Environmental and Experimental Botany 56 (2006) 182–189

Gas exchange analysis and chlorophyll a fluorescence in cotyledons ofthe xan1 sunflower mutant with defects in light energy utilization

Lucia Guidi a,∗, Elena Degl’Innocenti a, Marco Fambrini b,Claudio Pugliesi b, Gian Franco Soldatini a

a Dipartimento di Chimica e Biotecnologie Agrarie, Via del Borghetto 80, I-56124 Pisa, Italyb Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti 1/B, I-56124 Pisa, Italy

Received 13 September 2004; accepted 24 January 2005

Abstract

Mutants with a depigmentated phenotype and characterized by Mendelian inheritance can be useful to analyze the role of nuclear genes inchloroplast biogenesis and photosynthesis. In the important oil crop sunflower, we have recently demonstrated that under dim light conditionsthe xantha1 (xan1) mutant develops true leaves unable to express and retain PSII activity. In order to investigate if this mutation induces anotanioessX©

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cbsiTtdt

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rgan specific effect on photosynthetic activity, the light energy utilization of cotyledons (7, 15 and 18 days after sowing) was characterizedhrough gas exchange and chlorophyll a fluorescence. The xan1 cotyledons are unable to fix CO2 at normal rates. However, rubisco specificctivity is not different in the wild-type (wt) and the xan1 seedlings, indicating that biochemical reactions of the photosynthetic process wereot altered. The photochemical potential of PSII, measured as the dark-adapted quantum efficiency of PSII (Fv/Fm) was strongly decreasedn xan1 cotyledons and is attributable to a significant increase in F0 values. The light-adapted quantum efficiency (ΦPSII) and the efficiencyf excitation capture of PSII (Φexc.), were significantly depressed in xan1 cotyledons as compared to the wt. Under light conditions, the mostvident result is the strong decrease of ΦPSII, which is linked to a decrease in excitation energy pressure and also to a strong increase in qNP. Inummary, with analogy to results previously obtained in leaves, the xan1 cotyledons have a reduced number of photosynthetic units and highensibility of the PSII reaction centres to photodamage even in low light conditions. We conclude that the loss-of-function of the sunflowerAN1 nuclear gene affects the light energy utilization of chloroplasts in an organ-independent way.2005 Elsevier B.V. All rights reserved.

eywords: Helianthus annuus; Cotyledons; Fluorescence analysis; Photosynthesis; Photosystem II activity; Rubisco assay

. Introduction

Photosynthesis involves light energy harvesting and itsonversion into chemical energy by means of four mem-rane polypeptide complexes (PSII, Cyt b6/f, PSI and ATPynthase). The products ATP and NADPH are then utilizedn the Calvin–Benson cycle to drive CO2 photoassimilation.he interactions between the different component subsys-

ems are very complicated and the whole process involvesevelopmental aspects, regulatory mechanisms and acclima-ion responses (Horton, 2000).

∗ Corresponding author. Tel.: +39 050 9719320; fax: +39 050 598614.E-mail address: guidilu@agr.unipi.it (L. Guidi).

The biogenesis of photosynthetic machinery involvesthe light-dependent conversion of nongreen plastids togreen, photosynthetically competent plastids (Thomson andWhatley, 1980) and the primary reactions of photosynthesisrequire chloroplasts with fully differentiated thylakoid mem-brane system (Vothknecht and Westhoff, 2001).

In species such as sunflower, the development of photo-synthetic competence appears first in cotyledons, then laterin leaves. In addition to storage function, cotyledons haveassimilatory activity and the importance of this competenceis corroborated by the delayed development of primary leavesas long as functioning cotyledons are left on the devel-oping seedling (Kutschera, 1992; La Rocca et al., 1996;Jucknischke and Kutschera, 1998).

