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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

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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
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  Physiologia Plantarum 134: 609–623. 2008  Copyright  ª  Physiologia Plantarum 2008, ISSN 0031-9317 Senescence progression in a single darkened cotyledondepends on the light status of the other cotyledon in Cucurbita pepo  (zucchini) seedlings: potential involvementof cytokinins and cytokinin oxidase/dehydrogenase activity Kalina Ananieva a , Evgue´ni D. Ananiev b, *, Snejana Doncheva a , Katya Georgieva a , Nikolina Tzvetkova c ,Miroslav Kamı´nek d , Va´clav Motyka d , Petre Dobrev d , Silvia Gajdos ˇova´ d,e and Jiri Malbeck d a Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113, Bulgaria b Department of Plant Physiology, Faculty of Biology, St. Kl. Ohridski University of Sofia, 8 Dragan Tsankov bld., 1164 Sofia, Bulgaria c University of Forestry, 10 Kliment Ohridski av., Sofia 1756, Bulgaria d Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova´  135, CZ-16502 Prague 6, Czech Republic e Department of Plant Physiology, Faculty of Science, Charles University, Vinic ˇ na´  2, CZ-12844 Prague 2, Czech Republic Correspondence *Corresponding author,e-mail: ananiev@bio21.bas.bgReceived 20 June 2008; revised 18 July2008doi: 10.1111/j.1399-3054.2008.01161.x Darkness mediates different senescence-related responses depending on thetargeting of dark treatment (whole plants or individual leaves) and on the organsthat perceive the signal (leaves or cotyledons). As no data are available on thepotential role of darkness to promote senescence when applied to individualcotyledons, we have investigated how darkness affects the progression of sene-scence in either a single or both individually darkened cotyledons of young10-day-old  Cucurbita pepo   (zucchini) seedlings. Strong acceleration of senes-cence was observed when both cotyledons were darkened as judged by thedamage in their anatomical structure, deterioration of chloroplast ultrastructurein parallel with decreased photosynthetic rate and photochemical quantumefficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAAwere strongly reduced. In a single individually darkened cotyledon, the structureandfunctionofthephotosyntheticapparatusaswellasthecontentsofendogenousCKsandIAAweremuchlessaffectedbydarkness,thussuggestinginhibitoryeffectof the illuminated cotyledon on the senescence of the darkened one. Apparently,the effect of darkness to accelerate/delay senescence in a single darkenedcotyledon depends on the light status of the other cotyledon from the pair. Theclose 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 essentiallyinvolved in the mechanisms of downregulation of endogenous CK levels. Ourresults indicated that CKX-regulated CK signaling could be a possible regulatorymechanism controlling senescence in individually darkened cotyledons. Introduction Leaf senescence is a programmed and highly regulateddevelopmental process comprising the final stage of plant/organdevelopmentleadingtodeathandabscission(Noode´n1988a,Smart1994).Theonsetofleafsenescencerepresents an age-dependent genetically controlled  Abbreviations –   CK, cytokinin;  cis -Z,  cis- zeatin;  cis- ZR,  cis- zeatin riboside; CKX, cytokinin oxidase/dehydrogenase; DHZ,dihydrozeatin; FW, fresh weight; iP, N 6 -(2-isopentenyl)adenine; iPR, N 6 -(2-isopentenyl)adenine 9-riboside; Z,  trans -zeatin; ZR, trans -zeatin 9-riboside; ZOG,  trans -zeatin O-glucoside; ZROG,  trans -zeatin 9-riboside O-glucoside; ZRMP,  trans -zeatin 9-riboside-5 # -monophosphate; (abbreviations for cytokinins according to Kamı´ nek et al. 2000). Physiol. Plant. 