effect of crude extracts and cytokinin fractions isolated from the mycelial biomass of medicinal mushrooms on the biological properties of cells of hepatocyte-like cells of the HepG2 line (human hepatocellular carcinoma). The objects of the research were pure mushroom cultures of Hericium coralloides, Fomitopsis officinalis, Trametes (Coriolus) versicolor, Pleurotus ostreatus and Morchella esculenta. Cytokinin fractions from the extracts were isolated by centrifugation followed by fractionation and purification using ion exchange chromatography. Qualitative and quantitative analysis of cytokinins was performed by high-performance liquid chromatography. GGT activity was determined using the kit "Filisit" (Ukraine), glucose level – glucose oxidase method, with modifications for the cellular culture medium. The analysis of mycelial biomass of medicinal macromycetes revealed the presence of transzeatin, zeatin riboside, zeatin-O-glucoside and isopentenyladenine, that showed high activity in relation to cytokinin synthesis. Inhibition of glucose diffusion from the cultivation medium with the use of crude extracts and cytokinin fractions of medicinal mushroom and a decrease in GGT activity, more pronounced with the action of cytokinin fractions, compared with crude extracts, was noted. The difference between the effects of crude extracts and cytokinin fractions indicated the complex nature of the action of biologically active substances of medicinal mushroom. Presented results regarding the effects of crude extracts and cytokinin fractions of medicinal mushroom showed a normalizing effect on the main metabolic parameters which change in tumor cells, as a mechanism of biochemical anaplasia.

Global View on the Cytokinin Regulatory System in Potato.

Cytokinins (CKs) were earlier shown to promote potato tuberization. Our study aimed to identify and characterize CK-related genes which constitute CK regulatory system in the core potato (Solanum tuberosum) genome. For that, CK-related genes were retrieved from the sequenced genome of the S. tuberosum doubled monoploid (DM) Phureja group, classified and compared with Arabidopsis orthologs. Analysis of selected gene expression was performed with a transcriptome database for the S. tuberosum heterozygous diploid line RH89-039-16. Genes responsible for CK signaling, biosynthesis, transport, and metabolism were categorized in an organ-specific fashion. According to this database, CK receptors StHK2/3 predominate in leaves and flowers, StHK4 in roots. Among phosphotransmitters, StHP1a expression largely predominates. Surprisingly, two pseudo-phosphotransmitters intended to suppress CK effects are hardly expressed in studied organs. Among B-type RR genes, StRR1b, StRR11, and StRR18a are actively expressed, with StRR1b expressing most uniformly in all organs and StRR11 exhibiting the highest expression in roots. By cluster analysis four types of prevailing CK-signaling chains were identified in (1) leaves and flowers, StHK2/3→StHP1a→StRR1b/+; (2) shoot apical meristems, stolons, and mature tubers, StHK2/4→StHP1a→StRR1b/+; (3) stems and young tubers, StHK2/4→StHP1a→StRR1b/11/18a; and (4) roots and tuber sprouts, StHK4→StHP1a→StRR11/18a. CK synthesis genes StIPT3/5 and StCYP735A are expressed mainly in roots followed by tuber sprouts, but rather weakly in stolons and tubers. By contrast, CK-activation genes StLOGs are active in stolons, and StLOG3b expression is even stolon-confined. Apparently, the main CK effects on tuber initiation are realized via activity of StLOG1/3a/3b/7c/8a genes in stolons. Current advances and future directions in potato research are discussed.

Cytokinin-Regulated Expression of Arabidopsis thaliana PAP Genes and Its Implication for the Expression of Chloroplast-Encoded Genes.

Cytokinins (CKs) are known to regulate the biogenesis of chloroplasts under changing environmental conditions and at different stages of plant ontogenesis. However, the underlying mechanisms are still poorly understood. Apparently, the mechanisms can be duplicated in several ways, including the influence of nuclear genes that determine the expression of plastome through the two-component CK regulatory circuit. In this study, we evaluated the role of cytokinins and CK signaling pathway on the expression of nuclear genes for plastid RNA polymerase-associated proteins (PAPs). Cytokinin induced the expression of all twelve Arabidopsis thaliana PAP genes irrespective of their functions via canonical CK signaling pathway but this regulation might be indirect taking into consideration their different functions and versatile structure of promoter regions. The disruption of PAP genes contributed to the abolishment of positive CK effect on the accumulation of the chloroplast gene transcripts and transcripts of the nuclear genes for plastid transcription machinery as can be judged from the analysis of pap1 and pap6 mutants. However, the CK regulatory circuit in the mutants remained practically unperturbed. Knock-out of PAP genes resulted in cytokinin overproduction as a consequence of the strong up-regulation of the genes for CK synthesis.

