dehydroascorbate reductase affects leaf growth ... · dehydroascorbate reductase affects leaf...

Dehydroascorbate Reductase Affects Leaf Growth,Development, and Function1

Zhong Chen and Daniel R. Gallie*

Department of Biochemistry, University of California, Riverside, California 92521–0129

Ascorbic acid (Asc) is a major antioxidant in plants that detoxifies reactive oxygen species (ROS) and maintains photosyntheticfunction. Expression of dehydroascorbate reductase (DHAR), responsible for regenerating Asc from an oxidized state,regulates the cellular Asc redox state, which in turn affects cell responsiveness and tolerance to environmental ROS. Because ofits role in Asc recycling, we examined whether DHAR is important for plant growth. Suppression of DHAR expressionresulted in a preferential loss of chlorophyll a, a lower steady state of Rubisco as measured by the amount of the large subunitof Rubisco (RbcL), and a lower rate of CO2 assimilation. As a consequence, a slower rate of leaf expansion and reduced foliardry weight were observed. In addition, an accelerated rate of loss of chlorophyll, RbcL, light-harvesting complex II, andphotosynthetic functioning was observed in mature leaves, resulting in premature leaf aging. Reduced growth rate asmeasured by plant height and leaf number was consistent with the DHAR-mediated reduction of photosynthetic function.Increasing DHAR expression maintained higher levels of chlorophyll, RbcL, light-harvesting complex II, and photosyntheticfunctioning, resulting in delayed leaf aging. The effect of DHAR expression on leaf aging inversely correlated with the level oflipid peroxidation, indicating that DHAR functions to protect against ROS-mediated damage. These observations support theconclusion that through its Asc recycling function, DHAR affects the level of foliar ROS and photosynthetic activity during leafdevelopment and as a consequence, influences the rate of plant growth and leaf aging.

Ascorbic acid (Asc) is a major antioxidant that servesmany functions in plants. Asc is involved in the detoxi-fication of reactive oxygen species (ROS), e.g. super-oxide, singlet oxygen, ozone, and hydrogen peroxide(H2O2), which are produced during aerobic metabolicprocesses such as photosynthesis or respiration (Asadaand Takahashi, 1987). Asc also participates in theregeneration of a-tocopherol (vitamin E) from thetocopheroxyl radical (Asada, 1994). In addition, Asc func-tions as a cofactor for enzymes such as prolyl and lysylhydrolases, violaxanthin deepoxidase, and ethylene-forming enzyme (Davies et al., 1991; McGarvey andChristoffersen, 1992; Smith et al., 1992; Eskling et al.,1997) as well as for 2-oxoacid-dependent dioxygenasesrequired for the synthesis of abscisic acid (ABA) andgibberellic acid (Arrigoni and De Tullio, 2000, 2002;Smirnoff, 2000). Asc is involved in the regulation ofcell elongation and progression through the cell cycle(for review, see Smirnoff, 1996; Horemans et al., 2000).

Once used, Asc is oxidized to the monodehydroas-corbate (MDHA) radical that can be reduced to Asc inthe chloroplast or cytosol by MDHA reductase

(MDHAR) in an NAD(P)H-dependent reaction (Asada,1999). In the chloroplast, the MDHA radical can alsobe reduced to Asc by thylakoid-associated, reducedferredoxin that is more effective than reduction byMDHAR (Miyake and Asada, 1994; Asada, 1999).MDHA produced in the thylakoid lumen by viola-xanthin deepoxidase or following donation of electronsfrom Asc to PSII or PSI (Mano et al., 1997; Mano, 1999),however, is not available as a substrate for reductionby ferredoxin or MDHAR but rapidly disproportion-ates to Asc and dehydroascorbate (DHA) when the pHof the lumen is low (Asada, 1999). DHA is then reducedto Asc by DHA reductase (DHAR) in a reaction requir-ing glutathione. Because the apoplast contains little glu-tathione or DHAR, DHA, which predominates in theapoplast, must reenter the cell for reduction to Asc. Inthe absence of sufficient DHAR, however, DHA under-goes irreversible hydrolysis to 2,3-diketogulonic acid.

Given that Asc is present in most cellular compart-ments and that several pathways exist to ensure thatAsc is recycled, it might be expected that perturbationsin one recycling pathway would be offset by the ac-tivity of the remaining pathways to maintain the cel-lular Asc redox state. However, changes in DHARexpression result in substantial alterations in the cyto-solic and apoplastic Asc redox state; overexpression ofDHAR in tobacco (Nicotiana tabacum) leaves increasedthe Asc redox state (i.e. was more reduced), whereassuppression of DHAR had the opposite effect (Chenet al., 2003; Chen and Gallie, 2004, 2005). These obser-vations suggest that DHAR is expressed in rate-limitingamounts and that DHAR contributes significantly toestablishing the cellular Asc redox state, at least inleaves.

1 This work was supported by the National Research Initiative ofthe U.S. Department of Agriculture Cooperative State Research,Education and Extension Service (grant no. 2002–35100–12469) andby the University of California Agricultural Experiment Station.

* Corresponding author; e-mail [email protected]; fax 951–827–4434.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Daniel R. Gallie ([email protected]).

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H2O2 functions as a signaling intermediate down-stream of ABA to promote stomatal closure (Price et al.,1994, 2000; Murata et al., 2001; Schroeder et al., 2001a,2001b; Zhang et al., 2001). Guard cells of plants ex-pressing DHAR exhibit a higher (i.e. more reduced)Asc redox state, reduced levels of H2O2, decreased re-sponsiveness to H2O2 or ABA signaling, and greaterstomatal opening (Chen and Gallie, 2004). Suppressionof DHAR expression results in higher levels of H2O2 inguard cells and enhanced stomatal closure under nor-mal growth conditions or following water stress. Thus,the level of DHAR expression is important for appro-priate signaling in guard cells, because it establishesthe efficiency of H2O2 scavenging. Increasing DHARexpression also provided enhanced tolerance to envi-ronmental ROS, e.g. ozone, despite the increase instomatal conductance (Chen and Gallie, 2004).

