The developmental transition to flowering represses ascorbate peroxidase activity and induces...

Plant Science 158 (2000) 115–127

The developmental transition to flowering represses ascorbateperoxidase activity and induces enzymatic lipid peroxidation in

leaf tissue in Arabidopsis thaliana

Zhenzhen Ye, Roxana Rodriguez, Augustine Tran, Hoang Hoang,Darryn de los Santos, Shawn Brown, Robert Luis Vellanoweth*

Department of Chemistry and Biochemistry, California State Uni6ersity Los Angeles, Los Angeles, CA 90032-8202, USA

Received 7 February 2000; received in revised form 31 May 2000; accepted 3 June 2000

Abstract

Leaf senescence in many plant species is associated with increased oxidative damage to cellular macromolecules by reactiveoxygen species (ROS). Since ROS levels and their damage products in many plants are known to increase during senescence, itis possible that these changes are due to a decline in the levels of certain antioxidant enzymes. Using specific assays, we find thatthe developmental transition to bolting and flowering is associated with up to a 5-fold decline in ascorbate peroxidase activity andan increase in chloroplastid superoxide dismutase. As expected, these changes are associated with a measured increase in lipidperoxidation products. By HPLC separation of the products, we identified the different positional isomers and find thatstereospecific lipid peroxidation occurs after the bolting transition. The product distribution suggests that enzyme-mediated lipidperoxidation, via a lipoxygenase, is responsible for the observed increase. Surprisingly, though consistent with the knowninduction of antioxidant defenses by hydrogen peroxide, the activity of APX rebounds with further development (reproductionand seed setting) and this increase (up to 5-fold) is associated with declines in lipid peroxidation and with the onset of visiblesenescence symptoms. Thus, in Arabidopsis, ROS increases are associated with the developmental transition to flowering, perhapsdue to programmed declines in APX activity, and apparently lead to the oxidative activation of lipoxygenase and subsequent lipidperoxidation. The reactivation of APX at later stages appears to help reduce the lipid peroxidation rate, although the senescenceprogram continues unabated. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Ascorbate peroxidase; Lipid peroxidation; Reactive oxygen species; Senescence; Arabidopsis

www.elsevier.com/locate/plantsci

1. Introduction

During the final developmental phase in the lifeof an annual plant, reproduction and seed devel-opment are accompanied by senescence of theleaves. This genetically programmed aging event isan active process characterized by the degradationof leaf cell components. Metabolism in the leavesis drastically altered. During vegetative growth,the primary function of the leaf is to provide fixedcarbon to the rest of the plant through the process

of photosynthesis. However, as senescence pro-ceeds, leaf photosynthetic capacity is diminishedthrough a destruction of chlorophyll, a proteolyticattack on the abundant CO2-fixing enzyme ribu-lose bisphosphate-1, 5-carboxylase, and a declinein de novo synthesis of the photosynthetic appara-tus [1]. Protein and nucleic acid levels declinethrough enhanced proteolysis and nuclease attackand membrane integrity is lost. A critical role forreactive oxygen species (ROS) such as superoxideanion (O2

�−), hydrogen peroxide (H2O2), and hy-droxyl radical (HO�) throughout all stages ofsenescence has been implicated in many plants,with the primary target of damage being the mem-brane lipids [2]. Although apparently a destructive

* Corresponding author. Tel: +1-323-3432148; fax: +1-323-3436490.

E-mail address: [email protected] (R.L. Vellanoweth).

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 68 -9452 (00 )00316 -2

Z. Ye et al. / Plant Science 158 (2000) 115–127116

process, the onset of senescence requires the activetranscription and translation of new gene productsand is thus under direct genetic control. It isdefinitely not a simple case of down-regulation ofgenes that define a juvenile state, though suchrepression forms part of the process, but ratherprogression into this final life phase requires theexpression of new genes [3–5]. Some of thesesenescence-associated genes have begun to be elu-cidated, and the known products of the moreabundantly expressed genes are those expected:nucleases [6,7], proteases [3,4], and nitrogen reas-similation products [8], as well as some surprises[9]. The primary adaptive reason for the induceddegradation is to mobilize leaf nitrogen for trans-port to the developing seeds. With the induction ofthe senescence program, the leaf no longer pro-vides new fixed carbon to the rest of the plant andthus senescence involves a redistribution of re-sources from somatic tissue to the developingprogeny, a perfect example of parental altruism.

Several models have been put forth to explainthe highly ordered and controlled nature of leafsenescence. In many leguminous plants, the agingprogram appears to be controlled by the develop-ing seeds [10]. Removal of floral structures extendsthe longevity of the leaves, suggesting the involve-ment of a diffusible substance that initiates leafsenescence. The nature of this diffusible regulatoris still unknown. Plant-specific hormones also ap-pear to institute senescent phenotypes in manysystems. Since most studies using plant hormonesinvolve their exogenous addition to various or-gans, attached or detached, in many instancestheir endogenous role remains unclear. A commonmolecular theme in plant senescence is evident inmore downstream events, however. In most spe-cies examined, ROS and their damage productsincrease during leaf aging. Thompson has sug-gested that O2

−� levels rise in senescing plant tis-sues due to an increase in lipoxygenase activity,placing ROS production and damage at the samelocation, cellular membranes [2]. Others have sug-gested, both in plants [11] and in animals [12] thatage-related ROS increases result from leaks in thenormal electron transfer reactions of metabolicpathways. Whatever their source, ROS are inti-mately associated with the degradative aspects ofsenescence physiology in all plants that have beenstudied.

In this report, we test the hypothesis that age-dependent increases in ROS are the result of aprogrammed downregulation of antioxidant en-zymes in Arabidopsis. Given the increasingly im-portant role of H2O2 as a signaling molecule, bothin plant and animal systems, we decided to focuson those enzymes that can modulate H2O2 levels.We show that the transition from vegetativegrowth to reproductive growth (bolting) is relatedto the longevity of rosette leaves as measured bychlorophyll level and that this transition is associ-ated with ascorbate peroxidase (APX) decline andlipoxygenase (LOX) activation.

