Amounts Of Cytokinins Research Articles

The Role of Cytokinins and Abscisic Acid in the Growth, Development and Virulence of the Pathogenic Fungus Stagonospora nodorum (Berk.).

Cytokinins (CKs) and abscisic acid (ABA) play an important role in the life of both plants and pathogenic fungi. However, the role of CKs and ABA in the regulation of fungal growth, development and virulence has not been sufficiently studied. We compared the ability of two virulent isolates (SnB and Sn9MN-3A) and one avirulent isolate (Sn4VD) of the pathogenic fungus Stagonospora nodorum Berk. to synthesize three groups of hormones (CKs, ABA and auxins) and studied the effect of exogenous ABA and zeatin on the growth, sporulation and gene expression of necrotrophic effectors (NEs) and transcription factors (TFs) in them. Various isolates of S. nodorum synthesized different amounts of CKs, ABA and indoleacetic acid. Using exogenous ABA and zeatin, we proved that the effect of these hormones on the growth and sporulation of S. nodorum isolates can be opposite, depends on both the genotype of the isolate and on the concentration of the hormone and is carried out through the regulation of carbohydrate metabolism. ABA and zeatin regulated the expression of fungal TF and NE genes, but correlation analysis of these parameters showed that this effect depended on the genotype of the isolate. This study will contribute to our understanding of the role of the hormones ABA and CKs in the biology of the fungal pathogen S. nodorum.

Effect of low-temperature stress on the growth of plants of Secale cereale (Poaceae) and endogenous cytokinin content in roots and shoots

Phytohormones play a key role in the regulation of plant acclimation to low temperature. To elucidate the role of cytokinins in rye plant response to chilling, we studied the dynamics of these hormones in shoots and roots under short-term and prolonged cold stress. The 7-day-old plants were exposed to cold stress (2 °C) for 2 h (alarm phase of response) or for 6 h for two days (acclimation phase of response). Endogenous content of cytokinins was analyzed by HPLC-MS method. Low temperature had a differential effect on the content of individual cytokinins and their localization in rye plants. During the short-term stress, a decrease in the content of active cytokinins (trans-zeatin and trans-zeatin riboside) in the roots and an increase in the shoots were shown. Prolonged low-temperature stress declined the amount of cytokinins except trans-zeatin riboside, which was detected in both roots and shoots. Significant rise in trans-zeatin riboside content in roots and shoots in this period evidenced an important role of this cytokinin during cold acclimation of rye plants.

In vitro evaluation of free flavonoids inCassia fistula Linn. calli of different agesunder varying concentrations of plant growthregulators (PGRs)

Cassia fistula Linn, a paramount medicinal plant consumed by huge population all over the world. Its bioactive metabolites include flavonoids, phenolics, and polyphenols. Major objective of current study is to find right media combination and age of callus for maximum accumulation of free flavonoids in Cassia fistula cultures. In this study, the calli were induced in MS + 1.0mg/L 2,4-D + 0.05 mg/L BAP with 50 mg/L ascorbic acid. These calli were then transferred to basic MS medium with 1.0 mg/L2,4,5-T and varied BAP concentrations. Four and 8 weeks old calli were used to find out the right concentration of BAP and age for upscaling of free flavonoids. The samples were extracted and HPTLC analyzed for free flavonoids at 260 nm along with the leaf explants. The study revealed that the amount and number of free flavonoids in 4 weeks old calli increased as the amount of cytokinin (0.5 to 1.0 mg/L BAP) increased but decline with further addition of BAP (2.0 mg/L BAP). In aged calli number and amount of free flavonoids exhibit a tremendous increase when compared to the calli in the same medium at 4 weeks. This is inferred that accruing age of callus, reduced quantities of plantgrowth regulators (optimized for callus growth) accumulate free flavonoids. The best medium for free flavonoid production is MS+ 1.0 mg/L 2,4,5-T with 1.0 mg/L BAP may be recommended for producing a high amount of free flavonoids in C. fistula in vitro.The results were justified with the results of quantification.