One means by which Asc reduces photoinhibition isby promoting conversion of violaxanthin to zeaxan-thin in the xanthophyll cycle to dissipate excess exci-tation energy as part of nonphotochemical quenching(NPQ). The importance of the Asc pool size in support-ing growth was shown with the Arabidopsis (Arabidop-sis thaliana) vitamin C1 (vtc1) mutant that is defective inGDP-Man pyrophosphorylase and accumulates only25% to 30% of the wild-type level without altering theAsc redox state (Conklin et al., 1996, 1999; Veljovic-Jovanovic et al., 2001). In addition to being hypersen-sitive to ozone, sulfur dioxide, or UVB light, vtc1 plantsexhibit slower shoot growth, smaller leaves, andreduced shoot fresh weight and dry weight (Veljovic-Jovanovic et al., 2001). vtc1 plants exhibited no signif-icant difference in the light saturation curves for CO2assimilation or chlorophyll fluorescence under thesegrowth conditions, suggesting that the effect on growthwas not due to decreased photochemical efficiency orreduced photosynthetic capacity (Veljovic-Jovanovicet al., 2001). No change in the amount of H2O2 and onlya slight reduction in NPQ were observed in vtc1 leaves(Veljovic-Jovanovic et al., 2001). Expression of cyto-solic ascorbate peroxidase (APX) increased and that ofchloroplast APX isoforms was either unchanged orslightly decreased, suggesting that the level of Asc isinvolved in the regulation of the compartmentaliza-tion of the antioxidant system in Arabidopsis (Veljovic-Jovanovic et al., 2001). The vtc2 mutant has only 10% to30% of wild-type levels of Asc and is slightly deficientin the feedback deexcitation component of NPQ thatcauses the dissipation of excess light as heat (Muller-Moule et al., 2003). The vtc2 mutant experienceschronic photooxidative stress in high light and photo-bleaches when transferred from low to high light ac-companied by increased lipid peroxidation andphotoinhibition (Muller-Moule et al., 2003, 2004).

Because changes in DHAR expression result insubstantial changes in the Asc redox state not seen inthe vtc mutants, we examined whether the efficiency ofAsc recycling as determined by DHAR influencedplant growth and leaf function. Plants suppressed inDHAR expression exhibited a slower rate of leaf expan-

sion, slower shoot growth, delayed flowering time,and reduced foliar dry weight. These phenotypes cor-related with reduced leaf function as measured by thepreferential loss of chlorophyll a, a reduced level ofRubisco large subunit (RbcL), and a lower rate of CO2assimilation in mature leaves. The effect of DHAR ex-pression on leaf aging inversely correlated with thelevel of lipid peroxidation, indicating that the effi-ciency of Asc recycling was important in regulatingROS-mediated damage. These results suggest thatDHAR contributes to plant growth by maintaining pho-tosynthetic functioning through efficient Asc recyclingthat limits ROS-mediated damage that slows leaf aging.

RESULTS

Chlorophyll Pool Size, CO2 Assimilation, and Plant

Growth Rate Correlate with the Level of FoliarDHAR Activity

To investigate how the level of DHAR activitycorrelates with leaf function at the whole plant level,DHAR activity and protein levels were measured inevery second leaf of mature tobacco plants just prior toflowering. DHAR activity was highest in the youngestleaves and declined with leaf age (Fig. 1A). DHARprotein levels also were highest in the youngest leavesand declined with leaf age, although the decline inDHAR protein was not as great as the decline in DHARactivity, suggesting that DHAR activity may be post-translationally regulated. The changes in DHAR activ-ity largely correlated with the change in the chlorophylla and b pool sizes (Fig. 1B) as well as the rate of CO2assimilation (Fig. 1C). The only exception to this wasthat DHAR activity was near maximum in the youn-gest leaves when the chlorophyll a and b pool size andthe rate of CO2 assimilation were not yet at maximum.

In its antioxidant function, Asc acts to maintain pho-tosynthetic function through the detoxification of ROSas well as to maintain electron flow through PSI andPSII (Asada, 1999). Because DHAR contributes sub-stantially to the regulation of the Asc redox state (Chenet al., 2003; Chen and Gallie, 2004), we examinedwhether alterations in DHAR expression affectedcomponents of the photosynthetic machinery or pho-tosynthetic activity. To investigate this possibility, weused DHAR-overexpressing (DOX) or DHAR-suppressed(DKD) tobacco that have been described previously(Chen et al., 2003; Chen and Gallie, 2004). DOX lineswere generated following the introduction of a wheat(Triticum aestivum) DHAR (DHARTa) cDNA under thecontrol of the cauliflower mosaic virus (CaMV) 35Spromoter. A wheat DHAR cDNA was used becauseintroduction of a tobacco DHAR cDNA under thecontrol of the CaMV 35S promoter resulted only insuppression of endogenous tobacco DHAR expres-sion. Although the nucleotide sequence of the wheatDHAR cDNA has diverged enough that it did notsilence endogenous tobacco DHAR expression, at the

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protein level, wheat DHAR shares 73% identity and85% similarity with the tobacco ortholog. Therefore,expression of wheat DHAR was used to generatetobacco with increased DHAR activity (i.e. the aggre-gate of wheat DHAR activity and endogenous tobaccoDHAR), whereas expression from the tobacco DHARtransgene was used to generate tobacco with reducedDHAR activity. In each case, it is the level of totalDHAR activity present within a line that is correlatedwith changes in leaf growth and function. The wheatand tobacco DHAR genes used encode a cytosolicDHAR. Of the nine independent DOX lines initiallycharacterized (three of which have been previouslydescribed; Chen et al., 2003) and six DKD lines (one ofwhich has been previously described; Chen and Gallie,2004), a reduction in DHAR activity correlated withreduced chlorophyll content and rate of growth,whereas an increase in DHAR activity correlatedwith higher chlorophyll content (data not shown).Detailed growth measurements were then performedon one representative DOX and DKD line.

To examine leaf function and growth at different leafages, expanding, mature, and presenescent leaves ofDOX, DKD, and control plants were used for the anal-ysis. An expanding leaf is defined as one that hasachieved approximately 50% of its final size but hasnot reached a maximum rate of CO2 assimilation (e.g.leaf 4, Fig. 1C). A mature leaf is defined as a fullyexpanded leaf that exhibits a maximum rate of CO2assimilation (e.g. leaf 6, Fig. 1C). Thus, each leaf typecollected from the three lines examined was definedby its developmental stage, in the same position withregard to the apical leaf whorl, and of similar chrono-logical age. Presenescent leaves exhibited reduced ratesof CO2 assimilation relative to the maximum exhibitedby a mature leaf (e.g. leaf 18, Fig. 1C) but still con-tained chlorophyll, albeit at significantly reducedlevels. Presenescent leaves were defined according totheir position (numbering from the plant base) so thatleaves of equivalent chronological age could be col-lected from all lines to permit a comparison of variousparameters of leaf aging, e.g. chlorophyll content, levelof Rubisco or light-harvesting chlorophyll-proteincomplex, lipid oxidation, and leaf dry weight. All mea-surements were made at the same time each day dur-ing the course of an experiment to avoid any possiblediurnal effects.