134, 2008  609  transitionfromafunctionallyactivephotosyntheticorganto a degradative phase of senescence (Noode´n et al.1997, Smart 1994). The degradation of the chloroplastand its components is one of the most prominentphenomena of senescence resulting in the decrease inphotosynthetic activity (Krupinska and Humbeck 2004,Noode´n et al. 1997).Senescence can also be triggered by many environ-mental stresses, such as extreme temperatures, drought,nutrient deficiency, insufficient light/shade or totaldarkness (Smart 1994). The ability of darkness to accel-erate senescence has been studied in detached adultleaves (Biswal et al. 1983, Oh et al. 1997, Weaver et al.1998) and intact plants (Biswal and Biswal 1984, Kleber- Janke and Krupinska 1997). However, data have shownthat in  Arabidopsis   leaf senescence is not induced but isinfactinhibitedwhenawholeplantisdarkened,whereassenescence is strongly accelerated in individually dark-ened leaves (Weaver and Amasino 2001). These resultsdemonstrate that the light status of the rest of the plantcontrols the senescence progression of the individualleaf. Thus, darkness induces two separate senescence-related responses summarized in a model proposed byWeaver and Amasino (2001). One of them (promotionof senescence) is induced locallyat the cell, tissue and/ororgan level, whereas the other (repression of senescence)is expressed when darkness is experienced at the levelof the entire plant. Recent studies have increased ourknowledgeaboutthemolecularandcellularmechanismsof foliar senescence in whole darkened plants and in-dividually darkened leaves in  Arabidopsis   (Keech et al.2007,vanderGraaffetal.2006).Itwasshownthatduringage-dependent senescence, the upregulated genes ex-ceeded the number of downregulated genes, whereas inindividually darkened leaves and detached senescingleaves the fractions were similar or opposite. Most of theup- or downregulated genes (75%) in individually dark-ened leaves were common for age-dependent senes-cence(van der Graaff et al. 2006). Regarding cellularandmetabolic changes during dark-induced senescence,data have shown that while the photosynthetic capacityof whole darkened  Arabidopsis   plants remained un-changed, the mitochondrial respiration decreased, thussuggesting a ‘stand-by mode’ of metabolism maintainingthe activity of the photosynthetic apparatus (Keech et al.2007). This result underlies the mechanism of delayedsenescence in whole darkened plants observedearlier byWeaver and Amasino (2001). In contrast, in individuallydarkened leaves, a rapid decline in photosynthetic ac-tivity was found while the mitochondrial respirationwas increased, thus indicating accelerated senescence.The changes in chloroplasts and mitochondria provokedby the two treatments were accompanied with anunequal degradation of chloroplasts and mitochondriain the senescing leaves (Keech et al. 2007).The control of senescence by darkness may bemediated by plant hormones that represent one of themost significant internal factors influencing senescence(Gan 2004). There is a great deal of evidence suggestingthat cytokinins (CKs) are the major senescence-inhibitinghormones (Gan 2004, Mok 1994, Noode´n and Letham1993, Van Staden et al. 1988). A close correlation existsbetween senescence progression and a decline inendogenous CKs (Gan and Amasino 1996, Van Stadenetal.1988).ItiswelldocumentedthatCKs,eitherdirectlyor indirectly through a signaling pathway, can inhibit thetranscription of senescence-related genes and thus pre-venttheonsetofsenescence(Buchanan-Wollaston1997,Gan 2004, Gan and Amasino 1995). A detailed tran-scription analysis of three types of senescence processesin  Arabidopsis   (age-dependent senescence and dark-induced senescence in individually darkened attachedleavesordark-incubateddetachedleaves)hasshownthatthe expression profiles of CK homeostasis genes aremostly consistent with the depletion of CKs during senes-cence, whereas the expression of CK-signaling genesis much more complex (van der Graaff et al. 2006). Ina recent study, we have shown that, as in leaves, naturalsenescence of   Cucurbita pepo   (zucchini) cotyledons wasassociatedwithagradualdecreaseintheconcentrationof bioactive CKs and CK riboside phosphates, whereas thelevels of storage CK  O  -glucosides and