Differential expression of genes in olive leaves and buds of ON- versus OFF-crop trees

Alternate bearing (AB) refers to the tendency of trees to have an irregular crop load from 1 year (ON) to the next year (OFF). Despite its economic importance, it is not fully understood how gene networks and their related metabolic pathways may influence the irregular bearing in olive trees. To unravel molecular mechanisms of this phenomenon in olive (cv. Conservalia), the whole transcriptome of leaves and buds from ON and OFF-trees was sequenced using Illumina next generation sequencing approach. The results indicated that expressed transcripts were involved in metabolism of carbohydrates, polyamins, phytohormones and polyphenol oxidase (POD) related to antioxidant system. Expression of POD was increased in leaf samples of ON- versus OFF-trees. The expression pattern of the greater number of genes was changed more in buds than in leaves. Up-regulation of gene homologues to the majority of enzymes that were involved in photorespiration metabolism pathway in buds of ON-trees was remarkable that may support the hypotheses of an increase in photorespiratory metabolism in these samples. The results indicated changes in expression pattern of homologous to those taking part of abscisic acid and cytokinin synthesis which are connected to photorespiration. Our data did not confirm expression of homologue (s) to those of chlorogenic acid metabolism, which has been addressed earlier that have a probable role in biennial bearing in olive. Current findings provide new candidate genes for further functional analysis, gene cloning and exploring of molecular basses of AB in olive.

Overexpression of Maize ZmMYB59 Gene Plays a Negative Regulatory Role in Seed Germination in Nicotiana tabacum and Oryza sativa.

MYB transcription factors are involved in many biological processes, including metabolism, stress response and plant development. In our previous work, ZmMYB59 was down-regulated by deep sowing during maize seed germination. However, there are few reports on seed germination regulated by MYB proteins. In this study, to examine its functions during seed germination, Agrobacterium-mediated transformation was exploited to generate ZmMYB59 overexpression (OE) tobacco and rice. In T2 generation transgenic tobacco, germination rate, germination index, vigor index and hypocotyl length were significantly decreased by 25.0–50.9, 34.5–54.4, 57.5–88.3, and 21.9–31.3% compared to wild-type (WT) lines. In T2 generation transgenic rice, above corresponding parameters were notably reduced by 39.1–53.8, 51.4–71.4, 52.5–74.0, and 28.3–41.5%, respectively. On this basis, antioxidant capacity and endogenous hormones were determined. The activities of catalase, peroxidase, superoxide dismutase, ascorbate peroxidase of OE lines were significantly lower than those of WT, suggesting that ZmMYB59 reduced their oxidation resistance. As well, ZmMYB59 overexpression extremely inhibited the synthesis of gibberellin A1 (GA1) and cytokinin (CTK), and promoted the synthesis of abscisic acid (ABA) concurrently. Taken together, it proposed that ZmMYB59 was a negative regulator during seed germination in tobacco and rice, which also contributes to illuminate the molecular mechanisms regulated by MYB transcription factors.

Root-derived trans-zeatin cytokinin protects Arabidopsis plants against photoperiod stress.

Recently, a novel type of abiotic stress caused by a prolongation of the light period-coined photoperiod stress-has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signalling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signalling system to protect from the negative consequences of strong photoperiod stress.

MicroRNA and regulation of auxin and cytokinin signalling during post-mowing regeneration of winter wheat (Triticum aestivum L.)