Overexpression of wheat DHAR resulted in a sub-stantial increase in DHAR activity in expanding, ma-ture, and presenescent leaves (Fig. 2A). The level ofendogenous DHAR protein (DHARNt) remained unaf-fected in expanding and mature leaves of DOX plantsas quantitated from the western analysis but was 77%higher in presenescent leaves than in control leaves(Fig. 2B). This is consistent with the prolonged main-tenance of leaf function in DOX plants (see below). Incontrast, DKD tobacco exhibited substantially reducedlevels of DHAR activity in leaves at the samedevelopmental stages (Fig. 2A). The level of endoge-nous DHARNt in expanding, mature, and presenescent

Figure 1. DHAR activity, chlorophyll pool size, and rate of CO2

assimilation during leaf development. A, DHAR activity was measuredfrom every second leaf of adult tobacco just prior to flowering. DHARprotein levels were measured by western analysis. B, Chlorophyll a (¤)and b (s) levels and rate of CO2 assimilation (C) were determined fromevery second leaf of adult tobacco. Leaf 2 was the youngest leaf andleaf 20 the oldest leaf tested.

Dehydroascorbate Reductase Affects Growth Rate

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leaves of DKD plants was reduced to 47%, 35%, and44%, respectively, of the level in the correspondingleaves of control plants (Fig. 2B), data that are in goodagreement with the reduction in DHAR activity (Fig.2A). Although the level of DHAR activity declinedwith leaf age in DOX, DKD, and control plants, the levelof activity was always higher in DOX leaves and lowerin DKD leaves relative to the control (Fig. 2A).

The level of lipid peroxidation, as measured bythiobarbituric acid reactive substances (TBARS) assay,increased with leaf age; however, it was consistentlyhigher in mature and presenescent leaves of DKDplants than in the corresponding leaves of DOX plants(P , 0.05 and P , 0.005; n 5 3, respectively) whengrown in moderate light (approximately 500 mmol m22

s21; Fig. 2A). Lipid peroxidation in presenescent DKDleaves was also significantly higher than in the corre-sponding leaves of control plants (P , 0.05; n 5 3), in-dicating that the extent of lipid peroxidation may beaffected by the level of foliar DHAR activity. Althoughthe level of lipid peroxidation was consistently lowerin DOX leaves relative to the control, the difference wasnot significant. No significant difference in lipid per-oxidation was observed in expanding leaves amongthe three lines, suggesting that the differences in lipidperoxidation accumulated over time. Although totalAPX activity declined with leaf age in DOX, DKD, andcontrol plants, no consistent change in its activity withrespect to DHAR activity was observed, in good agree-ment with previous results (Chen et al., 2003). Thelevel of Rubisco, as measured by the amount of RbcLand the level of light-harvesting complex II (LHCII)declined with leaf age, but their relative abundance inexpanding or mature leaves of DOX, DKD, and controlplants was not substantially different. In presenescentleaves, however, the level of RbcL and LHCII in DKDplants was reduced to 64% and 38%, respectively, ofthe level present in the corresponding leaves of controlplants, and the level of LHCII in DOX plants was 29%higher than in the control (Fig. 2B). The reduction inthe amount of LHCII in DKD plants was not observedin vtc2 mutant plants (Muller-Moule et al., 2004). Thecorrelation between DHAR activity and the abundanceof RbcL and LHCII suggests that the level of DHARactivity may be important in maintaining these pro-teins during leaf aging.

To examine whether changes in the level of DHARactivity influenced the induction of a senescence-related gene, expression of tobacco CP1, the orthologof the senescence-associated gene SAG12 of Arabidop-sis, was examined in DOX, DKD, and control plantsusing reverse transcription (RT)-PCR. Little to no CP1

Figure 2. DHAR activity correlates with ROS-mediated damage andthe level of RbcL and LHCII. A, The level of DHAR activity, lipidperoxidation (as measured by the TBARS assay), and total APX wasmeasured in expanding, mature, and presenescent leaves of control (C),DOX, and DKD tobacco. The average and SD from three replicates arereported. B, The level of DHAR (DHARNt, DHARTa), RbcL, and LHCII

were measured by western analysis. C, RT-PCR analysis of CP1expression (top) in mature leaves, presenescent leaves, senescent stage1 leaves (approximately 2 weeks older than presenescent leaves), andsenescent stage 2 leaves (approximately 4 weeks older than presenes-cent leaves) of control, DOX, and DKD plants. RT-PCR analysis of actinexpression (bottom) was also performed as a control using the same leafsamples.

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expression was detected in mature or presenescentleaves from DOX, DKD, and control plants (Fig. 2C),supporting the conclusion that the presenescent leavesused in this study had not initiated senescence. CP1expression was detected in senescent stage 1 DKDleaves, which were approximately 2 weeks older thanthe presenescent leaves used in this study (Fig. 2C). NoCP1 expression was detected, however, in senescentstage 1 leaves of DOX or control plants. CP1 expressionwas also detected in senescent stage 2 DKD leaves(approximately 4 weeks older than the presenescentleaves used in this study), whereas expression was justdetectable in senescent stage 2 DOX or control leaves(Fig. 2C). These data suggest that a reduction in thelevel of DHAR activity accelerates the onset of senes-cence.