098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2005.01.013

L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189 183

Despite both cotyledons and leaves containing photosyn-thetically active plastids, chloroplast development in the twoorgans follows different paths. For example, in Phaseolusvulgaris, a very early development of cotyledonary plastidswith small grana has been observed 16 days after flower-ing. This ultrastructural aspect is transitory but not describedduring the development of primary leaves (Opik, 1968).Analogously, in the embryonic cotyledons of Glycine maxpeculiar stages of plastid differentiation have been observed(Saito et al., 1989). Moreover, during the initial seedlinggrowth, cotyledons exert a specific control of chloroplastdevelopmental ontogenesis of leaf chloroplasts (Whatley,1977).

Several light-regulated chloroplast genes show differ-ent spatial and temporal patterns of expression in dif-ferent seedling organs (Saito et al., 1990; Chang andWalling, 1992; Kretsch et al., 1995; Sato-Nara et al.,2004). In the plant model Arabidopsis thaliana, somegenes with organ-specific roles have been reported (Pilgrimet al., 1998; Yamamoto et al., 2000; Privat et al.,2003). Furthermore, at the protein level, Kim and Apel(2004) have identified a difference between cotyledonsand true leaves for the substrate-dependent import of theA type of NADPH-dependent protochlorophyllide oxido-reductase.

Mutations that affect pigmentation and/or seedling viabil-iripcpS

daerw(coo2

efagrolefes

2. Materials and methods

2.1. Plant material

The xantha1 mutant (xan1) analysed in this study is a spon-taneous and monogenic mutant, found in a selfed progenyof the inbred line AC/2224 (Dipartimento di Biologia dellePiante Agrarie, Sezione di Genetica, Pisa, Italy). This nuclearmutant is lethal and can be maintained only by selfing het-erozygous (XAN1/xan1) plants (Fambrini et al., 2004).

2.2. Growth conditions

Achenes from homozygous (XAN1/XAN1) wild-type (wt)plants and from heterozygous plants (XAN1/xan1) were ger-minated in the dark in Petri dishes, on filter papers moistenedwith distilled water, at 23 ± 1 ◦C. After 3 days, the germinatedseeds were transferred to 8 cm diameter pots containing amixture of soil and sand. Wild-type (XAN1/XAN1) and mutant(xan1/xan1) seedlings were grown for two to three weeks ina growth chamber at 23 ± 1 ◦C, under a 16-h photoperiodand dim light (3.5 �mol m−2 s−1, PPFDs). Irradiance wasprovided by a mercury vapour lamp (Osram HQI-TS 250W/NDN, Wembley, England). Dim light was used to mini-mize photooxidative damage in the mutant seedlings.

2

uFGWciCS

wtt(ts

2

wtfhF(

ty are easily scored and despite their pleiotropic nature, rep-esent the ordinary material to study genetic factors involvedn plastid development and photosynthetic performance. Inarticular, mutants characterized using chlorophyll fluores-ence kinetics have been used to elucidate essential chloro-lastic functions in light energy utilization (Leister, 2003;tern et al., 2004).

We have recently characterized a nuclear and recessiveepigmentated lethal mutant (xantha1; xan1) of Helianthusnnuus, which dies at the cotyledonary stage (Fambrinit al., 2004). The xan1 leaves were unable to express andetain full PSII activity and the values of Fv/Fm and qPere consistent with the high-chlorophyll-fluorescence

hcf) mutants. An extensive physiological and biochemicalharacterization of leaf plastids suggested an involvementf the XAN1 protein in the biogenesis and maintenancef the thylakoid membrane system (Fambrini et al.,004).

In this study, we extend the characterization of theffects of the mutation in a photosynthetic organ differentrom the true leaves. In line with this, here we present annalysis of xan1 cotyledons photosynthesis by means ofas exchange analysis and chlorophyll a fluorescence. Ouresults show that the photosynthetic activity in cotyledonsf the mutant seedlings is impaired. Together with the ear-ier data previously obtained about true leaves (Fambrinit al., 2004), these results clearly demonstrate that theunction of the XAN1 gene is not organ-dependent butssential to ensure photosynthetic competence in sunflowereedlings.