biologically inac-tive CK 7- and 9-glucosides were increased (Ananievaet al. 2004). The transfer of whole 1-week-old seedlingsto the dark for 2 or 5 days resulted in promotion of cotyledon senescence as judged by the reduction in thecontent of bioactive CKs, CK riboside phosphates andstorage CK  O  -glucosides. During the recovery of thecotyledons after return to light regime, bioactive CKs andtheir nucleotides increased. In this respect, zucchinicotyledons differ from  Arabidopsis   cotyledons where se-nescence was accelerated after dark treatment of wholeadult plants (Weaver and Amasino 2001). Thus, the se-nescence mechanisms found in  Arabidopsis   could bedifferent from other monocarpic plants, such as zucchinibecause of the lack of correlative control of leaf se-nescence by the developing reproductive organs (Limet al. 2007).In the past years, progress has been made on theinvolvement of CK oxidase/dehydrogenase (CKX; EC1.4.3.18/1.5.99.12), the major CK degrading enzyme,in CK signaling of senescence. Few existing data indicatean enhancement of CKX activity during senescence of leaf segments in the dark associated with a decline of endogenous CKs content (Conrad et al. 2007, Kamı´neket al. 1997a). However, overexpression of CKX genes in 610  Physiol. Plant. 134, 2008  transgenics may result in retardation of leaf senescence(Werner et al. 2001, 2003). Recent data have shown thecomplexity of CKX gene expression in  Arabidopsis  including upregulation of   Atckx5   and downregulationof   Atckx6   during natural senescence and dark-inducedsenescence in individually darkened attached leaves ordark-incubated detached leaves (van der Graaff et al.2006).Thus,the roleofCKXinsenescenceremainsasyetunclear.A complex web of signal interactions, both synergisticand antagonistic, exists between CKs and auxins in thecontrol of many developmental processes in plants(Coenen and Lomax 1997, Coenen et al. 2003, Eklo¨f etal.1997,Nordstro¨metal.2004).Althoughsomeresultsare contradictory, it is clearly documented that auxin canregulate CK pool sizes and vice versa (Eklo¨f et al. 1997,2000, Miyawaki et al. 2004, Nordstro¨m et al. 2004). Themechanisms of the cross talk between the two planthormones include interactive control of gene expression,posttranslational effects and modulation of enzymeactivity (Coenen and Lomax 1997). However, data onthe interactions of these two plant hormones in theregulation of plant senescence are quite limited (Gan2004).It has been shown that the ability of cotyledons tosenesce in darkness is similar rather to the dark-inducedsenescence of detached leaves but not to attached trueleaveswhose senescence is repressed when whole plantsare placed in darkness (Biswal and Biswal 1984, Weaverand Amasino 2001), indicating that dark-induced senes-cence can be organ specific.The present study extends our previous investigationson dark-mediated responses in zucchini cotyledons aftertransferofwholeplantstodarkness(Ananievaetal.2004,2007). As no data are available on the potential role of darkness to promote senescence when applied to indi-vidual cotyledons, investigation of dark-induced senes-cence in individually darkened cotyledons (both ora single one from the pair) allowed us to assess whetherthe light status of one cotyledon could contribute to thesenescence progression in the other one, thus providinginsights into the mechanisms coordinating senescencein the pair of cotyledons. As the seedlings used in ourexperiments were young (10 days old) having two coty-ledons at an equal stage of development and avery smallstill developing primary leaf, age as a separate inducerof senescence did not interfere with the response. Thus,the response of individually darkened cotyledons wasonly because of the dark treatment. Present experimentsare based on the experimental scheme proposed byWeaver and Amasino (2001) and further developed byKeechet al. (2007) for individually darkened  Arabidopsis  leaves. Senescence progression was assessed by thechanges in the structure and function of