2. Materials and methods

2.1. Plant material and growth conditions

Arabidopsis thaliana (Columbia) seeds (LehleSeeds, Inc) were sown in 2:1:1 pottingsoil:perlite:vermiculite following a 5-day imbibedincubation in 0.1% agarose at 4°C. Nearly syn-chronous germination was encouraged by a 3-dayincubation of the pots at 4°C before transfer togrowth chambers. The plants were irrigatedweekly with a nutrient solution [13] and kept in agrowth chamber or environmental room at 23°Cunder a 12 h photoperiod with a light fluence of250 mmole/m2 per s. Plants were evenly spacedevery 4 cm to encourage similar growth rates andsizes.

2.2. Extract preparations and enzyme acti6ityassays

Extracts were prepared by freezing leaf tissue inliquid N2 and pulverizing to a fine powder beforehomogenization in with a Tissue Tearor in abuffer solution [14]. The homogenate was cen-trifuged at 14 000 rpm for 20 min at 4°C and thesupernatant collected and stored at −80°C untilfurther analysis. Protein concentration was deter-mined spectrophotometrically by the Bradforddye-binding assay using bovine serum albumin asstandard [15]. Protein extracts (equal amounts perlane) were subjected to Laemmli discontinuouspolyacrylamide gel electrophoresis under nondena-turing, nonreducing conditions. Gels for superox-ide dismutase (SOD) and ascorbate peroxidase(APX) were run for 4 h at 4°C in a potassium

Z. Ye et al. / Plant Science 158 (2000) 115–127 117

phosphate buffer (pH 7.0) with a constant currentof 23 mA per gel. SOD activity was determined bythe negative staining procedure using nitrobluetetrazolium. For SOD isozyme identification, thegels were incubated prior to activity staining in 5mM H2O2 or 2 mM KCN. Of the three isozymesthat may be present, the Mn form is insensitive toeither treatment, while the CuZn isozyme is sensi-tive to CN−. The Fe isozyme is sensitive to H2O2

[16]. APX activity was determined as described [14].This assay is based on the ability of ascorbate toreduce NBT, in the presence of tetramethylethylene-diamine to formazan. Ascorbate peroxidase pre-vents the formation of formazan in the presence ofH2O2, and thus the ascorbate peroxidase activity isseen as an achromatic band on a purple-bluebackground. Activities were quantified usingMolecular Analyst software (BioRad, Inc.) on dig-ital images recovered from a CCD camera drivenGelDoc 1000 system. The linear range of activitywas determined by loading increasing amounts ofcell extract on the gels and comparing the pixeldensity of the bands with the amounts loaded.

2.3. Lipid peroxide and chlorophyll measurements

Lipids were extracted from growing plant rosetteleaves at various stages following Bligh and Dyer’smethod [17]. Lipid peroxides were quantified by theFOX method [18] using the PeroXoquant™ Quan-titative Peroxide Assay from Pierce Chemical Co.Samples (50 ml) were incubated with 500 ml ofworking reagent which consists of 1 volume of 25mM of Fe2+ and 2.5 M H2SO4, and 100 volumesof 4 mM BHT and 125 mM xylenol orange inmethanol. After a 20 min incubation at roomtemperature, the absorbances at 560 nm were readon an HP 8452A diode array spectrophotometer.Freshly prepared hydrogen peroxide solutions wereused as standard.

A portion of total lipid extracts was used forchlorophyll determination. The lipid extracts werediluted with N,N-dimethylformamide (DMF). Ab-sorbances at 648 and 664 nm were recorded usingDMF as blank. The total chlorophyll content wascalculated as described [19].

2.4. Lipid peroxide characterization

Standards were prepared by autoxidation oflinoleic and linolenic acids and subsequent reduc-

tion to hydroxyl forms. In 10 ml of 0.2 mMtBuOOH in 0.5% ethanol, linoleic or linolenic acidwas dissolved at 4 mM final concentration. Theautoxidation was initiated by adding FeSO4 to 0.2mM. The reaction proceeded for 10 min at roomtemperature. Reduction was fulfilled by introducinga molar excess of NaBH4 in 5 N NaOH. Thereaction mixture was acidified to pH 4 with 5 N HCland extracted by 1 ml hexane: diethyl ether (70:30,v/v). After centrifugation, the upper layer wascollected and used for HPLC injection.

Hydroperoxy fatty acids were isolated and re-duced to their corresponding hydroxyl forms ac-cording to Degousee et al. [20]. Briefly, fatty acidsfrom Arabidopsis were prepared by homogeniza-tion of 0.5 g of rosette leaves in 1 ml 5%(w/v)NaBH4 in 0.2 N NaOH. After acidifying the ho-mogenate to pH 4 with 70% HClO4, 2.2 ml ofCHCl3: CH3OH (50:50, v/v) were used to separateaqueous and organic phase. The lower phase wascollected after centrifugation for 5 min at 11 000rpm 4°C. The upper phase was further extracted by1.1 ml chloroform. All lower phases were combinedfollowing a 5 min 11 000 rpm 4°C centrifugation.Solvent was removed and the residue dissolved in0.5 ml of absolute ethanol and 0.5 ml of 3.5 NNaOH. Following a 15 min reflux and cooling, 0.5ml 3.5 N HClO4 was used to acidify sample to pH4. Phases were then separated with 0.3 ml hexane:diethyl ether (70:30, v/v). The upper layer wascollected after a 10 min 10 000 rpm centrifugationat 4°C. After filtering through a 0.45mm NY PPfilter, the sample was used for HPLC analysis.