.3. Gas exchange analysis

Measurements of gas exchange were made on cotyledonssing an open system (CMS-400, Walz, Effeltrich, Germany).or details of the experimental procedures, see Soldatini anduidi (1992). During gas exchange measurements (GK-022,alz), the temperature was 25 ± 2.8 ◦C, RH 65 ± 12%, CO2

oncentration 350 �mol mol−1 and O2 21%. Responses torradiance (0–1500 �mol m−2 s−1, PPFD) of photosyntheticO2 assimilation in the seedlings were calculated using themith equation:

ΦCO2 × I × Amax√Amax + ΦCO2 + I2

here ΦCO2 is the quantum yield for CO2 uptake, Amax ishe CO2 assimilation rate at light saturation level and I ishe irradiance (Tenhunen et al., 1976). Stomatal conductanceGw), transpiration rate (E) and intercelluar CO2 concen-ration (Ci) were measured at 350 �mol mol−1 CO2 and ataturating light.

.4. Chlorophyll a fluorescence

Modulated chlorophyll a fluorescence measurementsere made with a PAM-2000 fluorometer (Walz, Effel-

rich, Germany) on cotyledons, which were dark-adaptedor 40 min. The fluorometer was connected to a leaf-clipolder (2030-B, Walz) with a tri-furcated fiberoptic (2010-, Walz) and to a computer with data acquisition softwareDA-2000, Walz). For details of measurements, see Guidi

184 L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189

et al. (2002). The saturation pulse method was used forthe analysis of quenching components (Schreiber et al.,1986), which was carried out at a photon fluence of about35 �mol m−2 s−1. Quenching components qP and qNP werecalculated as defined by Schreiber et al. (1986). The term(1 − qP) was used as an estimate of the reduction state ofthe primary quinone electron acceptor (QA). The actual PSIIefficiency (ΦPSII) was calculated as (F ′

m − Fs)/F ′m (Genty et

al., 1989). The values of Φexc.(= F ′v/F

′m), and the apparent

electron transport rate, ETR = ΦPSII × PFD × a × f, assum-ing the absorptivity of photosynthetic active radiation, a,in the leaves as 0.84, and setting the light distribution fac-tor between photosystems I and II, f, as 0.5 were computedaccording to Schreiber et al. (1986). Relative excessive PPFDwas obtained as (Fv/Fm − ΦPSII)(Fv/Fm) (Bilger et al., 1995).The product of Φexc.(= F ′

v/F′m) and (1 − qP) is the fraction

of excitation energy not dissipated in the antennae that can-not be utilized for photochemistry, and may lead to damagingsinglet oxygen formation (Demmig-Adams et al., 1996).

To measure the fluorescence-PPFD response, cotyle-dons of the wt and the xan1 mutant were held horizon-tally in the leaf-clip holder. The PPFD on the cotyledons,provided by a halogen lamp (2050-H, Walz, Effeltrich,Germany), was adjusted from darkness to saturating lightin steps of 50–200 �mol m−2 s−1 for both plants. For thexan1 mutant, the maximal light intensity reached wasa1eewnttt

2

omTD4iT�a5upwb3pL

2.6. Statistical analysis

The data were derived from three independent experi-mental trials. Data were subjected to a one-way analysis ofvariance (ANOVA) with mutation as a factor. For comparisonof the means, the Student’s t-test was used. For the responseof the fluorescence parameter to increasing irradiance levels,polynomial second-order curves were fitted.