Winter wheat not only provides adequate fresh forage grass in winter, but also ensures ample grain production in summer. The mechanisms underlying the regeneration of winter wheat after mowing or grazing are not well understood. In this study, the miRNA expression profile of winter wheat was determined using RNA sequencing and the endogenous auxin and cis-zeatin concentrations, as well as the expression of related miRNA-targeted genes, were measured. During the post-mowing regeneration of winter wheat, the concentrations of endogenous indole-3-acetic acid (IAA), methyl indole-3-acetate (ME-IAA), and indole-3-carboxaldehyde (ICA) decreased, while those of cis-zeatin (cZ) increased. Moreover, 15 novel miRNAs and three known miRNAs were found to be involved in the synthesis and signalling transduction of auxins and cytokinins (CKs). Among these miRNAs, miR1153-y, miR5059-x, miR2916-x, novel-miR1532–3p, novel-miR1060–3p, and novel-miR0890–3p, were found to be negatively correlated with the expression of their target genes including auxin response GH3.7, auxin response factor (ARF), type-A two-component response regulator (A-ARR), aldehyde dehydrogenase (ALDH), and O-glucosyltransferase (CISZOG). Furthermore, miR1153-y was identified as mediating the cleavage of GH3.7 by RACE assay. In turn, these genes inhibited the biosynthesis and signalling of IAA and activated CK signal transduction, resulting in the rapid regeneration of mowed winter wheat. This study revealed that some miRNAs exert a positive regulatory effect on the post-mowing regeneration of winter wheat by controlling the synthesis and signal transduction of IAA and CK, and our founding will aid developments in biotechnology aimed at improving the post-mowing regeneration ability of winter wheat.

CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola

We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.

From Survival to Productivity Mode: Cytokinins Allow Avoiding the Avoidance Strategy Under Stress Conditions.

Growth retardation and stress-induced premature plant senescence are accompanied by a severe yield reduction and raise a major agro-economic concern. To improve biomass and yield in agricultural crops under mild stress conditions, the survival must be changed to productivity mode. Our previous successful attempts to delay premature senescence and growth inhibition under abiotic stress conditions by autoregulation of cytokinins (CKs) levels constitute a generic technology toward the development of highly productive plants. Since this technology is based on the induction of CKs synthesis during the age-dependent senescence phase by a senescence-specific promoter (SARK), which is not necessarily regulated by abiotic stress conditions, we developed autoregulating transgenic plants expressing the IPT gene specifically under abiotic stress conditions. The Arabidopsis promoter of the stress-induced metallothionein gene (AtMT) was isolated, fused to the IPT gene and transformed into tobacco plants. The MT:IPT transgenic tobacco plants displayed comparable elevated biomass productivity and maintained growth under drought conditions. To decipher the role and the molecular mechanisms of CKs in reverting the survival transcriptional program to a sustainable plant growth program, we performed gene expression analysis of candidate stress-related genes and found unexpectedly clear downregulation in the CK-overproducing plants. We also investigated kinase activity after applying exogenous CKs to tobacco cell suspensions that were grown in salinity stress. In-gel kinase activity analysis demonstrated CK-dependent deactivation of several stress-related kinases including two of the MAPK components, SIPK and WIPK and the NtOSAK, a member of SnRK2 kinase family, a key component of the ABA signaling cascade. A comprehensive phosphoproteomics analysis of tobacco cells, treated with exogenous CKs under salinity-stress conditions indicated that >50% of the identified phosphoproteins involved in stress responses were dephosphorylated by CKs. We hypothesize that upregulation of CK levels under stress conditions desensitize stress signaling cues through deactivation of kinases that are normally activated under stress conditions. CK-dependent desensitization of environmental stimuli is suggested to attenuate various pathways of the avoidance syndrome including the characteristic growth arrest and the premature senescence while allowing normal growth and metabolic maintenance.

Exogenous spermidine regulates starch synthesis and the antioxidant system to promote wheat grain filling under drought stress

Polyamines (PAs) markedly affect the cereal’s grain filling characteristics under drought stress. But little is known that the regulatory mechanism that governs this process. So, in the present study, drought was imposed, and exogenous spermidine (Spd) was applied at anthesis. The grain filling characteristics of and gene expression in the grains were measured. The research purpose of this study was to investigate whether and how the PAs regulated the grain filling in wheat under drought stress. In the present study, external Spd markedly relieved the inhibitory action of drought stress on grain filling. Moreover, RNA-seq analysis revealed that the effects of drought and exogenous Spd on grain filling in wheat were strongly related to protein processing in the endoplasmic reticulum, phenylalanine metabolism, hormone signaling, and starch and sucrose metabolism. In addition, under drought stress, the exogenous Spd significantly promoted the synthesis of cytokinin (CTK) and starch in wheat grains of plants. In contrast, exogenous Spd significantly decreased ethylene (ETH) synthesis in the grains of plants under drought stress. Beside this, exogenous Spd application notably promoted the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and decreased the malondialdehyde (MDA) content in the grains of plants under drought stress. In summary, the promoting action of Spd on the grain filling characteristics in wheat under drought stress was notably related to an increase in endogenous zeatin (Z) + zeatin riboside (ZR) contents, the synthesis of starch from sucrose, the activities of antioxidant enzymes and a decrease in ETH synthesis in the grains.