Isolated leaf fatty acids were characterized byanalytical HPLC using a Rainin Dynamax instru-ment equipped with a 4.6×250 mm Microsorb-MVcolumn packed with 5 mm silica gel. Mobile phasesolvent consisted of hexane: diethyl ether: aceticacid (70:30:0.5, v/v/v). Hydroxyl fatty acidstereoisomers were separated by isocratic elution ata flow rate of 0.5 ml/min. Absorbance at 234 nmwas monitored with a photodiode array PDA-2detector.

3. Results

3.1. Antioxidant enzyme acti6ities during theArabidopsis life span

Numerous studies in several species have demon-strated a role for reactive oxygen species in


the progress of leaf senescence [2]. Their buildup ispurported to lead to membrane leakage and

organellar disintegration during late stages ofsenescence. It is possible that a programmeddownregulation of antioxidant capacity mightcontribute to the observed ROS increase. Wefocused our studies first on enzyme activity, asopposed to mRNA levels, since it is at this levelthat changes would affect ROS metabolism. Wethus prepared enzyme extracts from Arabidopsisleaves at each important stage in the plant’s lifehistory and measured the activities of twoantioxidant enzymes. The stages includedpre-bolting, i.e. before the transition from thevegetative (juvenile) phase to the reproductivestage, short bolt (B1 cm), medium bolt (\4 cm),long bolt (\10 cm), flowering, seeding, andsenescing (with rosette leaves at least 25% yellow).Equal amounts of protein isolated from thevarious stages were loaded onto a native PAGEgel and stained for SOD activity. An invertedimage of one such gel is shown in Fig. 1. Slightvariations in Mn-SOD activity as the life spanprogressed were evident in this example, whileFe-SOD consistently displayed an increase at themid-bolt stage followed by a return to pre-boltlevels prior to senescence. The situation was evenmore dramatic for ascorbate peroxidase (APX).Fig. 2a shows the results of a similar experimentfor APX activity. The major band for APXactivity declined precipitously as the plants enteredthe reproductive phase of life. Thus, from pre-bolt(39 days after planting) to short bolt (43 dap)APX levels declined more than fivefold. Thedecline was only transient, however, as the APXlevel rebounded to pre-bolt levels at the long boltstage (50 dap). APX levels were then found tovary only slightly as the plants continued their lifespan through senescence. Data from six separateexperiments were compiled and are shown in Fig.2b. The APX activity at each stage is reported asthe six-experiment average of the % of the highestactivity measured within any series. The standarddeviation of the data is shown as error bars in thefigure. The transition to bolting at the meristemwas associated with an average two to threefolddecrease in leaf APX activity. A very carefulselection of plants at identical developmentalstages showed that this decline can reach fivefold(Fig. 2). In all cases, the APX level subsequentlyincreased to the highest levels measured as thebolts reached lengths of 10 cm. This dip andsubsequent rebound of APX activity occurred

Fig. 1. Leaf superoxide dismutase activity as a function of lifehistory stage. Inverted image of a native PAGE gel stainedfor SOD activity. Extracts were prepared from plants of theindicated age in days and the following stages: PB, pre-bolt;SB, short bolt; MB, medium bolt; LB, long bolt; FS, flower-ing and seeding; and SN, senescing. The positions of migra-tion of Mn- and Fe-SOD are indicated.

Fig. 2. Leaf ascorbate peroxidase activity as a function of lifehistory stage. (a) Inverted image of a native PAGE gel stainedfor APX activity. Extracts were prepared from plants of theindicated age and stage. The arrow denotes an invariantlystained activity. (b) The chart depicts a compilation of APXactivity from six independent determinations. The data areexpressed as the average percent of maximum activity for agiven generation. The error bars indicate the standard devia-tion in the data.


Fig. 3. Leaf lipid peroxide level as a function of life history stage. Lipid peroxides (columns) were measured using the FOX assayand are expressed as mg of ROOH per mg of leaf tissue. The curve indicates the level of chlorophyll at the indicated stages. SN1,leaf was approximately 20% yellow; SN2, 40% yellow; SN3, 60% yellow; SN4 80% yellow; and SN5, 100% yellow.

within a span of 10 days, or roughly one-eighththe total life span. As the plants entered senes-cence, only a slight decrease in APX was mea-sured. Thus, in Arabidopsis it appears that APXactivity does decline in an apparently programmedmanner, but its timing precedes considerably theonset of visible symptoms of senescence.

3.2. Leaf lipid peroxide le6els as a function of lifespan

The primary purpose of leaf APX is to scavengehydrogen peroxide before it can react with cellularbiomolecules and cause damage. Our observationsthat leaf APX declines as Arabidopsis plants enterthe reproductive phase suggests that H2O2 levelsmight increase during the 10-day period of lowactivity and lead to biomolecular damage, a hall-mark of senescence in virtually all species [21]. Totest this possibility, we measured the levels ofwhole-cell lipid peroxides at the same stages usedabove. Total lipid was extracted and lipid perox-ides were determined using the ferrous oxidation-xylenol orange (FOX) method [18]. This assaymethod was used due to its higher specificity to-wards lipid peroxides than the TBARS test. Foreach stage, only the 5th and 6th leaves to emergefrom the rosette were used for extract prepara-tions. Twenty samples were collected for each

stage in order to study the deviation in the data.The data in Fig. 3 show that lipid peroxides beganto increase at the bolting transition, as expectedfrom the observed decline in APX, and reached amaximum twofold higher level by the floweringstage. As the plants entered senescence though,indicated by the decline in leaf chlorophyll at 64dap, lipid peroxide levels also declined. By thefinal stage of senescence (83 dap, leaves completelyyellow but not dry), lipid peroxides were evenlower than at the young, pre-bolt stage. Since thedata are reported as mg lipid peroxide per mg ofleaf tissue, the level of lipid peroxides on a per cellbasis would be even lower since leaf mass haddeclined due to senescence processes. The APXdecline was therefore associated with an increasein lipid peroxidation during the 10-day period oflow APX. Lipid peroxides remained elevated,however, for a further 10–15 days, suggesting thatsome mechanism other than simple chemical at-tack of H2O2 on membrane lipid double bondswas responsible for the elevated lipid peroxidelevels.