3. Results

3.1. Gas exchange and rubisco activity

All gas exchange parameters reported in Table 1 weredetermined at a light saturation level, which was differentin the wt and xan1 cotyledons. Indeed, while the wt showeda level of light intensity for saturation of the CO2 assimilationin the range of 1200–1500 �mol m−2 s−1, photosynthesis inthe xan1 mutant saturated at 200 �mol m−2 s−1. The wt CO2fixation rate at light saturation was significantly higher thanin the xan1 cotyledons (Table 1). Stomatal conductance wassimilar in the mutant and wt, while the transpiration rate wassignificantly lower in the mutant cotyledons. On the con-trary, intercellular CO2 concentration (Ci) was significantlyhigher in xan1. Collectively, data from gas exchange analysisipo

f

3

dostc

TCaHcfl

AGEC

TCNl*

bout 600 �mol m−2 s−1, while it reached values of about200 �mol m−2 s−1 in the wt. The halogen lamp wasquipped with a heat-reflecting filter to reduce heat gen-ration by the lamp. PPFD on cotyledons was monitoredith a microquantum sensor installed on the leaf-clip holderext to the spot where fluorescence was measured. Afterhe cotyledon was exposed to the desired PPFD for 20 min,he chlorophyll a fluorescence of PSII was measured usinghe PAM-2000.

.5. Rubisco assay

Rubisco activity was determined according to the methodf Sawada et al. (1990) and Holaday et al. (1992) with minorodifications, using an extraction buffer containing 250 mMris–HCl (pH 7.8), 50 mM MgCl2, 2.5 mM EDTA and 37.5 gTT. The extract was centrifuged at 10,000 × g for 10 min at◦C and the supernatant was immediately used to determine

nitial rubisco activity in an assay buffer containing 50 mMris–HCl (pH 7.8), 10 mM MgCl2, 30 mM NaHCO3, 5 mM-MeSH and 5 mM ATP. The following compounds werelso added: 5 units of 3-phosphoglyceric phosphokinase,

units of glyceraldehyde-3-phosphate dehydrogenase, 1nit of glycero-3-phosphate dehydrogenase, 6 units of triosehosphate isomerase and 0.15 mM NADH. The reactionas initiated by the addition of 5 mM of ribulose-1,5-isphosphate and the oxidation of NADH was monitored at40 nm. Rubisco content was expressed on the total solublerotein determined using the Bio Rad kit II assay (Bio-Radab., Hercules, USA).

ndicate that, in the xan1, the photosynthetic rate was com-romised and the alteration was linked to a poor utilizationf CO2.

The specific activity of rubisco was not significantly dif-erent in wt and xan1 cotyledons (Table 2).

.2. Chlorophyll a fluorescence analysis

Chl fluorescence induction transients were used as a non-estructive technique to monitor the photosynthetic statusf intact cotyledons from the xan1 and the wt sunflowereedlings. The xan1 cotyledons had a higher F0 comparedo the wt at 7, 15 and 18 days of growth (Fig. 1A). This indi-ated that, apart from the cotyledon age, an increased frac-

able 1O2 fixation rate at light saturation level (Amax; �mol CO2 m−2 s−1), stom-tal conductance (Gw; mmol H2O m−2 s−1), transpiration rate (E; mmol

2O m−2 s−1) and intercellular CO2 concentration (Ci; �mol mol−1) inotyledons of wild-type and the xan1 mutant of sunflower grown at a photonuence of 3.5 �mol m−2 s−1 of PPFD for 15 days

Wild-type xan1 P

max 1.51 (0.32) 0.19 (0.01) *

w 31.0 (8.48) 24.9 (0.07) NS0.87 (0.08) 0.50 (0.01) *

i 278 (5.66) 325 (0.71) **

he measurements were carried out at 25 ◦C, 65% RH, 350 �mol mol−1

O2 concentration and 21% O2. Data are the mean of three replicates.umbers in brackets indicate the standard deviation of the mean. In the

ast column the significance of Student’s t-test is reported. NS: P > 0.05;P < 0.05; **P < 0.01.