To determine whether the level of DHAR activitymay affect the pool size of chlorophyll a and b, chlo-rophyll levels were measured in expanding, mature, orpresenescent leaves of DOX, DKD, and control plants.The level of chlorophyll a was significantly higher inexpanding leaves of DOX plants than in control plants(P , 0.005; n 5 4) as it was in mature or presenescentleaves (P , 0.05 and P , 0.005; n 5 4, respectively; Fig. 3),whereas it was significantly lower in DKD mature and

presenescent leaves (P , 0.005 and P , 0.005; n 5 4,respectively) but not in expanding DKD leaves (P 50.085; n 5 4) relative to the control. However, the levelof chlorophyll a was significantly lower in expandingDKD leaves relative to expanding DOX leaves (P , 0.005;n 5 4). Although a small increase in the level of chlo-rophyll b was observed in expanding DOX leaves rela-tive to the control, this difference was only significantin presenescent leaves (P , 0.005; n 5 4; Fig. 3). Nosignificant difference in the level of chlorophyll b wasobserved in expanding DKD leaves relative to the con-trol (P 5 0.797; n 5 4), but its level was significantlyreduced in mature or presenescent leaves of DKDplants relative to the control (P , 0.05 and P , 0.05;n 5 4, respectively). The chlorophyll a to b ratio wassignificantly lower in expanding, mature, and prese-nescent leaves of DKD plants than in DOX leaves (P ,0.05, P , 0.05, and P , 0.005; n 5 4, respectively). Thechlorophyll a to b ratio was also significantly lower inpresenescent DKD leaves than in control leaves (P ,0.01; n 5 4; Fig. 3).

The chlorophyll pool size is determined by rates ofits synthesis and degradation. To investigate whetherthe level of DHAR activity influences the rate of chlo-rophyll loss, young leaves from DOX, DKD, and controlplants were dark treated, and the rate of chlorophyllloss was followed over time. Although the chlorophyllpool size in DOX leaves was larger than in controlleaves, the rate of chlorophyll loss from DOX leaves (i.e.loss of 38.1 mg/g fresh weight/d) was only slightlylower than that from control leaves (i.e. loss of 42.4mg/g fresh weight/d; Fig. 4). The rate of chlorophyllloss from DKD leaves was more than 2-fold greater (i.e.loss of 87.1 mg/g fresh weight/d) than that from con-trol leaves despite the use of DKD leaves with an initialchlorophyll pool size similar to control leaves.

To examine whether the observed differences amongDOX, DKD, and control leaves correlated with differ-ences in CO2 assimilation, gas exchange was measuredfrom every second leaf. The rate of CO2 assimilation inexpanding and mature leaves of DOX plants was sim-ilar to those of control plants despite a higher rate oftranspiration (Fig. 5A). The rate of stomatal conduc-tance was too high to measure differences in thesesame leaves between DOX and control plants. In older,fully expanded leaves as well as in presenescent leaves,a higher rate of CO2 assimilation was observed in DOXplants. Although this correlated with a higher rate oftranspiration, a lower substomatal CO2 concentrationwas also observed, suggesting a higher rate of CO2 as-similation (Fig. 5A). The rate of CO2 assimilation wasconsistently lower in all leaves of DKD plants relative tothe control (Fig. 5A). In mature and presenescentleaves, this correlated with reduced rates of transpi-ration and stomatal conductance previously reportedfor DKD plants (Chen and Gallie, 2004), but the higherlevel of substomatal CO2 concentration in these DKDleaves indicated that the internal concentration of CO2was not limiting and thus suggested that DKD leaveswere less efficient in assimilating CO2. In young DKD

Figure 3. DHAR activity correlates with the chlorophyll pool size andchlorophyll a/b ratio. Chlorophyll a, chlorophyll b, and the chlorophylla/b ratio were measured in expanding, mature, and presenescent leavesof control (C), DOX, and DKD tobacco. The average and SD from fourreplicates are reported.





leaves, the rate of transpiration was similar to that ofcontrol leaves, and therefore the observed reduced rateof CO2 assimilation in the young DKD leaves supportedthe conclusion of less efficient CO2 assimilation. Thesedata suggest that the reduced rate of CO2 assimilationin young DKD leaves is likely not a consequence ofchanges in the level of RbcL or LHCII but do correlate

with changes in the chlorophyll pool size and chloro-phyll a/b ratio. The reduced rate of CO2 assimilationin older DKD leaves and higher rate of CO2 assimilationin older DOX leaves correlate with similar changes inthe levels of RbcL, LHCII, and chlorophyll pool size aswell as the chlorophyll a/b ratio (Figs. 2 and 3).

The reduced rate of CO2 assimilation in DKD leavescorrelated with a reduced leaf dry weight of expand-ing and mature leaves relative to the control (P , 0.005and P , 0.01; n 5 3, respectively), although the dif-ference was not significant in presenescent leaves (P 50.11; n 5 3; Fig. 5B). Leaf dry weight in expanding,mature, and even presenescent DKD leaves was signif-icantly lower than DOX leaves (P , 0.01, P , 0.05, andP , 0.05; n 5 3, respectively; Fig. 5B). No significantdifference in leaf dry weight was observed betweenexpanding, mature, or presenescent leaves of DOX andcontrol plants (P 5 0.19, P 5 0.83, and P 5 0.08; n 5 3,respectively; Fig. 5B), correlating with their similarrates of CO2 assimilation during leaf growth (Fig. 5A).To examine whether the differences in CO2 assimila-tion observed in DOX and DKD plants are accompaniedby other changes in growth, parameters of plantgrowth were measured. Plant growth as measured byheight over time was little altered in DOX plants rela-tive to the control, and no change in leaf number wasobserved (Fig. 6). DOX plants flowered at the same time

Figure 4. Suppression of DHAR expression accelerates loss of chloro-phyll. Chlorophyll (a 1 b) was measured every 4 d from dark-treatedleaves of control (C, ¤), DOX (n), and DKD tobacco (:). The average andSD from three replicates are reported.

Figure 5. DHAR activity affects the effi-ciency of CO2 assimilation and leaf dryweight. A, The rates of CO2 assimilation,transpiration, and stomatal conductance,and the substomatal CO2 concentrationwere measured from every second leaf ofcontrol (C, ¤), DOX (n), and DKD tobacco(:). Leaf 2 was the youngest leaf and leaf18 the oldest leaf tested. Expanding leavesinclude leaves 2 to 4, mature leaves in-clude leaves 6 to 8, and presenescentleaves include leaves 16 to 18. B, Thedry weight as measured as percent of freshweight was determined for expanding,mature, and presenescent leaves. The av-erage and SD from three replicates arereported.