3.3. Isomeric composition of leaf lipid peroxides

The reaction of H2O2 with plant membranelipids would be expected to generate a multitudeof products which vary both in the position of the


added peroxy group on the fatty acid chain and inthe number of double bonds and chain lengthreflecting the natural diversity of cellular lipids.The major lipid fraction in Arabidopsis leaves iscomposed of linoleic and linolenic acids, however[22]. Thus, a determination of the peroxy positionin cellular lipids is manageable by HPLC isomericcomposition analysis. A set of standards was firstprepared by autoxidation and enzymatic genera-tion of lipid hydroperoxides to identify the peakson the chromatogram. Fig. 4a and b show resultsfrom t-butly hydroperoxide-catalyzed autoxida-tion of linoleic and linolenic acid, respectively,

while a mixture of the two is shown in Fig. 4. Theconjugated diene of the reduced products wasfollowed by A234. Compared with published work[20,23] under similar HPLC conditions, the peakswere identified and are listed in the Fig. 4 legend.The amounts of the positional isomers were notequal. Thus, 13-HODE and 9-HODE representedabout 85% of the four identified isomers fromlinoleic acid while 16-HOTE and 9-HOTE ac-counted for about 75% of the four isomers fromlinolenic acid. Other peaks that eluted prior to 12min were not identified but were also present inthe pre-autoxidation material. Standards were alsoprepared from enzymatic peroxidation of linoleicacid using soybean lipoxygenase (LOX) and sub-jected to HPLC separation. Unlike autoxidation,13-HODE was the predominant product withmore than 90% of the total as shown in Fig. 5.Next, lipid peroxides from different-aged Ara-bidopsis leaves were prepared and analyzed byHPLC following reduction to their correspondinghydroxyl forms. The chromatographic profile (Fig.6) between 15 and 25 min showed considerablyless complexity in the leaf-derived sample than theautoxidation standards (Fig. 4a and b) and wasrather similar to the enzymatically generated stan-dard (Fig. 5). In the natural sample, the secondpeak, 13-HOTE, showed the greatest increase inamount following the transition from a pre-boltedstage to the reproductive phase. The level of 13-HOTE slowly began to decrease as the plantsentered senescence, paralleling the kinetics of lipidperoxide changes found by the FOX assay (Fig.3). In addition to the major increase in 13-HOTE,smaller increases were observed for 13-HODE,13-HODE trans, 16-HOTE, and 9-HODE. Theseisomers followed the same kinetics as 13-HOTEand thus contributed to the overall pattern of lipidperoxidation changes during the life span.

3.4. Leaf longe6ity and flowering

We have made casual observations in our labthat the timing of the developmental transition tothe reproductive state was associated with thetiming of visible senescence symptoms in leaves.We have observed this phenomenon in the Colum-bia ecotype, whereas others have reported thatlate-flowering mutants exhibit wild-type leaf senes-cence timing in the Landsberg ecotype, suggestinga lack of association between flowering and

Fig. 4. HPLC separation of reduced hydroperoxy fatty acidstandards generated by autoxidation of (a) linoleic and (b)linolenic acid. A mixture of the two is shown in panel c. Theisomers indicated by letters are: a, 13-HODE, 13-hydroxy-9,11(Z,E)octadecadienoic acid; b, 13-HOTE, 13-hydroxy-9,11,15(Z,E,Z)octadecatrienoic acid; c, 12-HOTE,12-hydroxy-9,13,15(Z,E,Z)octadecatrienoic acid; d, 13-HODEtrans, 13-hydroxy-9,11(E,E)octadecadienoic acid; e, 9-HODE,9-hydroxy-10,12(Z,E)octadecadienoic acid; f, 16-HOTE, 16-hydroxy-9,12,14(Z,Z,E)octadecatrienoic acid; g, 9-HOTE, 9-hydroxy-10,12,15(E,Z,Z)octadecatrienoic acid; and h,9-HODE tran, 9-hydroxy-10,12(E,E)octadecadienoic acid.


Fig. 5. HPLC separation of products generated by incubation of linoleic acid with soybean lipoxygenase and reduced to theirhydroxy derivatives. The products indicated by letter are: a, 13-HODE; and e, 9-HODE.

4. Discussion

In the present investigation of antioxidant de-fense enzyme activities during the Arabidopsis lifespan, we have evidence for a developmentallyregulated depression of leaf APX activity as plantsenter the reproductive state (Fig. 2). The resultsshow that APX activity is dramatically decreasedin activity just after bolting. At least seven APXisozymes are known in Arabidopsis [25]. Becauseof our extraction method, we expect that the oneband we detect on the APX activity gel is at leastone of the cytoplasmic types, of which there aretwo. The other forms, two chloroplastic and threemembrane bound, would be expected to be foundin the membrane fraction, although we cannot ruleout the possibility that the chloroplast forms con-tribute to the observed band. The mitochondriallylocalized Mn-SOD, exhibits only minor changes inactivity throughout the life span, displaying a con-sistent slight increase at later stages. The chloro-plastid Fe-SOD, on the other hand, increasesseveral-fold at the mid-bolt stage and declinesprior to senescence. Interestingly, although cer-tainly expected considering the role of H2O2 in the

longevity [3]. We therefore decided to test therelationship directly by physically removing boltsfrom Columbia wild-type plants and comparingthe longevity of their leaves to those of plants leftto flower and set seed normally. We followedsenescence by measuring chlorophyll content inthe 5th and 6th leaves. Data for the 5th leaf areshown in Fig. 7. This experiment was repeated 4times with similar results for both leaves. Theresults show that plants physically prevented fromflowering delay senescence considerably, inagreement with experiments of the same typeconducted in many other plant species [10,24]. Wehave also found that allowing the bolts to re-growinitiated senescence almost immediately (data notshown). It is noteworthy that we could notdemonstrate a flowering-senescence coupling whendark-induced senescence occurred in thelate-flowering state. We found that copiousvegetative growth, complete with the appearanceof many new leaves, occurs in delayed-floweringplants and the resultant shading of leaves 5 and 6causes the chlorophyll loss. If shading by newgrowth is minimized, then the results that wereport here were consistently observed.