Mechanism of exogenous cytokinins inducing bulbil formation in Lilium lancifolium in vitro.

The cytokinin pathway promotes the initiation of bulbil formation, and iPA may an important type of cytokinin during bulbil formation in Lilium lancifolium. Bulbils are important vegetative reproductive organs in triploid Lilium lancifolium. We previously showed that cytokinins are involved in bulbil formation, but how cytokinins participate in bulbil formation is not clear. In this study, bulbil formation was divided into three stages on the basis of anatomical and histological observations: the bulbil initiation stage, bulbil primordium-formation stage and bulbil structure-formation stage. The results indicated that iPA was the most critical cytokinin during the bulbil initiation. qRT-PCR revealed that increased iPA content during bulbil initiation was mainly due to increased expression of cytokinin synthesis genes (IPT1/5) and cytokinin activation genes (LOG1/3/5/7) and significantly decreased expression of the cytokinin degradation gene CKX4. Exogenous 6-BA and lovastatin affected the cytokinin pathway and promoted or inhibited bulbil initiation by increasing or decreasing the content of endogenous iPA, respectively. In summary, we demonstrate that cytokinins positively regulate bulbil formation and provide preliminary insight into the regulatory mechanisms by which the cytokinin pathway promotes bulbil initiation.

Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins.

The biosynthesis of aromatic cytokinins in planta, unlike isoprenoid cytokinins, is still unknown. To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike active cytokinins-bases, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2′-deoxyribo- and 5′-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2′-deoxyribo- and 5′-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5′-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be “activated” by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid (true hormonal activity) and aromatic (almost no activity) cytokinins indicate a significant difference in the biosynthesis of these compounds.

Morphological changes, alteration of photosynthetic parameters and chlorophyll production induced by infection with alfalfa dwarf virus in Medicago sativa plants

In Argentina, infections by alfalfa dwarf virus (ADV) affect the cultivation of alfalfa, which is globally one of the most important forage plants. The main objective of this study was to improve current understanding of the underlying mechanisms related to the dwarfism phenotype developed during viral infection. Hydrogen peroxide production, callose deposition and PR5 gene expression levels were evaluated to determine if ADV induces plant defence responses. At the morphological level, higher epicuticular wax production and an increase in proliferation of cells from the fundamental parenchyma were observed when plants were infected by ADV. Infected plants had reduced photosynthesis, stomatal conductance and transpiration/evaporation rate, but interestingly the production of chlorophylls was induced. Finally, using transcriptional analysis, ADV was observed to negatively affect the expression of genes related to synthesis of auxins, cytokinins and brassinosteroids. These results suggest that ADV infection induces a hormonal imbalance leading to an increase in chlorophyll pigment synthesis, stomatal closure and generation of tissue deformation. Overall, the results show a morphological, physiological and photosynthetic characterization of dwarf plants affected by ADV.

Light Regulation of Axillary Bud Outgrowth Along Plant Axes: An Overview of the Roles of Sugars and Hormones.

Apical dominance, the process by which the growing apical zone of the shoot inhibits bud outgrowth, involves an intricate network of several signals in the shoot. Auxin originating from plant apical region inhibits bud outgrowth indirectly. This inhibition is in particular mediated by cytokinins and strigolactones, which move from the stem to the bud and that respectively stimulate and repress bud outgrowth. The action of this hormonal network is itself modulated by sugar levels as competition for sugars, caused by the growing apical sugar sink, may deprive buds from sugars and prevents bud outgrowth partly by their signaling role. In this review, we analyze recent findings on the interaction between light, in terms of quantity and quality, and apical dominance regulation. Depending on growth conditions, light may trigger different pathways of the apical dominance regulatory network. Studies pinpoint to the key role of shoot-located cytokinin synthesis for light intensity and abscisic acid synthesis in the bud for R:FR in the regulation of bud outgrowth by light. Our analysis provides three major research lines to get a more comprehensive understanding of light effects on bud outgrowth. This would undoubtedly benefit from the use of computer modeling associated with experimental observations to deal with a regulatory system that involves several interacting signals, feedbacks, and quantitative effects.