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as control plants (Table I). DKD plants exhibited a sig-nificantly reduced rate of growth (P , 0.005 at weeks5–7; n 5 8) but caught up during late growth (P 5 0.130and P 5 0.161 at weeks 8 and 9, respectively; n 5 8).DKD plants had significantly fewer leaves duringgrowth (P 5 0.05, P , 0.005, and P , 0.05 at weeks5, 6, and 7, respectively; n 5 8) but eventually pro-duced the same number of leaves as the control (Fig. 6).Consistent with this, DKD plants flowered significantlylater (Table I) than control plants (P 5 0.001; n 5 8).The average leaf internode distance of fully grown DKD

plants was slightly less than that of control plants,although the difference was not significant (P 5 0.9;n 5 18). Although no significant difference in leaf areawas observed between DOX and control plants, leavesfrom DKD plants were consistently smaller in sizerelative to the control (P 5 0.0014; Fig. 6). These dataindicate that the growth rate but not final plant heightis reduced in DKD tobacco, suggesting that efficiency ofAsc recycling is important in maintaining maximumgrowth. Because increasing DHAR expression did notincrease plant growth, the endogenous level of DHARactivity appears to be sufficient to support a maximumgrowth rate.

The Level of DHAR Activity Correlates with theMaintenance of Chlorophyll a and Leaf Growth Rate

The previous analyses provided insight into howDHAR activity affects growth at the level of the wholeplant. However, it is not valid to assume that expand-ing, mature, and presenescent leaves from a singleplant are representative of the developmental stagesthat a specific leaf undergoes during its development,as not all leaves on a given plant are biochemically orfunctionally equivalent (e.g. leaf size, chlorophyll con-tent). Therefore, to investigate how DHAR activity af-fects growth of a specific leaf, we followed the growthof the sixth true leaf (as numbered from the plant base)in DOX, DKD, and control plants. We first measured thearea of the sixth true leaf when the leaf was fullyexpanded, a value that remained constant for a givenline when the plants were grown under identical con-ditions. From the area of the sixth true leaf in its fullyexpanded state, sixth true leaves could be analyzedduring their growth when they were 8%, 50%, or 100%of the fully expanded size. DHAR activity and proteinlevel on a fresh weight basis declined during leaf ex-pansion in control plants as it did in DOX and DKDplants (Fig. 7, A and B). Correlating with the decline inDHAR was a decrease in the Asc pool size and redoxstate (i.e. more oxidized) during the expansion of con-trol leaves (Fig. 7A). The decrease in protein and Ascpool size may be explained in part by the dilution dur-ing the cell expansion that occurs during leaf growth.In contrast, dilution does not account for the oxidationof the Asc redox state. As reported previously (Chenand Gallie, 2004), increasing DHAR expression in DOXplants results in an increase in the Asc pool size andredox state, both of which decline during leaf expansion

Figure 6. The level of DHAR activity affects the rate of plant growth.Plant height and leaf number were measured in control (C, ¤), DOX (n),and DKD tobacco (:) beginning at 4 weeks after germination until theappearance of the inflorescence. Leaf area was measured following thevegetative to floral transition when all leaves had reached their finalsize. Leaf 1 was the youngest leaf and leaf 9 was the oldest leafmeasured. The average and SD from eight replicates are reported.

Table I. DHAR is required for correct internode length, leaf growth,and flowering time

Internode Length Leaf Expansiona Flowering Time

cm cm2/d d

Control 9.32 6 0.68 36.5 60.25 6 1.09DOX 9.42 6 0.70 36.6 60.13 6 1.27DKD 8.89 6 0.74 29.7 64.38 6 2.18

aExpansion from the sixth true leaf was measured.





although always remaining higher than control leaves(Fig. 7A). Suppressing DHAR expression in DKD plantsresults in higher levels of DHA (Chen and Gallie,2004), consistent with reduced Asc recycling, althoughthe total ascorbate pool size (i.e. Asc 1 DHA) showsslight to no reduction depending on leaf age (Fig. 7A).As with control and DOX leaves, the Asc pool size andredox state in DKD plants declined with leaf expansionand were consistently lower than in control leaves. Therelative level of RbcL was not substantially differentbetween DOX and control plants during leaf expansion.The relative level of RbcL was also not substantiallydifferent between DKD and control plants when leaveswere 8% of their fully expanded size, but when DKDleaves reached 50% or 100% of their fully expandedsize, the level of RbcL was reduced to 87% and 64% ofthat present in control leaves (Fig. 7B). No consistentchange in the relative level of LHCII was observed in

DOX, DKD, and control leaves during their expansion(Fig. 7B).

The pool size of chlorophyll a and b in control leavesdeclined concomitantly during leaf expansion such thatthe chlorophyll a/b ratio was largely unaltered (Fig. 8A).Although the pool size of chlorophyll a in DKD leaveswas not significantly different from control leaves whenthe leaves were 8% of their fully expanded state (P 50.437; n 5 4), it was significantly lower than the controlwhen the leaves reached 50% or 100% of the fullyexpanded state (P , 0.05 and P , 0.05; n 5 4; Fig. 8A).In contrast, the pool size of chlorophyll a in DOX leaves

Figure 7. The level of DHAR activity correlates with the foliar Ascredox state and level of RbcL and LHCII during leaf expansion. A, Thelevel of DHAR activity, Asc (black bars), DHA (white bars), and the Ascredox state (Asc/DHA) were measured in the sixth true leaf during itsgrowth from 8%, 50%, and 100% of fully expanded size of control (C),DOX, and DKD tobacco. The average and SD from three replicates arereported. B, The level of DHAR, RbcL, and LHCII were measured in thesame leaves as in A by western analysis.

Figure 8. The level of DHAR activity correlates with the chlorophyll apool size, the chlorophyll a/b ratio, and leaf dry weight during leafexpansion. A, Chlorophyll a, chlorophyll b, and the chlorophyll a/bratio were measured in the sixth true leaf during its growth from 8%,50%, and 100% of fully expanded size of control (C), DOX, and DKD

tobacco. B, The dry weight as measured as the percent of fresh weightwas determined at the same stages of leaf growth as in A. The averageand SD from four replicates are reported.

Chen and Gallie




was significantly higher than in control leaves at 8%,50%, and 100% of the fully expanded state (P , 0.05,P , 0.05, and P , 0.01, respectively; n 5 4), althoughthe pool size of chlorophyll b was not significantlydifferent from the control (Fig. 8A). Thus, althoughthe chlorophyll a/b ratio in DOX and DKD leaves wasnot significantly different when the leaves were 8% oftheir fully expanded state (P 5 0.189; n 5 4), it wassignificantly lower in DKD leaves when the leavesreached 50% or 100% of the fully expanded state (P ,0.05 and P , 0.01; n 5 4; Fig. 8A). These data suggestthat increasing DHAR activity increases the pool sizeof chlorophyll a from early leaf development up tosenescence, whereas decreasing DHAR activity doesnot affect the initial pool size of chlorophyll a but doesresult in a preferential loss of chlorophyll a duringsubsequent leaf expansion and aging.