L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189 185

Table 2Specific activity of rubisco enzyme in cotyledons of wild-type and the xan1mutant of sunflower

Wild-type 0.367 (0.002)xan1 0.383 (0.004)P NS

Rubisco activity was expressed as �mol CO2 min−1 mg−1 prot. Seedlingshave been grown for 15 days at an irradiance of 3.5 �mol m−2 s−1. Dataare the mean of three replicates. Numbers in brackets indicate the standarddeviation of the mean. In the last row the significance of Student’s t-test isreported. NS: P > 0.05.

tion of the excitation energy received by the light-harvestingpigment-proteins was not used to drive electron transportbut was re-emitted as fluorescence. In the xan1 mutant, verylow variable fluorescence was detected indicating that PSIIwas completely inactive and that linear electron transport

FaoP4dad

flow was interrupted. Therefore, the maximum photochemi-cal efficiency of PSII (measured as Fv/Fm) was strongly andsignificantly reduced in the mutant cotyledons (Fig. 1C).

The parameters derived from quenching analysis carriedout at an actinic light of about 35 �mol m−2 s−1 are reportedin Figs. 2 and 3. The apparent relative electron transport rate(ETR) (Fig. 2A) was lower in the wt, but this was largelydue to the actinic light utilized during the quenching analy-sis. ETR was strongly and significantly reduced in the xan1cotyledons. A similar pattern was recorded for the quantumyield of linear electron transfer via PSII (ΦPSII; Fig. 2B) andfor Φexc. (Fig. 2C), which reflects the intrinsic efficiency ofopen PSII reaction centres in the light-adapted state. Thefraction of light absorbed by PSII antennae that was neitherutilized in photosynthetic electron transport nor dissipatedthermally was estimated from Φexc. × (1 − qP) and labelledas “excess energy”. In the mutant, this excess was statisticallyhigher than in the wt (Fig. 2D).

The fraction of QA, the primary electron acceptor of PSII,present in a reduced state (1 − qP; Fig. 3A) increased inthe mutant, together with the non-photochemical quenchingcoefficient, qNP, which significantly increased in the xan1cotyledons, except for the value recorded at 15 days of growth(Fig. 3B).

Figs. 4 and 5 show PPFD response curves for some of thefluorescence parameters measured at the end of the experi-mctuwwpiwSipav

t

ig. 1. Minimal (F0; A) and maximal (Fm; B) chlorophyll fluorescencend Fv/Fm ratio (C) in cotyledons of wild-type (white) and xan1 (gray)f sunflower seedlings grown at a photon fluence of 3.5 �mol m−2 s−1 ofPFD. The measurements were carried out on cotyledons dark-adapted for0 min and 7, 15 and 18 days after the sowing. The bars indicate the standardeviation of the means. The significance of the difference between wild-typend the xan1 mutant, calculated with the Student’s t-test is reported for theifferent growth phases. ***P < 0.001; **P < 0.01; NS: P > 0.05.

dAolplpo(inmsr

ent (15 days after sowing). ETR values showed that mutantotyledons were characterized by a lower apparent electronransport rate than the wt (Fig. 4A) and also the ΦPSII val-es were strongly depressed in the mutant in comparison tot (Fig. 4B). The reductions of ETR and ΦPSII in the mutantere detected at even relatively low light intensities. Anotherrominent difference between the wt and the mutant was vis-ble in Φexc., which decreased more rapidly in the mutantith increasing irradiance in comparison to the wt (Fig. 4C).trongly increased relative excessive PPFDs at increasing

rradiances were observed in the mutant cotyledons as com-ared to the wt (Fig. 4D). Marked differences between the wtnd the mutant were evident for this parameter also even atery low light intensities.

