Light promotes an increase of cytokinin oxidase/dehydrogenase activity during senescence of barley leaf segments

Following a study of the relationship between cytokinin oxidase/dehydrogenase (CKX) and senescence in darkened barley leaf segments, we have now investigated the influence of light on the in vitro activity of CKX. Seedlings of Hordeum vulgare L. were grown for 8 d under a light/dark regime of 18 h white light and 6 h darkness. Then apical parts of 7 cm length were cut from the first foliage leaves and their bases were placed in water. In segments kept in the dark, the CKX activity measured by cleavage of N₆-(Δ₂-isopentenyl)adenine rose from 0.1 pkat (g FW)⁻¹ to 0.8 pkat (g initial FW)⁻¹ within the first 4 d of incubation. In contrast, in segments kept under the light/dark regime it reached a value of 8.6 pkat (g initial FW)⁻¹ over the same time period. The chlorophyll a content declined slightly slower during light/dark cycling than in darkness. In contrast to segments and isolated laminae, corresponding attached laminae exhibited less CKX activity after 2 d under light/dark conditions than after 2 d in the dark. The activity in attached laminae of first foliage leaves of plants growing in light/dark cycling increased strongly only when the plants were older than 4 weeks. In line with this, the CKX activity in attached laminae of flag leaves of barley growing in fields increased in a late developmental state. The senescence of darkened isolated laminae of Zea mays L. and Phragmites australis (Cav.) Trin. ex Steudel was associated with an enhancement of CKX activity too. Because in most cases a positive correlation between CKX activity and senescence was found, it is likely that the enzyme promotes senescence by destroying cytokinins, which help to keep Poaceae leaves green. Light may promote not only cytokinin degradation but also the formation of bioactive cytokinins in leaf segments.     

N9-substituted derivatives of kinetin: effective anti-senescence agents

The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55 years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and ¹H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Degradation of ribulose-1,5-bisphosphate carboxylase and chlorophyll in senescing barley leaf segments triggered by jasmonic acid methylester, and counteraction by cytokinin

Barley ( Hordeum vulgare L. cv. Salome) primary leaf segments responded to the application of a putative plant growth regulator, ± jasmonic acid methylester (JA-Me). with accelerated senescence, as indicated by the loss of chlorophyll and the rapid decrease in activity and immunoreactive protein content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39). The senescence-promoting action of JA-Me differed in light and in darkness; e.g. the initial rates of chlorophyll and RuBP carboxylase breakdown were markedly higher in light than in darkness in the presence of 4.10⁻⁵ M JA-Me. Cytokinin (benzyladenine, 4.10⁻⁵ M ) stopped the loss of chlorophyll and RuBP carboxylase during senescence; however, the rapid drop induced by JA-Me in the early phase of leaf segment senescence could not be prevented by concomitant or previous addition of BA. On the other hand, BA added 24 h after JA-Me application resulted in a recovery of chlorophyll and RuBP carboxylase at the later stages, indicating a possible rapid inactivation of JA-Me in the tissues. The activities of a number of other chloroplastic and cytosolic enzymes were not significantly altered in JA-Me-treated leaf segments compared with controls floated on water. Time-dependent chlorophyll decrease in isolated chloroplasts did not change upon JA-Me addition to the isolated organelles. It is suggested that JA-Me acts on chloroplast senescence by promoting cytoplasic events which eventually bring about the degradation of chloroplast constituents.     

Cytokinin oxidase/dehydrogenase in Pisum sativum plants during vegetative development. Influence of UV-B irradiation and high temperature on enzymatic activity

Cytokinin oxidase/dehydrogenase (EC 1.5.99.12) specific activity was determined in leaves and roots of two P. sativum cultivars (cv. Scinado and cv. Manuela) during vegetative development and the effect of UV-B irradiation or elevated temperature was assessed. The measurement of CKX activity during development showed localisation of this enzyme to roots. The reduction in CKX activity in leaves after UV-B irradiation and the increased levels of the enzyme in high temperature-treated plants suggests that the enzymes from the CKX gene family have a different expression during stress responses provoked by different factors and probably are tissue specific. Differences regarding cytokinin oxidase/dehydrogenase activity stress response were observed between the two pea cultivars.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co²⁺, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

Micro-scale chlorophyll analysis and developmental expression of a cytokinin oxidase/dehydrogenase gene during leaf development and senescence

Chlorophyll content is a baseline measurement in many plant studies. With the miniaturization of many molecular and biochemical assays the sample size needed for these assays has been greatly reduced. Chlorophyll is commonly determined by spectrophotometry. Traditionally this analysis has been carried out in 10 mm cuvettes with volumes of 0.2–2.0 ml. The NanoDrop™ spectrophotometer requires a sample volume of 1–2 μl which provides the opportunity to analyze far smaller samples. Chlorophyll analyses are a critical part of any study of senescence and the cytokinins are considered to play a key role in regulating senescence. The tissue specific levels of cytokinins are thought to be regulated, at least in part, by expression of the cytokinin degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Here an assay is described for the spectral determination of chlorophyll using the NanoDrop™ spectrophotometer that allows the determination of chlorophyll from part samples of clover leaves while leaving sufficient tissue for quantitative real-time PCR study of CKX expression. The expression of TrCKX2 increased during leaf development along the stolon of Trifolium repens but was stable during the onset and progression of senescence suggesting that TrCKX2 was not an initiator of leaf senescence but may have facilitated the progression.     