the photosyn-thetic apparatus as well as in endogenous levels of CKsandIAA.Inaddition,westudiedtheroleofCKXincontrolof CK levels, which can be involved in the senescence-related responses of individually darkened cotyledonsthrough the corresponding signaling pathways. Materials and methods Plant material and growth conditions Seeds of   Cucurbita pepo   L. (zucchini), cv. Cocozelle,were germinated on moistened filter paper for 96 h indarkness at 28  C. The 4-day-old etiolated seedlings weregrown further on a nutrient solution (Yamagishi andYamamoto 1994) in a growth chamber at a photon fluxdensityof 100  m mol m 2 2 s 2 1 , 26    2  C,relative humid-ity 70% and a 12/12 h day/night photoperiod. Dark-induced senescence Cotyledons of 8-day-old seedlings were individuallydarkened (either a single cotyledon or both cotyledonsfrom the pair) for 2 days by covering with paper mittens,which were dark only at the inner surface. Samples fromthe following four variants were analyzed: (1) controlcotyledons (harvested from 10-day-old plants grownunder controlled light regime), (2) both darkened coty-ledons, (3) a single darkened cotyledon and (4) the othercotyledon from the pair that remained under the initiallight regime. Net photosynthesis Net photosynthetic rate was measured with an infraredgas analyzer (Li 6400; Li Cor, Lincoln, NE) at quantumflux density of 500  m mol m 2 2 s 2 1 photosyntheticallyactive radiation (PAR). Data were statistically processedusing S YSTAT  7.0. Each value represents the mean of twodifferent experiments  SE . Chlorophyll fluorescence measurements Chlorophyll fluorescence emission from the upper leaf surfacewasmeasuredwithapulseamplitudemodulationfluorimeter (PAM 101-103; Walz, Effeltrich, Germany).The initial fluorescence yield in weak modulated mea-suringlight[0.075  m mol m 2 2 s 2 1 photosyntheticphotonfluxdensity(PPFD)],F o ,andmaximumtotalfluorescenceyield induced by a saturating white light pulse (1 s, over3500  m mol m 2 2 s 2 1 PPFD, by Schott KL 1500 lightsource), F m , were determined. The leaf disk (10 mmdiameter) was then illuminated with continuous red light Physiol. Plant. 134, 2008  611  (actiniclight,100  m mol m 2 2 s 2 1 PPFD).Simultaneously,with the onset of actinic light illumination, modulationfrequency was switched from 1.6 to 100 kHz. After 15-min actinic light, the short saturating light pulse was usedto obtain the fluorescence intensity F # m  with all PSIIreactioncentersclosed.Inductionkineticswereregisteredand analyzed with a microcomputer program  FIP  4.3elaborated by Tyystja¨rvi and Karunen (1990). The quan-tum efficiency of PSII electron transport was calculatedaccording to the formula:  F PSII ¼  F # m 2 F Þ = F # m . Thefraction of the absorbed light energy which was not usedfor photochemistry (LNU) was calculated as suggested byCornic (1994): LNU  ¼  1 – [ F PSII/(F v  /F m )]. Each valuerepresents the mean of two different experiments withthree replicates each  SE . Light and transmission electron microscopy Cotyledon segments (1–2 mm) were fixed in 4% (v/v)glutaraldehyde in 0.1  M   Na cacodilate buffer, pH 7.2 for2 h at 4  C and postfixed in 1% (w/v) OsO 4  in the samebuffer. The segments were dehydrated through a gradedethanol series (25, 50, 75, 96 and 100); ethanol-propylene oxide (1:1, v/v); propylene oxide and pro-pyleneoxide-DurcupanACM(1:1,v/v),andembeddedinDurcupan ACM (Fluka AG, Buchs, Switzerland). Forlight microscopy, semithin sections were double stainedwith methylene blue and Fuchsin and examined withaNikonE600lightmicroscope(Nikon,Tokyo,Japan).Forelectron microscopy, ultrathinsectionswere stainedwithuranyl acetate and lead citrate and examined with a JEM1200EXtransmissionelectronmicroscope(Tokyo,Japan)at 80 kV. Determination of the number of chloroplasts From 10 randomly chosen semithin cross-sections, 10fields over the image screen were analyzed using Micro-scope Eyepiece Camera (CCD-EYE-USB-2 Digital Video;Microscopes Inc., St Louis, MO). The number of chlo-roplasts per palisade cell was estimated by