induction of antioxidant defense genes [26], theactivity of APX rebounds with further develop-ment (reproduction and seed setting) and this in-crease (up to fivefold) precedes the onset of visiblesenescence symptoms. The mechanism of APXdecline is unknown, but considering that APXmRNA levels are known to be induced by oxida-tive stress, a transcriptional mode is likely. Wecarried out an add-back experiment looking for

evidence of an APX inhibitor in the short boltextracts, but found none (data not shown). Ofcourse, many assumptions are made in such anexperiment and a negative result does not rule outthe possibility of a specific APX inhibitor. Anaction directly on the protein is possible, though,as at least two reports have suggested that APXactivity can be altered without affecting APXmRNA levels [27,28] and the APX mRNA is

Fig. 6. HPLC separation of leaf fatty acids isolated from Arabidopsis plants at the indicated stage. The indicated products areas listed in Fig. 4 legend.


Fig. 7. Leaf chlorophyll level versus days after 5th leafemergence. Bolting occurred at 15 days. Chlorophyll wasmeasured spectrophotometrically following DMF extractionof leaf material. Control plants were allowed to flower and setseed normally while ‘Cut’ plants had their bolts continuouslyremoved as they appeared. The standard deviation of the datawas less than 10%.

tosolic APX levels remain fairly high, perhapskeeping H2O2 levels in check. The situation maydiffer in the mitochondria, however, as it wasrecently reported that mitochondrial APX levelsdecline during pea leaf senescence [30]. In ourstudy, the maximum lipid peroxidation rate isachieved by the long bolt stage when flowers haveformed and seeds are setting. This stage is alsowhen APX activity has returned to a level similarto that of the pre-bolt stage. For at least six moredays, total lipid peroxide is elevated before itbegins to fall. The delay suggests that H2O2, whichpresumably goes unscavenged during the APXactivity dip, is not the sole source of lipid peroxi-dation. The complete characterization of the lipidperoxides by HPLC (Fig. 6) revealed that thepredominant product during this period is 13-HOTE, not the racemic mixture of all isomersexpected from random, nonspecific chemical at-tack by H2O2. This suggests that a specific enzyme,a 13-LOX specific for linolenic acid, is activated atthe same time that APX levels decline and cata-lyzes the peroxidation observed. Thus, in Ara-bidopsis rosette leaves, ROS increases areassociated with the developmental transition toflowering, due to programmed declines in APXactivity, and may lead to the oxidative activationof a linolenic acid-specific 13-LOX and subsequentlipid peroxidation. The reactivation of APX atlater stages coincides with a reduced lipid peroxi-dation rate, although the senescence program con-tinues unabated.

A fundamental feature of plant lipoxygenases istheir conversion from an inactive form, where thenon-heme Fe center is in the high-spin ferrousstate, to an active form, where the Fe is oxidizedto the ferric state. This Fe oxidation can occur byreaction with H2O2 or organic peroxides [31,32]. Itthus seems plausible that the decline in APX al-lows an increase in H2O2 concentration that acti-vates a pre-existing LOX to attackmembrane-resident fatty acids, generating the ob-served increase in 13-HOTE. We quantified allhydroperoxy isomers over three generations ofplants and considered 13-HODE and 13-HOTE tobe enzymatically derived (although some smallfraction is undoubtedly of non-enzymatic origin)and all other isomers to be non-enzymaticallyderived. As a function of life history stage, theratio of enzymatic to non-enzymatic lipid peroxi-dation changed considerably. In the pre-bolt stage,

somehow prevented from being translated duringthe hypersensitive response [29].

Since the substrate for APX is H2O2, we ex-pected that peroxidation reactions would increasefrom substrate buildup during this transitory, lowAPX period. This assumes, of course, a limitedrole for catalase in controlling H2O2 levels in thecytoplasm. Most of the catalase activity is local-ized to glyoxysomes and peroxisomes, while APXis the major H2O2 scavenger in the cytosol andchloroplast. Given the importance of the chloro-plast in senescence physiology and the importanceof the cytosol as the medium through which sig-nals are transmitted to the nucleus, we decided tofocus our studies first on APX. We have measuredcatalase activity throughout the Arabidopsis lifespan. In Arabidopsis the catalase gene family iscomprised of three genes that encode up to sixdifferent isozymes. In our hands, three isozymes ofcatalase are evident and there does appear to besome temporal regulation of two of these; theyincrease slightly as plants enter senescence. How-ever, in comparison to the large drop in APXactivity, the changes in catalase activity are quitesmall. We thus measured lipid peroxidation, ahallmark of senescence in many other plants [2],and found that it increases with a kinetics thatcorresponded to the APX decline. Interestingly,while senescence in other plants involves consider-able ROS production and oxidative damage, inArabidopsis we find that lipid damage productsdecrease steadily throughout leaf senescence. Asthese plants undergo the senescence program, cy-