To examine how alterations in the efficiency of Ascrecycling resulting from changes in DHAR expressionaffected CO2 assimilation as a leaf ages, gas exchangewas measured in expanding leaves every 3 d. The rateof CO2 assimilation in DOX leaves was similar to con-trol leaves during leaf expansion but declined at aslower rate as the leaf aged, correlating with a higherstomatal conductance (Fig. 9). The rate of CO2 assim-ilation was consistently lower in DKD leaves through-out its expansion and subsequent aging. The reducedlevel of CO2 assimilation in DKD leaves could not beexplained by a corresponding reduction in stomatalconductance but did correlate with a higher level ofsubstomatal CO2 concentration (Fig. 9), suggesting lessefficient assimilation of CO2.

The rate of leaf expansion was followed for the samecohort of leaves and revealed that DOX and controlleaves expanded at the same rate (P 5 0.91; n 5 8),

whereas the expansion of DKD leaves was significantlydelayed relative to the control (P 5 0.028; n 5 8; Fig. 10;Table I). The reduced rate of CO2 assimilation andslower rate of expansion of DKD leaves correlated witha reduced leaf dry weight throughout leaf expansion(Fig. 8B) that was similar to the reduced leaf dryweight observed for all leaves of adult plants (Fig. 5B).No significant difference in leaf dry weight during leafexpansion was observed between DOX and controlleaves (Fig. 8B), correlating with their similar rates ofCO2 assimilation during early leaf growth (Fig. 9).

DISCUSSION

In this study, we present evidence suggesting thatthe level of foliar DHAR activity influences the rate ofleaf aging, and, as a consequence, the rate of plantgrowth. We were able to take advantage of the fact thatDHAR is expressed in limiting amounts with regard tothe Asc redox state (Chen et al., 2003; Chen and Gallie,2004) to test whether alterations in DHAR expressionwould perturb plant growth. Suppressing DHAR ex-pression resulted in less efficient Asc recycling and asa consequence, a lower (i.e. more oxidized) Asc redoxstate. The effect that suppressing DHAR expressionhad on Asc recycling increased with leaf age and cor-related with an increase in ROS (Chen and Gallie, 2004),lipid peroxidation, and slower leaf and plant growth.This reduced growth rate correlated with a smallerchlorophyll pool size and a lower chlorophyll a/b ratioresulting from the preferential loss of chlorophyll aduring leaf expansion. The lower chlorophyll a/b ratiocorrelated with a reduced rate of CO2 assimilation inall leaves. Although reducing DHAR expression hasbeen shown to reduce stomatal conductance, which

Figure 9. The level of DHAR activity af-fects the efficiency of CO2 assimilationduring leaf expansion. The rates of CO2

assimilation, transpiration, and stomatalconductance, and the substomatal CO2

concentration were measured in the sixthtrue leaf during its growth from control(C, ¤), DOX (n), and DKD tobacco (:).Measurements were taken every third day,starting when the leaves were 8% of fullyexpanded size until the leaves were 100%of fully expanded size (approximately day9) and continued for an additional 9 d. Theaverage and SD from three replicates arereported.





might be expected to limit CO2 diffusion into the leafinterior, a reduced rate of CO2 assimilation was alsoobserved in expanding and newly expanded leaves inwhich stomatal conductance was not substantiallyaffected. Moreover, despite the lower stomatal con-ductance in older DKD leaves, the substomatal CO2 con-centration was higher than it was in control leaves,indicating that CO2 diffusion into the leaf interior wasnot limited but rather CO2 was not being used effi-ciently. The preferential loss of chlorophyll a in DKDleaves may have been a consequence of the higherlevel of ROS present and may account for the reducedphotosynthetic activity observed in DKD leaves. In ad-dition to the lower chlorophyll a/b ratio in older DKDleaves, the reduced rate of CO2 assimilation correlatedwith reduced levels of RbcL and LHCII. Although it isnot known which of these may be rate limiting in olderleaves, these data suggest that the efficiency of Ascrecycling as determined by the level of DHAR activityplays an important role in leaf aging. The reduced rateof leaf expansion observed in DKD plants is likely aconsequence of the accelerated loss of photosyntheticactivity in mature leaves that may have prematurelyreduced their photosynthate that they could provideto sink tissues. This would also explain the slowerplant growth rate as measured by plant height and rateof leaf production over time. Changes in the level ofDHAR expression affect the Asc pool size and redoxstate within the cytosol and apoplast (Chen and Gallie,2005), and as Asc is readily transported throughout thecell (Horemans et al., 2000), changes in chloroplasticAsc would also be expected. Whether changes inchloroplastic or cytosolic Asc are responsible for the ob-served effects on leaf aging remain to be determined.

The observation that increasing DHAR expressioncorrelated with higher levels of RbcL and chlorophylland a higher rate of CO2 assimilation in presenescentleaves, whereas reducing DHAR expression in thesame leaves correlated with a lower level of RbcL and

LHCII, a smaller pool size of chlorophyll, and reducedrate of CO2 assimilation indicates that the level of foliarDHAR activity can affect the rate of loss of leaf func-tion. The progressive loss of DHAR as a function ofleaf age, correlating with a loss of chlorophyll and CO2assimilation rate, is consistent with a potential role forthis enzyme in influencing the rate of leaf aging. Thefact that reducing DHAR expression correlated withslower growth is also consistent with a role of DHARin influencing leaf aging. The observation that increas-ing the level of DHAR activity correlated with reducedlipid peroxidation whereas reducing DHAR activitycorrelated with increased lipid peroxidation particu-larly during leaf aging is consistent with previous obser-vations demonstrating an inverse relationship betweenthe level of DHAR expression and the foliar level ofROS (Chen and Gallie, 2004). It also suggests that thelevel of DHAR activity may affect leaf function andaging by regulating the level of ROS and, as a conse-quence, affect the amount of ROS-generated damagethat occurs to the photosynthetic machinery. ReducingDHAR expression in DKD leaves also resulted in anaccelerated onset of senescence as revealed by the pre-mature induction of expression of the SAG12 ortholog,CP1. These observations support the conclusion thatthrough its Asc recycling function, the level of DHARactivity affects the basal level of ROS during leaf devel-opment and, as a consequence, influences the rate ofleaf aging. In addition, the observation that increasingDHAR expression did not substantially increase leafexpansion or plant growth indicates that the endoge-nous level of DHAR expression provides a level of Ascrecycling sufficient to support maximum growth. Thisis in contrast to the role of DHAR in regulating guardcell function, where increasing DHAR expression re-sults in substantial changes in stomatal behavior as aresult of changes in ROS levels in the guard cells (Chenand Gallie, 2004).

