Salinity stress response of non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn.) shoots and roots grown in vitro

Common centaury (Centaurium erythraea Rafn.) is a plant species that can inhabit saline soils. It is known as a plant with high spontaneous regeneration potential in vitro. In the present work we evaluated shoots and roots salinity tolerance of non-transformed and three AtCKX transgenic centaury lines to graded NaCl concentrations (0, 50, 100, 150, 200 mM) in vitro. Overexpression of AtCKX genes in transgenic centaury plants resulted in an altered cytokinins (CKs) profile leading to a decline of bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms and/or CK nucleotides. Significant increment of fresh shoot weight was obtained in shoots of non-transformed and AtCKX1 transgenic line only on medium supplemented with 50 mM NaCl. However two analysed AtCKX2 transgenic lines reduced shoot growth at all NaCl concentrations. In general, centaury roots showed higher tolerance to salinity than shoots. Non-transformed and AtCKX1 transgenic lines tolerated up to 100 mM NaCl without change in frequency of regeneration and number of regenerated plants. Roots of two analysed AtCKX2 transgenic lines showed different regeneration potential under salt stress. Regeneration of transgenic AtCKX2-26 shoots even at 200 mM NaCl was recorded. Salinity stress response of centaury shoots and roots was also evaluated at biochemical level. Free proline, malondialdehyde and hydrogen peroxide content as well as antioxidative enzymes activities were investigated in shoots and roots after 1, 2, 4 and 8 weeks. In general, adition of NaCl in culture medium elevated all biochemical parameters in centaury shoots and in roots. Considering that all analysed AtCKX transgenic centaury lines showed altered salt tolerance to graded NaCl concentrations in vitro it can be assumed that CKs might be involved in plant defence to salt stress conditions.     

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l⁻¹ indole-3-acetic acid and 1.0 mg l⁻¹ 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g⁻¹ dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.     

Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation

Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7–GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7‐expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis‐zeatin (cZ) and N‐glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ‐deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone.     

Photosynthesis and protective mechanisms during ageing in transgenic tobacco leaves with over-expressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content

The content of cytokinins (CKs), the plant inhibitors of the final phase of plant development, senescence, is effectively controlled by irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). In transgenic tobacco, denoted as AtCKX, with over-expressed CKX causing lowered CK content, we investigated changes in the time courses of chlorophyll (Chl) and xanthophyll (violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein) contents. We also determined parameters of slow Chl fluorescence kinetics such as minimum Chl fluorescence yield in the darkadapted state F₀, maximum quantum yield of PS2 photochemistry (F_v/F_m), maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in photosystem 2 (PS2), F_v/F₀, non-photochemical quenching (NPQ), and effective quantum yield of photochemical energy conversion in PS2 (Φ₂). We used three different developmental leaf stages, old, mature, and young, and compared this with time courses of these characteristics in leaves with natural CK levels. The parameters F_v/F_m, F_v/F₀, and Φ₂ were unchanged during ageing in AtCKX plants in contrast to control ones where a significant decrease in old leaves was found. In control plants F₀ increased during ageing, but in the oldest leaf a considerable decrease was observed. This could indicate progressive damage to PS2 reaction centres and then detachment and rapid degradation of Chl. This is in agreement with time course of Chl content. NPQ decreased with age and was similar in both plant types. We observed a decline of xanthophyll contents in the oldest leaves in both plant types, but the contents were enhanced in AtCKX compared to control plants, especially of neoxanthin. The higher xanthophyll contents in the transgenic plants contribute to a better photoprotection and the fluorescence parameters indicated that photosynthetic apparatus was in better condition compared to control and it consequently postponed the onset of leaf senescence.     

Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars

Key message

The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin.

Abstract

Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.     

Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase

We studied changes in physiological parameters of whole leaves and in antioxidant protection of chloroplasts during ageing and senescence of tobacco (Nicotiana tabacum L. cv. Samsun NN) leaves with enhanced cytokinin oxidase/dehydrogenase activity (CKX) or without it (WT). Old leaves of CKX plants maintained higher pigment content and photosystem 2 activity compared to WT leaves of the same age. Chloroplasts of old CKX plants showed better antioxidant capacity represented by higher superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities.     