the ratio was approximately one. However, justafter the bolting transition, the ratio increased to4–6. As development proceeded, the ratio declinedto 2–3. The even distribution of isomers in thepre-bolt stage suggests that a substantial propor-tion of lipid peroxidation during vegetative growthis from random chemical attack. LOX-catalyzedperoxidation predominates after the bolting transi-tion and remains the major source of peroxidesthroughout the subsequent life of the plant. Thisregulated biochemical event may initiate the syn-thesis of a lipid derived secondary messenger. Jas-monic acid is one such wound-response messengerthat is purported to be involved in senescence andits synthesis via the octadecanoate pathway iscommenced with 13-lipoxygenase, apparently thesame that is activated in response to bolting [33].Other LOX-derived volatiles have also beendemonstrated, including a number of six-carboncompounds that appear to mediate a wound re-sponse [34]. We do not believe that the peroxida-tion observed in this study is a simple, artifactualwound response resulting from the preparation ofthe extracts. The leaves were immediately trans-ferred to liquid N2 after harvesting, pulverized,and extracted with organic solvents. Thus anywound response would be terminated or preventedfrom initiating. In addition, all extracts were pre-pared in exactly the same manner, but peroxida-tion was only observed at those stages where APXactivity had declined. Thus, it appears from thepresence of 13-HOTE that a LOX enzyme is acti-vated in a developmental manner, coincident withthe programmed APX inactivation, and leads tolipid peroxidation as the plant enters the reproduc-tive phase of life.

Like most organisms, the controlling factorsdetermining longevity in Arabidopsis are un-known. A number of studies suggest that theprimary regulator of Arabidopsis leaf senescence issomething other than ethylene or seed develop-ment. The senescence-promoting hormone ethyl-ene does not appear to be the dominant factor inleaf longevity since an analysis of ethylene-insensi-tive mutants reveals that senescence still proceeds,though slightly delayed [35–37]. While late-flower-ing mutants have been suggested to exhibit in-creased longevity of rosette leaves, Hensel et al. [3]have reported that in Landsberg erecta this issimply due to an increased number of leaves insuch mutants and that the longevity of individual

leaves is identical to the wild type plant. In thesame study, seed development was ruled out as themajor determinant in leaf senescence since malesterile mutants exhibited leaf longevity identical towild type plants. We have found that the longevityof the 5th and 6th leaves can be compromised bydark-induced senescence due to the growth of newrosette leaves over their surfaces, especially underconditions known to increase leaf number such asshort days. Our current study using the Columbiaecotype (Fig. 7), suggests that the transition toreproductive growth may indeed initiate senes-cence in Arabidopsis, as in other monocarpicplants. Removing the possibility for dark-inducedsenescence, we found that 5th and 6th leaflongevity was increased in plants whose floralmeristems were removed compared to uncut con-trols, suggesting that Arabidopsis is no differentfrom other plant species in displaying a causalrelationship between flowering and senescence.Nooden et al. [38] also investigated the possiblerelation of reproductive development to thelongevity of Arabidopsis leaves. Their comparisonwas to the rate of silique growth and they foundno relationship to leaf longevity. In light of ourpresent results, it is possible that the causal factorfor leaf senescence comes not from later reproduc-tive structures, such as siliques, but rather fromthe early inflorescence meristem. By physically re-moving this early meristem, we find that thelongevity of individual leaves is extended. Onepossible explanation for this result is that withreproductive structures eliminated, we have re-moved the sink in the seed-leaf sink-source pair.Leaf senescence might be expected to cease withnowhere for the degraded leaf nutrients to go. Itcan be noted from Fig. 7 that chlorophyll doesdrop even in cut plants before stabilizing at a levelhigher than uncut controls. However, if the earlyleaves are covered by later vegetative growth, wedo not find a difference in leaf longevity betweencontrol and cut plants. This suggests that completesenescence is possible even without developingseeds. Thus, as comparative studies of aging mech-anisms in numerous disparate organisms have re-vealed [21], there may be a number of differentways to initiate the aging phenotype. Even in plantleaves, we know that senescent events can bepromoted by ethylene, abscissic acid, ozone, anddarkness, besides natural senescence brought on


by time and development. As previously suggestedby others [3,36], an endogenous, age-related signalappears to initiate the natural senescence programin Arabidopsis leaves. Our results in this paperfurther suggest that a particular developmentalstage, the floral transition, contributes to senes-cence initiation. Considering the involvement ofROS in the generation of senescence physiology inmany other species, it is possible that the increasein ROS, caused by the bolting transition-induceddepression of APX activity, serves as the develop-mental signal to initiate the expression of thesenescence program. It is interesting to note thatthe late-flowering Arabidopsis mutant Giganteaexhibits both increased leaf longevity and in-creased tolerance to oxidative stressors [39], dueapparently to elevated levels of antioxidant en-zymes. The increased antioxidant capacity of thismutant may interfere with the programmed de-pression of APX activity we find with wild-typeplants.

Precedence for ROS control of gene expressionis found in the plant hypersensitive response (HR)to pathogenic attack. An incompatible interactionof an avirulent pathogen (bacterial or fungal) witha resistant host plant involves a highly localizednecrosis of the infected tissue. An elicitor moleculeproduced as a result of the infection is thought toinitiate a G protein-coupled signal transductionpathway that activates a plasma membrane-local-ized oxidase generating O2

�− and H2O2 in anNADPH-dependent reaction [40–42]. An addi-

tional contributor to the elevated H2O2 appears tobe a programmed decrease in cytosolic APXprotein in response to pathogen infection, as re-ported in tobacco [29]. This result was furthersubstantiated in experiments where APX activitywas decreased using antisense RNA; such plantsare hypersensitive to pathogen infection [43].Thus, programmed declines in APX activity, as wereport in this study, may constitute a generalmechanism for ensuring the production of H2O2.It is thought that the locally high levels of ROSproduced during the HR catalyze a strengtheningof the cell wall of the infected cells by formingcrosslinks, apparently to prevent the spread of thepathogen. High local concentrations of H2O2 alsoinitiate the programmed cell death pathway thatrequires the expression of new genes for the de-struction of the affected cells [42]. In adjacentcells, the levels of ROS are lower and thus do notreach the threshold level for programmed celldeath. However, the lower levels are sufficient toactivate the expression of a battery of genes, in-cluding superoxide dismutase (SOD) and glu-tathione S-transferase, that appear to provideprotection from the ROS [26].