Because no change in the photosynthetic capacity orin H2O2 levels was observed in the Arabidopsis vtc1mutant, it was suggested that the low Asc pool size didnot impair photosynthetic functioning or result inoxidative stress (Veljovic-Jovanovic et al., 2001). Theobservation that vtc1 plants continued to exhibit aslower shoot growth rate when grown under condi-tions of high CO2 that suppress oxidative stress result-ing from photorespiration supported the conclusionthat the slow growth phenotype was unrelated to ahigher oxidative load (Veljovic-Jovanovic et al., 2001).vtc2 mutants that contain only 10% to 30% of the wild-type level of Asc exhibit sensitivity to high light andphotobleach when transferred from low to high light(Muller-Moule et al., 2002, 2003). When grown in highlight, vtc2 plants contain about 40% of the wild-typelevel of Asc and are able to grow despite signs of oxi-dative stress, such as lower electron transport, a lowerrate of oxygen evolution, and lower PSII quantumefficiency (Muller-Moule et al., 2004). These mutantsdemonstrate that although some degree of photooxi-dative stress may result from the reduced levels of Asc,

Figure 10. The level of DHAR activity affects the rate of leaf growth.Leaf area was measured for the sixth true leaf during its growth incontrol (C, ¤), DOX (n), and DKD tobacco (:) every 3 d beginning at leafemergence. The average and SD from eight replicates are reported.

Chen and Gallie




a significant reduction in Asc does not prevent growthunder conditions of high light.

In contrast to vtc mutants, DKD plants showed re-duced photosynthetic function and reduced growth inmoderate light. Plants suppressed for DHAR are notsubstantially Asc deficient but rather have a decreasedAsc redox state (i.e. more oxidized) that may explainthe differences observed between the present plantsand the vtc mutants. However, the fact that perturba-tions to the Asc pool through changes in Asc biosyn-thesis (vtc mutants) or recycling (DHAR-silenced lines)can affect photosynthetic function under certain growthconditions suggests that the Asc pool is important toleaf function. The correlations between changes inDHAR activity and changes in the foliar level of H2O2(Chen and Gallie, 2004), rate of CO2 assimilation, andthe level of RbcL, LHCII, and chlorophyll are consistentwith the conclusion that the slow growth phenotypeexhibited by plants suppressed for DHAR is related toreduced photosynthetic functioning and increased ox-idative load. The reduced photosynthetic function inDKD leaves may lead to an earlier onset of carbonstarvation that can induce autophagy (Moriyasu andOhsumi, 1996; Brouquisse et al., 1998). The higher rateof chlorophyll loss in carbon-starved (i.e. dark-treated)DKD leaves relative to control leaves (Fig. 4) is consistentwith this possibility. The higher level of ROS (suchas H2O2) present in DKD leaves (Chen and Gallie,2004) may also promote leaf aging and senescencethrough increased signaling that induces expression ofsenescence-related genes known to be enhanced inleaves experiencing a higher oxidative load (Navab-pour et al., 2003). It is also possible that a lower (i.e.more oxidized) Asc redox state in plants suppressed forDHAR contributes to slower rate of cell division and/or elongation through mechanisms that are separatefrom a lower fixed carbon supply resulting from a lowerrate of photosynthesis. Asc promotes G1 to S progressionof cells within the onion (Allium cepa) root meristem andpericycle (for review, see Smirnoff, 1996) and reversedthe inhibition of cell division caused by lycorine treat-ment, which reduces Asc content (Arrigoni, 1994). How-ever, any such effect would be limited to growing tissuesand would not account for the observed reduction inchlorophyll, RbcL, or CO2 assimilation in expandingDKD leaves or the accelerated loss of chlorophyll, RbcL,LHCII, or CO2 assimilation from mature DKD leaves.Moreover, the prolonged maintenance of leaf function inplants overexpressing DHAR supports a role for thisenzyme in leaf aging. Thus, these observations supportthe conclusion that an important function of DHAR inleaves is to maintain photosynthetic function by limitingROS-mediated damage.

MATERIALS AND METHODS

Plant Transformation and Growth Conditions

Full-length wheat (Triticum aestivum) and tobacco (Nicotiana tabacum)

DHAR cDNAs (accession nos. AY074784 and AY074787, respectively) were

isolated as described previously (Chen et al., 2003). Transgenic tobacco (cv

Xanthi) expressing the His-tagged wheat DHAR from the CaMV 35S promoter

(in the binary vector, pBI101) was generated using Agrobacterium tumefaciens as

described (Chen et al., 2003). Transgenic tobacco plants suppressed for DHAR

were identified following the introduction of a tobacco DHAR construct in

pBI101.

All plants were grown in commercial soil in a glasshouse supplied with

charcoal-filtered air. Experiments were carried out and repeated from fall to

spring to avoid excessive heat or light. Plants were watered to saturation

twice/day (7 AM and 1 PM) to ensure even soil moisture. Plants were grown

under natural light conditions in a 10-h light and 14-h dark cycle. The average

temperature during the day was 25.9�C 6 0.6�C and 20.2�C 6 0.5�C during the

night. The average light intensity in the morning (9 AM) was 514 6 206 mmol

m22 s21 and in the afternoon (1 PM) was 1,191 6 244 mmol m22 s21.

To determine leaf area during leaf expansion, digital images were taken at

3-d intervals. Leaf area was calculated using Adobe Photoshop (version 6.0)

by normalizing total leaf size to an internal standard included in each image.