Silicon delays salt stress-induced senescence by increasing cytokinin synthesis in tomato

Silicon (Si) is beneficial for plant growth and development, and many studies have shown that Si can alleviate biotic and abiotic stress-induced plant damage. Although the alleviative effect of Si on plant damage induced by salt stress has been widely reported, the underlying mechanism is still not very clear. Here, the effect of Si on salt stress-induced leaf senescence and the role of cytokinin in this process were investigated. The results showed that added Si could significantly decrease chlorophyll degradation induced by salt treatment in tomato. Under salt stress, the effect of Si on the levels of indoleacetic acid and gibberellins in tomato was irregular and dependant on the Si treatment concentrations. Si addition increased the concentrations of cytokinins and abscicic acid, and the expressions of major cytokinin sysnthesis genes in leaf discs of tomato under salt stress. Cytokinin synthesis inhibitor abolished the ameliorative effect of Si on salt-induced leaf senescence of tomato. Si could alleviate salt-induced senescence in the leaves of wild type Arabidopsis thaliana but not in the ipt1,3,5,7 mutant, which has very little amount of cytokinin. These results suggest that Si can delay salt stress-induced plant senescence by increased cytokinin synthesis. This study may help us further explore the in planta mechanism for Si-mediated stress tolerance in plants.

Interaction of the causal agent of apricot bud gall Acalitus phloeocoptes (Nalepa) with apricot: Implications in infested tissues.

Apricot bud gall mite, Acalitus phloeocoptes (Nalepa), is a destructive arthropod pest that causes significant economic losses to apricot trees worldwide. The current study explores the ways to understand the mode of dispersal of A. phloeocoptes, the development and ultrastructure of apricot bud gall, and the role of phytohormones in the formation of the apricot bud galls. The results demonstrated that the starch granules in the bud axon were extended at the onset of the attack. During the later stages of the attack, the cytoplasm was found to deteriorate in infected tissues. Furthermore, we have observed that the accumulation of large amounts of cytokinin (zeatin, ZT) and auxin (indoleacetic acid, IAA) led to rapid bud proliferation during rapid growth period, while abscisic acid (ABA) controls the development of gall buds and plays a vital role in gall bud maturity. The reduction of gibberellic acid (GA3) content led to rapid lignification at the later phase of bud development. Overall, our results have revealed that the mechanism underlying the interaction of apricot bud gall with its parasite and have provided reliable information for designing valuable Apricot breeding programs. This study will be quite useful for pest management and will provide a comprehensive evaluation of ecology-based cost-effective control, life history and demographic parameters of A. phloeocoptes.

Hormon Combinations for Root Induction of Pear

Propagation and root-induction of in vitro plants are regulated by different types and concentrations of phytohormones. The main objective of our study was to observe the effects of different hormone combinations on rooting habit of micropropagated in vitro pear plants. Different amounts of cytokinin (BAP) (0.0; 0,01; 0.02; 0.03 mg l-1) and auxin (2,4 D) (1;2;3 mgl-1) were applied in 8 different combinations of two treatments. Differences among hormone combinations were recorded by rooting habit, number of roots and root length.