From our results and the above discussion, wepropose the following model for the coupling offloral initiation and senescence (Fig. 8). The transi-tion of the vegetative meristem to a reproductivemeristem is a consequence of an unknown plantage-dependent, photoperiod signal [44]. Either thatsame signal, or a different diffusible substancefrom the floral meristem, results in a downregula-tion of APX activity. Loss of APX would cause anaccumulation of H2O2, which may be exacerbatedby the observed increase in Fe-SOD shortly there-after. H2O2 is known to activate LOX in vitro, andan in vivo activation may lead to the observedaccumulation of 13-HOTE. This may represent thefirst step in the synthesis of a lipid-derived secondmessenger that leads to the activation of the earlysenescence-associated genes (SAGs). SubsequentSAGs (middle and late) would be activated in ahierarchical manner via transcription factor mem-bers of the early SAG classes. Thus, ROS need notbe present throughout all stages leading to senes-cence but only as early players in the initiation ofthe gene expression cascade leading to the eventualregulated dismantling of the leaves duringsenescence.

Fig. 8. Model for bolting-transition induced alterations inAPX activity and LOX-mediated lipid peroxidation leading tosenescence.


Acknowledgements

This work was supported by grants from theNational Science Foundation (DBI-9710796 andHRB-9628725) and from the National Institutes ofHealth Minority Biomedical Research SupportProgram (GM-08101-25S3).

References

[1] S. Gepstein, Photosynthesis, in: L.D. Nooden, A.C.Leopold (Eds.), Senescence and Aging in Plants, Aca-demic Press, 1988, pp. 85–109.

[2] J.E. Thompson, The molecular basis for mebrane deteri-oration during senescence, in: L.D. Nooden, A.C.Leopold (Eds.), Senescence and Aging in Plants, Aca-demic Press, 1988, pp. 51–83.

[3] L.L. H. ensel, V. Grbic, D.A. Baumgarten, A.B.Bleecker, Developmental and age-related processes thatinfluence the longevity and senescence of photosynthetictissues in Arabidopsis, Plant cell 5 (1993) 553–564.

[4] K.N. Lohman, S. Gan, M.C. John, R.M. Amasino,Molecular analysis of natural leaf senescence in Ara-bidopsis thaliana, Physiol. Plant 92 (1994) 322–328.

[5] L.M. Weaver, S. Gan, B. Quirino, R.M. Amasino, Acomparison of the expression patterns of several senes-cence-associated genes in response to stress and hor-mone treatment, Plant Mol. Biol. 37 (1998) 455–469.

[6] A. Blank, T.A. McKeon, Expression of three RNaseactivities during natural and dark-induced senescence ofwheat leaves, Plant Physiol. 97 (1991) 1409–1413.

[7] C.B. Taylor, P.A. Bariola, S.B. Delcardayre, R.T.Raines, P.J. Green, RNS2-a senescence-associatedRNase or Arabidopsis that diverged from the S–RNasesbefore speciation, Proc. Natl. Acad. Sci. USA 90 (1993)5119–5122.

[8] K. Kamachi, T. Yamaya, T. Hayakawa, T. Mae, K.Ojima, Changes in cytosolic glutamine synthetase and itsmessanger RNA in a leaf blade of rice plants duringnatural senescence, Plant Physiol. 98 (1992) 1323–1329.

[9] E. Himelblau, H. Mira, S.J. Lin, V.C. Culotta, L. Penar-rubia, R.M. Amasino, Identification of a functionalhomolog of the yeast copper homeostasis gene ATX1from Arabidopsis, Plant Physiol. 117 (1998) 1227–1234.

[10] A.C. Leopold, Senescence in plant development, Science134 (1961) 1727–1732.

[11] P.R. Rich, W.B. Bonner, The sites of superoxide aniongeneration inhigher plant mitochondria, Arch. Biochem.Biophys. 188 (1978) 206–213.

[12] B.N. Ames, M.K. Shinenaga, T.M. Hagen, Oxidants,antioxidants, and the degenerative diseases of aging,Proc. Natl. Acad. Sci. USA 90 (1993) 7915–7922.

[13] L.J. DeKok, M. Graham, Levels of pigments, solubleproteins, amino acids and sulfhydryl compounds in fo-liar tissue of Arabidopsis thaliana during dark-inducedand natural senescence, Plant Physiol. Biochem. 27(1989) 203–209.

[14] M.V. Rao, B.A. Hale, D.P. Ormrod, Amelioriation ofozone-induced oxidative damage in wheat plants grownunder high carbon dioxide, Plant Physiol. 109 (1995)421–432.

[15] M.M. Bradford, A rapid and sensitive method for thequatitation of microgram quantities of protein utilizingthe principle of protein-dye bunding, Anal. Biochem. 72(1976) 248–254.

[16] K. Asada, K. Yoshikawa, M. Takahashi, Y. Maeda, K.Enmanji, Superoxide dismutases from blue-green alga,Plectonema boryanum, J. Biol. Chem. 250 (1975) 2801–2807.

[17] E.G. Bligh, W.J. Dyer, A rapid method of total lipidextraction and purification, Can. J. Biochem. Physiol. 37(1959) 911–917.

[18] Z.Y. Jiang, A.C. Woodlard, S.P. Wolff, Lipid hydroper-oxide measurement by oxidation of Fe2+ in the pres-ence of xylenol orange. Comparison with the TBA assayandan iodometric method, Lipids 26 (1991) 853–856.

[19] J.F. Wintermans, A.D. Mots, Spectrophotmetric charac-teristicsof chlorophylls a and b and their pheophytins inethanol, Biochem. Biophys. Acta 109 (1965) 448–453.