counting 250cells per cross-section from each variant. Analysis of endogenous CKs and auxins Endogenous CKs and auxin were extracted in methanol/ formic acid/water (15/1/4, v/v/v) and purified using dual-mode solid phase extraction method, which allowsseparation of CKs from auxin and abscisic acid by se-quential elution from Oasis MCX column (Waters Co.,Milford, MA) (Dobrev and Kamı´nek 2002).Detection and quantification of CKs were carried outusing HPLC/MS system consisting of HTS-Pal auto-sampler (CTC Analytics, Zwingen, Switzerland), quater-nary HPLC pump Rheos 2200 (Flux Instruments, Basel,Switzerland), Delta Chrom CTC 100 Column oven(Watrex, Praha, Czech Republic) and TSQ QuantumUltra AM triple-quad high-resolution mass spectrometer(Thermo Electron, San Jose, CA). Dried extracts wereredissolvedin dilutedacetonitrile andfiltered. An aliquotwas injected on an HPLC column Synergi Hydro(Phenomenex, Torrance, CA) and analyzed by usingternary gradient elution (water/acetonitrile/acetic acid).The mass spectrometer operated in the positive MS/MSmode (single reaction monitoring) with monitoring of 2–4 transitions for each compound. The most intensiveion was used for quantification, the others to identityconfirmation. The quantification was made by usingmultilevel calibration graph with [ 2 H]labeled CKs asinternal standards. Cytokinin riboside phosphates weredetermined as corresponding ribosides following theirdephosphorylation by alkaline phosphatase.IAA was determined using two-dimensional HPLCas developed by Dobrev et al. (2005). The segmentcontaining IAA obtained in the first dimension wascollectedin the loop of the fluid processorandredirectedto the second HPLC dimension. IAAwas quantified usingfluorescence detection and external standardization.Detection limits of different CKs varied from 0.05 to0.1 pmol/sample. Results of the hormone analyses rep-resent the mean of four independent samples obtainedfrom two different experiments with two replicates each,and two HPLC MS/MS injections for each sample. Determination of CKX activity CKX from zucchini cotyledons [lyophilized samplesequivalent to ca. 5 g fresh weight (FW)] was extractedand partially purified using the method described byChatfield and Armstrong (1986) and modified by Motykaet al. (2003). The CKX activity was determined by  in vitro  assays based on the conversion of [2- 3 H]iP (prepared byDr Jan Hanusˇ; Isotope Laboratory, Institute of Experimen-tal Botany AS CR, Prague, Czech Republic) to [ 3 H]ade-nine. The assay mixture (50  m l final volume) included100 m M   MOPS-NaOH buffer containing 75  m M   2,6-dichloroindophenol (pH 7.0), 2  m M   substrate ([2- 3 H]iP,7.4 TBq mol 2 1 ) and enzyme preparation equivalent to175 mg tissue FW (corresponding to 0.28–0.36 mg pro-tein g 2 1 FW). After incubation for 1 h at 37  C, the reac-tion was terminated and the substrate was separatedfrom the product of the enzyme reaction by HPLC asdescribed elsewhere (Gaudinova´ et al. 2005). The sub-strate specificity of CKX was tested using tritiatedsubstrates [ 3 H]iP, [ 3 H] trans  -Z and [ 3 H] cis-  Z (2  m M   each)in the standard enzyme assay. Protein concentrations 612  Physiol. Plant. 134, 2008  were determined according to the method of Bradford(1976) using bovine serum albumin as a standard. Statistical analysis Statistics was performed using Student’s  t-  test. Results aremarked as statistically different when  P     0.05 (*), P     0.01 (**) or  P     0.001 (***). Results Changes in cotyledon anatomical structure andchloroplast ultrastructure The anatomical structure of control zucchini cotyledonswascharacterizedbytwowell-definedlayersofpalisademesophyll cells and spongy mesophyll with large in-tercellular spaces (Fig. 1A). In comparison with thecontrol cotyledons, darkness affected most strongly theanatomical structure of both individually darkenedcotyledons (Fig. 1B). The arrangement of the cells inthe palisade mesophyll layers was denser, the cell shapebecoming more elongated with strongly reduced inter-cellular