Novel potent inhibitors of A. thaliana cytokinin oxidase/dehydrogenase

The synthesis of a new group of 2-X-6-anilinopurines, including compounds with potential cytokinin-like activities, with various substitutions (X=H, halogen, amino, methylthio or nitro) on the phenyl ring is described. The prepared compounds have been characterized using standard physico-chemical methods, and the influence of individual substituents on biological activity has been compared in three different bioassays, based on the stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves and the dark induction of betacyanin synthesis in Amaranthus cotyledons. The biological activity of the prepared compounds was also assessed in receptor assays, in which the ability of the compounds to activate the cytokinin receptors AHK3 and AHK4/CRE1 was studied. Finally, the interactions of the compounds with the Arabidopsis cytokinin oxidase/dehydrogenase AtCKX2 (heterologously expressed) were investigated. Systematic testing led to the identification of two very potent inhibitors of AtCKX2: 2-chloro-6-(3-methoxyphenyl)aminopurine and 2-fluoro-6-(3-methoxyphenyl)aminopurine.     

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.     

Differential accumulation of xanthones in methyl-jasmonate- and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale

Cell-suspension cultures of Centaurium erythraea and Centaurium littorale (Gentianaceae) respond to methyl jasmonate and yeast extract with a differential accumulation of xanthones. Methyl jasmonate induced the formation of 1-hydroxy-3,5,6,7-tetramethoxyxanthone, the amount of which increased in both cell cultures around 10 h after addition. A substantial increase in the activity of phenylalanine ammonia-lyase (PAL) was not observed. When challenged with yeast extract the cell cultures accumulated l,5-dihydroxy-3-methoxyxanthone. This appeared rapidly after addition of yeast extract in C. erythraea but its amount in C. littorale increased only after a lag phase of 25 h. While PAL activity in C. erythraea was strongly suppressed a fourfold increase in its activity was found in C. littorale. Both elicited xanthones accumulated intracellularly. A scheme for xanthone biosynthesis in the two cell cultures is proposed.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications

1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

Response of cytokinin pool and cytokinin oxidase/dehydrogenase activity to abscisic acid exhibits organ specificity in peas

Changes in cytokinin pool and cytokinin oxidase/dehydrogenase activity (CKX EC: 1.5.99.12) in response to increasing abscisic acid (ABA) concentrations (0.5–10 μM) were assessed in the last fully expanded leaves and secondary roots of two pea (Pisum sativum) varieties with different vegetation periods. Certain organ diversity in CKX response to exogenous ABA was observed. Treatment provoked altered cytokinin pool in the aboveground parts of both studied cultivars. Specific CKX activity was influenced significantly basically in roots of the treated plants. Results suggest that ABA-mediated cytokinin pool changes are leaf-specific and involve certain root signals in which CKX activity presents an important link. This enzymatic activity most probably regulates vascular transport of active cytokinins from roots to shoots.     

Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels

Maturing maize kernels are a rich source of cytokinins and cytokinin oxidase/dehydrogenase activity, but the relationship between kernel development, cytokinin levels, the induction of cytokinin oxidase/dehydrogenase and the control of cell division is not known. Using polyclonal antibodies raised against recombinant maize cytokinin oxidase/dehydrogenase, we investigated the appearance of cytokinin oxidase/dehydrogenase (ZmCKX1) in both hybrid and inbred maize kernels as a function of time after pollination. Cytokinin oxidase/dehydrogenase was detected by five days after pollination (5 DAP) in a hybrid line, but significantly later in inbred lines. The bulk of the cytokinin oxidase/dehydrogenase detected was associated with the embryo and placental/chalazal region of the kernels rather than with the endosperm. We identified additional maize sequences in the database that appear to encode cytokinin oxidase/dehydrogenase gene family members and correspond closely with a subset of the ten cytokinin oxidase/dehydrogenase genes identified in the rice genome. Gene expression of Zmckx1 was examined by RT-PCR in immature kernels and compared with that of three putative maize cytokinin oxidase/dehydrogenase homologs. We conclude that the manipulation of kernel cytokinin levels to increase endosperm cell division will require a more detailed understanding of specific expression patterns and localization of multiple cytokinin oxidase/dehydrogenases within kernels.     

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.     

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50=55 degrees C for 30 min incubation). The following pH optimum and K(m) values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (K(m)=0.5 microM), pH 7.6 for isopentenyl adenosine (K(m)=1.9 microM), pH 7.9 for zeatin (K(m)=1.5 microM) and pH 7.3 for zeatin riboside (K(m)=2.0 microM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors).     

Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties,activity, and expression during tuber dormancy progression

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [³H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.