PRODUCTION OF PHYTOHORMONES OF BRADYRHIZOBIUM JAPONICUM AND AZOSPIRILLUM BRASILENSE UNDER THEIR SIMULTANEOUS CULTIVATION

Objective. Investigate the activity of biosynthesis of phytohormonal substances with nitrogen-fixing bacteria Bradyrhizobium japonicum and Azospirillum brasilense in pure and mixed culture.
 Methods. Microbiological, chromatographic, and mathematical.
 Results It has been established that the simultaneous cultivation of B. japonicum M-8 and A. brasilense 410 increases the content of gibberellins and cytokinins in the culture fluid of the test microorganisms. The content of gibberellic acid and isopentenylidene has increased most intensively in mixed culture compared with the pure culture of rhizobia. In the course of co-cultivation, the studied diazotrophs more intensively produced auxins compared to soybean rhizobia in pure culture, but less compared to pure culture of azospirilla. The highest level of abscisic acid that can inhibit the formation of nodules was found in A. brasilense 410 culture fluid, and it was lower when cultivating B japonicum M-8. However, the smallest amount of this phytohormone was found in the culture liquid of diazotrophs under their co-cultivation. The lowest ratio of auxin/cytokinin was found in B. japonicum M-8 and A. brasilense 410 culture fluid under their co-cultivation, which should positively influence the formation of a symbiotic system when interacting with soybean plants.
 Conclusion. A combination of cultivating rhizobia and azospirilla showed an increase in the amount of cytokinins and gibberellins in the culture fluid of the microorganisms, a decrease in the amount of abscisic acid and improvement in the auxin/cytokinin ratio compared to the values of the pure cultures of the nitrogen-fixing bacteria studied. An analysis of the quantitative parameters of the content of phytohormones suggests the feasibility of combining B. japonicum and A. brasilense in a mixed culture for the effective introduction of rhizobia in soybean agrocenosis.

Symbiotic Potential of Bradyrhizobium japonicum Strains with Different Growth Rates

To study genetic properties of soybean nodule bacteria with different growth rates, to research the ability of rhizobia to produce phytohormonal substances and estimate their symbiotic potential in the conditions of combined inoculation and mono-inoculation of soybean plants. Sequencing of the 16S-23S rDNA intergenic spacer of soybean rhizobia was performed on ABI 3130xl Genetic Analyzer. Determination of the qualitative and quantitative composition of phytohormones in culture liquid of Bradyrhizobium japonicum strains was performed by SDTLC-chromatography of high resolution. Symbiotic potential of the strains was estimated in the field experiment in Polissia area of Ukraine. It has been established that strains of soybean nodule bacteria with different growth rates by the structure of ITS region belong to different genetic groups: USDA 6, USDA 110 and USDA 123. The studied rhizobia differ in the levels of extracellular phytohormones synthesis: the slow-growing B. japonicum strain 46 produces more amounts of auxins, but the intensive-growing B. japonicum strain KB11 significantly exceeds in the amounts of cytokinins. It has been shown that an important factor of increasing soybean productivity is the formation of balanced symbiotic systems of the host plant with several complementary strains but serologically and genetically different strains of rhizobia of the same species. Combined inoculation of soybean seeds with two active strains B. japonicum 46 and B. japonicum KB11 allows more fully realizing the potential of symbiotic partners and receiving the higher yield.

THE CONTENT OF PLANT HORMONES IN CALLUS TISSUE DURING THE INDUCTION OF SOMATIC EMBRYOGENESIS IN EMBRYOS OF NORWAY SPRUCE (PICEA ABIES [L.] KARST.)

Somatic embryogenesis was induced in embryos of Picea abies by adding 2,4-D and BAP to the culture medium. The content of IAA, ABA, zeatin-ribosid and 2ip was determined at various stages of callus tissue formation using ELISA. The process of embryogenic callus formation depended on the content of IAA and ABA in cultured tissues. The embryos capable of forming embryogenic callus contained 5–6 times more IAA than those incapable of somatic embryogenesis. The ABA content in embryos capable of forming embryogenic callus was two times higher than in embryos incapable of embryogenesis. A sufficiently high level of ABA was observed in embryogenic callus. One of the conditions for the formation of embryogenic callus was a high content of cytokinins in cultured embryos. The data showed that embryogenic callus contained a significantly smaller amount of cytokinins than the cultured tissue of embryos. The level of phytohormones observed in embryogenic callus was low compared to the callus it was formed on. The concentration gradient of phytohormones or a major difference between embryogenic and nonembryogenic callus showed special embryo tissue sensitivity to the growth regulators that determined its competence and readiness to accept exogenous hormonal signal and respond to it in a certain way.

Spatiotemporal modelling of hormonal crosstalk explains the level and patterning of hormones and gene expression in Arabidopsis thaliana wild-type and mutant roots.