Effects of ABA on Senescence and Cellulase Activity of Detached Rice Leaves

The relationship of cellulase to detached leaf senescence of rice seedlings was investigated by examining the effect of ABA and 6-BA on changes in the level of cellulase of leaf segments during senescence. It was shown that the rise in cellulase activity increased with declining chlorophyll content, which was used as the senescence indicator during the senescence of detached rice leaves caused by ABA. The action of ABA took place only after a 48h lag period. ABA enhanced the cellulase secretion and increased the permeability of plasma membrane. A high level of cellulase activity in cell wall closely related to membrane permeability changes. The action of cellulase in the cell wall may cause depolymerization of β-1, 4-glucan in situ, thus speeding senescence. The 6-BA reverses completely or partly the increase in cellulase activity and tile permeability caused by ABA during the first two day, }) ut it antagonized hardly any of the ABA effect from the third day on, suggesting the onset of an irreversible stage in the senescence of detached rice leaves.     

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.     

Changes in cytokinin levels and metabolism in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.) explants during in vitro shoot organogenesis induced by <Emphasis Type="Italic">trans</Emphasis>-zeatin and dihydrozeatin

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.     

Cytokinin oxidase/dehydrogenase activity as a tool in gibberellic acid/cytokinin cross talk

Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic acid (GA₃) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster, had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and 5 μM GA₃. Changes in CK content and CKX activity in GA₃-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly influenced by the application of GA₃. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA₃-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk.     

New insights into the biology of cytokinin degradation

A survey of recent results is presented concerning the role of cytokinin degradation in plants, which is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. An overview of Arabidopsis CKX gene expression suggests that their differential regulation by biotic and abiotic factors contributes significantly to functional specification. Here, we show using reporter gene and semiquantitative RT-PCR analyses regulation of individual CKX genes by cytokinin, auxin, ABA, and phosphate starvation. Partially overlapping expression domains of CKX genes and cytokinin-synthesizing IPT genes in meristematic tissues and endo-reduplicating cells lend support for a locally restricted function of cytokinin. On the other hand, their expression in vascular tissue suggests a function in controlling transported cytokinin. Recent studies led to a model for the biochemical reaction mechanism of CKX-mediated catalysis, which was refined on the basis of the three-dimensional enzyme structure. Last but not least, the developmental functions of CKX enzymes are addressed. The recent identification of the rice OSCKX2 gene as an important novel breeding tool is highlighted. Together the results corroborate the relevance of metabolic control in determining cytokinin activity.     

Membrane Permeability--Regulation by Exogenous Sugars during Senescence of Oat Leaf in Light and Darkness

The effect of sugars (sucrose, glucose and fructose) on normalphysiological changes during senescence of foliar segments ofAvena sativa cv. Suregrain was studied. In general applicationof sugars raised tissue permeability both in the light and indarkness. This change was associated with increases in endogenoussugars, hydroperoxide content and lipoxygenase activity. Inthe light it was also associated with low catalase activity.Sugars did not influence superoxide dismutase activity. In thelight, sugars accelerated senescence, measured as decreasesin chlorophyll and increases in soluble amino acids. In darknesssugars delayed senescence. The effect of sugars in the lightseemed to result from an increase in photo-oxidations associatedwith the increase in permeability. The delaying effect on senescence,found in darkness, seemed to result from an increase in respiratoryactivity plus the lack of (or combined with the lack of) photo-oxidations. (Received March 18, 1985; Accepted June 3, 1986)     

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.     

Characterization and mapping of a novel mutant sms1 （senescence and male sterility 1） in rice

Plant senescence plays diverse important roles in development and environmental responses.However,the molecular basis of plant senescence is remained largely unknown.A rice spontaneous mutant with the character of early senescence and male sterility (sms) was found in the breeding line NT10-748.In order to identify the gene SMS1 and the underlying mechanism,we preliminarily analyzed physiological and biochemical phenotypes of the mutant.The mutant contained lower chlorophyll content compared with the wild type control and was severe male sterile with lower pollen viability.Genetic analysis showed that the mutant was controlled by a single recessive gene.By the map-based cloning approach,we fine-mapped SMS1 to a 67 kb region between the markers Z3-4 and Z1-1 on chromosome 8 using 1,074 F2 recessive plants derived from the cross between the mutant sms1 (japonica) × Zhenshan 97 (indica),where no known gene involved in senescence or male sterility has been identified.Therefore the SMS1 gene will be a novel gene that regulates the two developmental processes.The further cloning and functional analysis of the SMS1 gene is under way.