[20] N. Degousee, C. Triantaphylides, S. Starek, G. Iacazio,D. Martini, C. Bladier, R. Voisine, J.-L. Montillet,Measurement of thermally produced volatile alkanes: anassay for plant hydroperoxy fatty acid evaluation, Anal.Biochem. 224 (1995) 524–531.

[21] C.E. Finch, Longevity, senescence and the genome. TheUniversity of Chicago Press: Chicago, (1990).

[22] J. Browse, C.R. Somerville, Glycerolipids, in: E.M.Meyerowitz, C.R. Somerville (Eds.), Arabidopsis, 1994,pp. 881–912.

[23] H.W. Chan, G. Levett, Autoxidation of methyl-linoleate. Separationand analysis of isomerice mixturesof methyl-linoleate-hydroperoxidases and methyl hy-droxylinoleates, Lipids 12 (1977) 99–104.

[24] L.D. Nooden, A.C. Leopold, Senescence and aging inplants. Academic Press Inc., San Diego (1988).

[25] H.M. Jespersen, I.V. Kjaersgard, L. Ostergaard, K.G.Welinder, From sequence analysis of three novel ascor-bate peroxidases from Arabidopsis thaliana to structure,function and evolution of seven types of ascorbate per-oxidase, Biochem. J. 326 (1997) 305–310.

[26] I. Crute, J. Benyon, J. Dangl, E. Holub, B. Mauchi-Mani, A. Slusarenko, B. Staskawicz, F. Ausubel, Micro-bial pathogenesis in Arabidopsis, in: E.M. Meyerowitz,C.R. Somerville (Eds.), Arabidopsis, CSHL Press, 1994,pp. 705–747.

[27] R. Mittler, B. Zilinskas, Regulation of pea cytosolicascorbate peroxidase and other antioxidant enzymesduring the progression of drought stress and followingrecovery from drought, Plant J. 5 (1994) 397–405.

[28] F. Lopez, G. Vansuyt, F. Casse-Delbart, P. Fourcroy,Ascorbate peroxidase activity, not the mRNA level, isenhanced in salt-stressed Raphanus sati6us plants, PlantPhysiol. 97 (1996) 13–20.

[29] R. Mittler, X. Feng, M. Cohen, Post-transcriptionalsuppression of cytosolic ascorbate peroxidase expressionduring pathogen-induced programmed cell death in to-bacco, Plant Cell 10 (1998) 461–473.


[30] A. Jimenez, J.A. Hernandez, G. Pastori, L.A. delRio, F.Sevilla, Role of the ascorbate–glutathione cycle of mito-chondria and peroxisomes in the senescence of peasleaves, Plant Physiol. 118 (1998) 1327–1335.

[31] J. deGroot, G.A. Veldink, J.F.G. Vliegenthart, J. Bold-ingh, R. Wever, B. vanGelder, Demonstration by EPRspectroscopy of the functional role of iron in soyabeanlipoxygenase–1, Biochim. Biophys. Acta 377 (1976) 71–79.

[32] F. Macri, E. Braidot, E. Petrussa, A. Vianello, Lipoxy-genase activity associated to isolated soybean plasmamembranes, Biochim. Biophys. Acta 1215 (1994) 109–114.

[33] B. McGurl, G. Pearce, C. Ryan, Polypeptide signallingfor plant defence gene, Biochem. Soc. Symp. 60 (1994)149–154.

[34] N.J. Bate, S.J. Rothstein, C6-volatiles derived from thelipoxygenase pathway a subset of defense-related genes,Plant J. 16 (1998) 561–569.

[35] A.B. Bleecker, M.A. Estelle, C. Somerville, H. Kende,Insensitivity to ethylene conferred by a dominant muta-tion in Arabidopsis thaliana, Science 241 (1988) 1086–1089.

[36] V. Grbic, A.B. Bleecker, Ethylene regulates the timing ofleaf senescence in Arabidopsis, Plant J. 8 (1995) 595–602.

[37] S.A. Oh, J.H. Park, G.I. Lee, K.H. Paek, S.K. Park,H.G. Nam, Identification of three genetic loci con-

trolling leaf senescence in Arabidopsis thaliana, Plant J.12 (1997) 527–535.

[38] L.D. Nooden, J.W. Hillsberg, M.J. Schneider, Inductionof leaf senscence in Arabidopsis thaliana by long daysthrough a light-dosage effect, Physiol. Plant 96 (1996)491–495.

[39] J. Kurepa, J. Smalle, M. VanMontagu, D. Inze, Oxida-tive stress tolerance and longevity in Arabidopsis: thelate flowering mutant gigantea is tolerant to paraqaut,Plant J. 14 (1998) 759–764.

[40] C.J. Lamb, Plant disease resistance genes in signal per-ception and transduction, Cell 76 (1994) 419–422.

[41] L. Legendre, P.F. Heinstein, P.S. Low, Evidence forparticipation of GTP–binding proteins in elucidationofthe rapid oxidation burst in cultured soybean cells, J.Biol. Chem. 267 (1992) 20140–20147.

[42] R. Tenhaken, A. Levine, L.F. Brisson, R.A. Dixon, C.J.Lamb, Function of the oxidative burst in hypersenstivedisease resistance, Proc. Natl. Acad. Sci. USA 92 (1995)4158–4163.

[43] R. Mittler, E.H. Herr, B.L. Orvar, W. vanCamp, H.Willekens, D. Inze, B.E. Ellis, Transgenic tobacco plantswith reduced capability to detoxify reactive oxygen inter-mediates are hyperresponsive to pathogen infection,Proc. Natl. Acad. Sci. USA 96 (1999) 14165–14170.

[44] S.D. O’Neill, The photoperiodic control of floweringprogress toward understanding the mechanism of induc-tion, Photochem. Photobiol. 56 (1992) 789–801.

.

The developmental transition to flowering represses ascorbate peroxidase activity and induces...

Documents

Transcript of The developmental transition to flowering represses ascorbate peroxidase activity and induces...