• Patterning in Arabidopsis root development is coordinated via a localized auxin concentration maximum in the root tip, requiring the regulated expression of specific genes. However, little is known about how hormone and gene expression patterning is generated.• Using a variety of experimental data, we develop a spatiotemporal hormonal crosstalk model that describes the integrated action of auxin, ethylene and cytokinin signalling, the POLARIS protein, and the functions of PIN and AUX1 auxin transporters. We also conduct novel experiments to confirm our modelling predictions.• The model reproduces auxin patterning and trends in wild-type and mutants; reveals that coordinated PIN and AUX1 activities are required to generate correct auxin patterning; correctly predicts shoot to root auxin flux, auxin patterning in the aux1 mutant, the amounts of cytokinin, ethylene and PIN protein, and PIN protein patterning in wild-type and mutant roots. Modelling analysis further reveals how PIN protein patterning is related to the POLARIS protein through ethylene signalling. Modelling prediction of the patterning of POLARIS expression is confirmed experimentally.• Our combined modelling and experimental analysis reveals that a hormonal crosstalk network regulates the emergence of patterns and levels of hormones and gene expression in wild-type and mutants.

Effect of Synthetic Cytokinin Application on Pod Setting of Individual Florets within Raceme in Soybean

The application of synthetic cytokinin (6-benzylaminopurine, BA) to racemes of soybean genotype IX93-100 at 7 days after anthesis (DAA) enhanced pod-set percentage of the florets at the 5th position and above (numbered from the base on rachis). The endogenous cytokinin (trans-zeatin riboside) content of individual florets was measured at the 1, 3, 5, 7th position every 3 days after anthesis. Cytokinin was detected only from the florets at 9 DAA, and the content was higher in the more proximal florets while it became negligible in the 7th floret. These results suggest that an increase in the amount of cytokinin in individual florets might enhance the pod setting of the florets positioned at the middle or distal part within the raceme.

Excretion of cytokinins into the cultivation medium by suspension-cultured tobacco cells

The dynamics of individual cytokinins were determined in both the cells and the cultivation medium during the subculture interval of cell suspension cultures of Nicotiana tabacum L., line VBI-0. The amounts of cytokinins detected in the cultivation medium were less than 1 pmol ml–1 of suspension. In the late stationary phase, the levels of isopentenyladenosine, as well as that of dihydrozeatin and its riboside, increased significantly. However, when expressed per cell number, the levels of zeatin- and isopentenyladenine-type cytokinins in both the cells and medium were at a maximum at the beginning of the subculture interval and then gradually decreased. Cytokinins were excreted from the cells during the whole subcultivation period, and their concentrations in the cultivation medium were found to be approximately in proportion to their momentary levels inside the cells. The excretion might thus represent one of the mechanisms controlling endogenous cytokinin concentrations.

Effects of Light Spectral Quality on Morphogenesis and Source–Sink Relations in Radish Plants

The accumulation of dry matter and the content of major phytohormones in the aboveground and underground plant parts, as well as light curves and the diurnal course of photosynthesis in the leaves were studied in radish (Raphanus sativusL.) plants of different ages that were grown under red (RL) or blue (BL) light. As seen from the rapid increase in plant biomass, the development of storage organs (hypocotyl or tap root) started on the 14th day after the emergence of seedling of the BL plants and on the 21st day for the RL plants. Conversely, RL stimulated biomass accumulation in the aboveground parts (petioles and stems) already in the early stages of plant development. Light spectral quality only slightly affected the activity and the diurnal course of photosynthesis. The GA content was ten times higher in the aboveground parts of the RL plants than those of the BL plants. The hypocotyl of the BL plants contained much higher amounts of cytokinins and IAA than that of the RL plants. The specific responses of the source–sink relations to the light quality were related to the distribution of various phytohormones between the aboveground and underground parts of the plants: RL increased the content of gibberellins (GA) in the aboveground parts of plants, thus increasing their sink activity, whereas BL stimulated the synthesis of cytokinins and IAA in the hypocotyl and enhanced its development. Light quality-specific morphogenetic responses were reversed when plants were treated with exogenous GA or paclobutrazol, an inhibitor of GA synthesis. The treatment of the BL plants with exogenous GA stimulated petiole and hypocotyl elongation and induced stem formation. The treatment of the BL plants with paclobutrazol led to shortened petioles, the flattening of the storage organ, and the disappearance of the stem.