spaces. Furthermore, the spongy mesophyll wascharacterized by shrinkage of the cell size and enlargedintercellular spaces.Theanatomicalstructure ofa singledarkened cotyledon was much less affected (Fig. 1C).In accordance with the deterioration in the anatomicalstructure, the number of chloroplasts per palisadecell was most strongly reduced in both darkenedcotyledons (by 47%) (Fig. 2). A smaller reduction wasobserved in a single darkened cotyledon (by 33%),whereas in the cotyledon that remained under lightregime, the chloroplast number was close to the controlvalue (Fig. 2).At the ultrastructural level, the control chloroplastswere characterized by ellipsoidal shape and well-organized internal membrane system arranged intogranal and stromal thylakoids (Fig. 3A, B). In contrast,the chloroplasts from both darkened cotyledons weremore rounded in shape, the thylakoid membrane systemwas strongly reduced (Fig. 3C, D) and a large part of thestroma was deprived of granal structures (Fig. 3D). Inaddition, invaginations of the chloroplast envelope intothe stroma were observed in some chloroplasts (Fig. 3C),suggesting that the observed reduction in the number of chloroplasts was because of phagocytosis-like destruc-tion. The ultrastructural changes in a single darkenedcotyledon comprised a transition in the shape fromellipsoid to spherical observed in some chloroplasts,disorientation of granal thylakoids with respect to oneanother and an increased number of plastoglobuli(Fig. 3E, F). The anatomical cotyledon structure as wellas the chloroplast ultrastructure in the cotyledon, whichremained under light regime, was similar to the control(Figs 1D, 3G). Photosynthetic function Chloroplast senescence was monitored by the declinein the photosynthetic activityand the functional activityof PSII. The decrease in the net photosynthetic rate wasstronger in both darkened cotyledons (48%) comparedwith a single darkened cotyledon (34%) (Fig. 4A).Similar to the changes in net photosynthetic rate, theactual quantum yield of PSII electron transport in thelight-adapted state ( F PSII) was reduced in both dark-ened cotyledons and a single darkened cotyledon by 13and 8%, respectively (Fig. 4B). In accordance withthese changes, the fraction of the absorbed light energy,whichisnotusedforphotochemistry(LNU)increasedinboth cases and particularly in the pair of darkenedcotyledons (about two-fold) (Fig. 4C). The net photo-synthetic rate as well as  F PSII in the cotyledon, whichremained under light regime, remained close to thecontrol values. Content of endogenous CKs CKs were grouped according to their structure andphysiological activity into five functionally differentgroups (Ananieva et al. 2004): (1) bioactive CK basesand ribosides [ trans  -zeatin ( trans  -Z),  trans  -zeatin 9-riboside ( trans  -ZR), N 6 -(2-isopentenyl)adenine (iP), N 6 -(2-isopentenyl)adenine 9-riboside (iPR), dihydrozeatin(DHZ), dihydrozeatin 9-riboside (DHZR)] which exhibithigh CK activity in different bioasays (Skoog and Ghani1981) and are recognized by CK receptors (Yonekura-Sakakibara et al. 2004, Spichal et al. 2004); (2) storage CK O  -glucosides[ trans  -zeatin O  -glucoside(ZOG), trans  -zeatin9-riboside  O  -glucoside (ZROG), dihydrozeatin  O  -gluco-side (DHZOG), dihydrozeatin 9-riboside  O  -glucoside(DHZROG)] (Mok 1994); (3) irreversibly inactive  N  -gluco-sides [ trans  -zeatin 7-glucoside (Z7G),  trans  -zeatin 9-glucoside (Z9G), dihydrozeatin 7-glucoside (DHZ7G),dihydrozeatin 9-glucoside (DHZ9G), N 6 -(2-isopentenyl)adenine7-glucoside(iP7G),N 6 -(2-isopentenyl)adenine9-glucoside (iP9G)] (Letham et al. I983); (4) CK ribosidephosphates [ trans  -zeatin 9-riboside-5 # -monophosphate(ZRMP), dihydrozeatin 9-riboside-5 # -monophosphate(DHZRMP), N 6 -(2-isopentenyl)adenine 9-riboside-5 # -monophosphate (iPRMP)] representing the first productsof CK biosynthesis which were determined after theenzymatic hydrolysis to the corresponding ribosides; and Physiol. Plant. 134, 2008  613
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