Eight leaves at each developmental stage were measured, and the data were

processed using Microsoft Excel. Leaf area of whole plants was determined

using the same approach by measuring the area of every other leaf. For leaf

expansion studies, we used the sixth true leaf as numbered from the plant

base. The area of the sixth true leaf of DOX, DKD, and control plants measured

when the leaf was fully expanded remained constant for a given line when the

plants were grown under identical conditions. Knowing the area of the sixth

true leaf in its fully expanded state, corresponding leaves were collected from

subsequently grown plants when they were 8%, 50%, or 100% of the size of the

fully expanded state.

Western Analysis

Anti-DHAR antiserum raised against DHAR purified from wheat seed-

lings was used for western analysis. Protein extracts were resolved using

standard SDS-PAGE and the protein transferred to 0.22 mm polyvinylidene

difluoride membrane by electroblotting. Following transfer, the nitrocellulose

membranes were blocked in 5% milk in TPBS (0.1% Tween 20, 137 mM NaCl,

2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) followed by incubation

with primary antibodies diluted typically 1:1,000 to 1:2,000 in TPBS with 1%

milk for 1.5 h. The blots were then washed twice with TPBS and incubated

with goat anti-rabbit horseradish peroxidase-conjugated antibodies (Southern

Biotechnology Associates) diluted to 1:5,000 to 1:10,000 for 1 h. The blots were

washed twice with TPBS and the signal detected typically between 1 to 15 min

using chemiluminescence (Amersham). Each western was repeated three to

four times and representative results presented. The results from the western

analysis were quantitated using MicroComputer Imaging Device Elite image

processing software (version 7.0, Imaging Research).

Enzyme Assays

DHAR activity was assayed essentially as described (Hossain and Asada,

1984). Tobacco leaves were ground in extraction buffer (50 mM Tris-HCl, pH

7.4, 100 mM NaCl, 2 mM EDTA, 1 mM MgCl2) and soluble protein obtained

following a 5-min centrifugation at 13,000 rpm. DHAR was assayed from an

equal amount of protein as described (Bradford, 1976) in 50 mM K2HPO4/

KH2PO4 pH 6.5, 0.5 mM DHA, and 1 mM reduced glutathione and its activity

followed by an increase in A265. APX activity was determined as described (de

Pinto et al., 2000). Each experiment was repeated two to three times and

representative results presented.

Asc and DHA Measurements

Asc was measured as described (Foyer et al., 1983). Leaves were ground in

2.5 M HClO4 and centrifuged at 13,000 rpm for 10 min. Two volumes of 1.25 M

Na2CO3 were added to the supernatant and following centrifugation, 100 mL

was added to 895 mL 100 mM K2HPO4/KH2PO4, pH 5.6. Asc was determined

by the change in A265 following the addition of 0.25 units ascorbate oxidase.

The total amount of reduced and oxidized Asc (i.e. Asc and DHA) was

determined by reducing DHA to Asc (in a reaction containing 100 mM

K2HPO4/KH2PO4, pH 6.5, 2 mM reduced glutathione, and 0.1 mg recombinant

wheat DHAR protein incubated at 25�C for 20 min prior to measuring Asc).

The amount of DHA was determined as the difference between these two

assays.





TBARS Assay

TBARS were measured following the approach essentially described by

Larkindale and Knight (2002). A total of 0.5 g of leaf was ground in liquid

nitrogen in a 1.5-mL microfuge tube using a micropestle. Then, 0.5 mL of 0.5%

(w/v) thiobarbituric acid in 20% (w/v) trichloroacteic acid and 0.5 mL of

buffer (50 mM Tris-HCl, pH 8.0, 175 mM NaCl) was added. Following heating

at 95�C for 25 min and pelleting the cell debris, the absorbance of the

supernatant was measured at 532 nm, with the A600 subtracted to account for

nonspecific turbidity. The amount of malonaldehyde was calculated using an

exciting coefficient of 155 mM21 cm21, in agreement with a standard curve

relating malonaldehyde concentrations to absorbance. The measurements

were repeated two times and representative results presented.

Chlorophyll Measurements

Chlorophyll a and b were measured spectrophotometrically as described

(Jeffrey and Humphrey, 1975). In brief, leaf samples were ground in liquid

nitrogen and extracted with 90% (v/v) acetone. The A664 and 647 nm was

determined and used to calculated chlorophyll a and b content by the

equations: Chl a 5 11.93A664 2 1.93A647 and Chl b 5 20.36A647 2 5.50A664, re-

spectively. Each experiment was repeated two to three times and represen-

tative results presented.

Leaf Function Assays

For all experiments, plants were grown under daylight conditions of

approximately 500 mmol m22 s21 in the morning and 1,200 mmol m22 s21 in the

afternoon. For all experiments, measurements were collected at the same time

each day (between 10:30 AM and 11:30 AM) during the course of the experiment.

In situ rates of CO2 assimilation, transpiration, and stomatal conductance

were measured with TPS-1 portable photosynthesis system. For whole plant

assays, every second leaf on a plant was measured. Each experiment was

repeated two times and representative results presented.

RT-PCR Analysis

Total nucleic acid was isolated from whole leaves using the RNeasy Plant

Mini kit (Qiagen). Residual genomic DNA was removed by on-column

DNAse I digestion, using RQ1 RNase free DNase I (Promega). We used

0.5 mg total RNA for cDNA synthesis using Omniscript RT (Qiagen) with

oligo(dT)20 as the primer. PCR reactions contained 1 mL of the RT reaction, and

1.5 mg of forward and primers in a total reaction volume of 25 mL and

were performed with the following conditions: initial denaturing step, 94�C/

15 min; 35 cycles of 94�C/15 s, 52�C/30 s, 72�C/60 s, and a final extension step

of 72�C/5 min. PCR products were visualized on an ethidium bromide-

stained 1.2% agarose gel. Primers used were: actin (X63603) forward,

5#-CGCGAAAAG-ATGACTCAAATC-39 and reverse, 5#-AGATCCTTTCT-

GATATCCACG-3#; tobacco CP1 (AY881011) forward, 5#-CTTTATCAGAGC-

AAGAGCTTG-3# and reverse, 5#-TTTTGATGCGCATATATCCAC-3#.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AY074784 and AY074787.

ACKNOWLEDGMENTS

We thank Dr. Tadahiko Mae for the gift of anti-Rubisco antiserum and

Dr. Bruce Kohorn for the gift of anti-LHCII antiserum

Received June 20, 2006; accepted July 28, 2006; published August 4, 2006.

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