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture.

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9-beta-D-ribofuranosil-N6-(delta2-isopentenil) adenina ([9R]iP), 9-beta-D-ribofuranosyl-zeatin ([9R]Z) and 9-beta-D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72 h in liquid medium. Subcellular immunolocalization was performed in cells from explants grown for 35 d on agar solidified-medium and for 30 min, 4 and 16 h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4 h of culture in the cytoplasm, and at 16 h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35 d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.

Determination of Dihydrozeatin and Dihydrozeatin Riboside by Cathodic Stripping Voltammetry

The present study develops methods to quantify the phytohormones dihydrozeatin (DHZ) and dihydrozeatin ridoside (DHZR) using cathodic stripping and differential pulse voltammetry (DPS). Employing a supportive electrolyte with 0.04 M acetic/acetate buffer pH 4.5 and 40 s accumulation time at –0.60 V, the detection and determination limits for DHZ were 5.5 and 8.0 ng mL–1, respectively. For DHZR using a 0.04 M acetic/acetate buffer pH 4 with a 50 s accumulation time at –0.95 V the detection limit was 16.0 ng mL–1 with a determination limit of 24.0 ng mL–1. Both methods were successfully applied to determine minute amounts of cytokinins in apples.

Growth, transpiration, root‐born cytokinins and gibberellins, and nutrient compositional changes in sesame exposed to low root‐zone temperature under different ratios of nitrate: Ammonium supply

The growth of sesame (Sesamum indicum L.) was studied at three root temperature regimes (25/25, 20/10 and 15/15°C day/night) factorially combined with three NO3 ‐: NH4 + ratios (mM ratios, 10:0, 8:2, or 6:4), as a source of nitrogen (N), in the irrigation solution. The air temperature was kept constant at 30°C. Transpiration, nutrient composition, and level of root‐born cytokinins and gibberellins in the xylem exudate were monitored. The two low root temperature regimes, 15/15 and 20/10°C, restricted the growth of sesame, reduced transpiration and increased the accumulation of soluble carbohydrates in the shoot and in the roots compared to the 25/25°C regime. The NO3:NH4 + ratios had no effect on growth. Nutrient contents in the shoot at low root temperatures, particularly K+, NO3 ‐, and H2PO4 ‐ were decreased markedly, but Na+ increased relative to it's content in the 25/25°C regime. Increasing NH4 + proportion in the irrigation solution raised total N concentration in the plant tissues at all root temperatures. The amounts of cytokinins and gibberellins in the xylem exudate decreased at the low root temperature regimes relative to the 25/25°C regime. Low root temperature reduced xylem transport of nutrients and root born‐phytohormones, most probably because of reduced water flow through the plant relative to the 25/25°C regime.

Microcutting leaf area, weight and position on the stock shoot influence root vigour, shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro.

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA3 generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca++ and Mg++ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca++ or higher Mg++ contents. Both had comparable amounts of cytokinins in shoot and root tissues.

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of ‘Fiesta’ scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml−1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of ‘Fiesta’ scion grafted onto the rootstocks. Cytokinin content (ng shoot−1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants. Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.

Endogenous cytokinins in the vegetative and reproductive phases of development in the dioecious plant Rumex acetosella L.

The content of endogenous cytokinins has been analysed in leaf and inflorescence extracts of male and female R. acetosella plants, using gas chromatography. Plant parts were extracted at four stages of development: leaves of juvenile plants, leaves of adult plants at the time of flower initiation and in full bloom, and upper internodes of the inflorescence stalks. Cytokinins with characteristics similar to isopentenyl adenine and adenosin, zeatin, zeatin riboside, and a bound form of zeatin, were all found in the extracts. The total amount of cytokinins was higher in female than in male plants during all these stages.