In comparison to the wt, qNP in the mutant was foundo have increased at any given irradiances (Fig. 5A); theifferences were established even at very low irradiance.dditional information of qNP can be derived from in vivo flu-rescence traces by plotting 1/F ′

m and 1/F ′0 − 1/F ′

m versusight intensities (Fig. 5B and C). In a first approximation, thearameter 1/F ′

m is a measure of the combined nonradiativeosses in light-harvesting antennae, whereas the fluorescencearameter 1/F ′

0 − 1/F ′m is a measure for the rate constant

f excitation energy trapped by the PSII reaction centresHavaux et al., 1991). Consistent with this idea, a declinen 1/F ′

0 − 1/F ′m has been correlated with the decline in the

umber of functional PSII units (Park et al., 1995) and theagnitude of 1/F ′

m was found to depend on the antennaeize of PSII (Briantais, 1994; Lokstein et al., 1993). Aseported in Fig. 5B and C, 1/F ′

m was strongly decreased

186 L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189

Fig. 2. Electron transport rate (ETR; A), actual quantum yield of PSII photochemistry (ΦPSII; B), intrinsic PSII efficiency (Φexc.; C) and excess energy (D) incotyledons of wild-type (white) and xan1 mutant (gray) of sunflower seedlings grown at a photon fluence of 3.5 �mol m−2 s−1 of PPFD. The measurementswere carried on cotyledons at a light of about 35 �mol m−2 s−1 and 7, 15 and 18 days after the sowing. The bars indicate the standard deviation of the means. Thesignificance of the difference between wild-type and xan1 mutant, calculated with the Student’s t-test is reported for the different growth phases. ***P < 0.001;**P < 0.01.

Fig. 3. Reduction state of the primary PSII electron acceptor (1 − qP; A)and non-photochemical quenching coefficient (qNP; B) in cotyledons ofwild-type (white) and xan1 mutant (gray) of sunflower seedlings grownat a photon fluence of 3.5 �mol m−2 s−1 of PPFD. The measurements werecarried on cotyledons at a light of about 35 �mol m−2 s−1 and 7, 15 and 18days after the sowing. The bars indicate the standard deviation of the means.The significance of the difference between wild-type and xan1 mutant, cal-culated with the Student’s t-test, is reported for the different growth phases.***P < 0.001; **P < 0.01; NS: P > 0.05.

and 1/F ′0 − 1/F ′

m was practically undetectable in the mutantcotyledons. These results suggest a relative lower contribu-tion of antenna-associated processes to the amplitude of qNPin the mutant than in the wt.

4. Discussion

Exogenous and endogenous factors affect the conver-sion of proplastids into photosynthetically competent chloro-plasts. Light is the most important environmental cueto control the differentiation process while plastid- andnuclear-encoded genes are required to establish the tran-scription/translation apparatus (Rochaix, 2004). In addition,the pathway of chloroplast biogenesis depends on the organtype analysed. For example, in some species during seed-maturation a precocious differentiation into chloroplast fol-lowed by subsequent de-differentiation into proplastids canbe specifically observed in cotyledons (Opik, 1968). There-fore, if a recessive mutation affects this developmental aspect,a differential phenotype between cotyledon and primaryleaves is observed. In line with this, a cotyledon-specificalbino locus has been identified on Chromosome 1 of theArabidopsis genome (Yamamoto et al., 2000).

Recently, we have reported the characterization of a lethalmraI

utant of sunflower characterized by true leaves with aber-ant development of chloroplasts, deficient pigment contentnd reduced CO2 assimilation rate (Fambrini et al., 2004).n this report, we wanted to determine whether the loss of

L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189 187

Fig. 4. Response curves of electron transport rate (ETR; A), actual quantum yield of PSII photochemistry (ΦPSII; B), intrinsic PSII efficiency (Φexc.; C) andrelative excessive PPFD (D) in function of irradiance in cotyledons of wild-type (open circle) and xan1 (closed circles) of sunflower seedlings grown at a photonfluence of 3.5 �mol m−2 s−1 of PPFD. The measurements were carried on cotyledons 15 days after the sowing.

function of the XAN1 gene induces the deleterious alterationon the photochemistry efficiency in cotyledons as well.

The xan1 mutant of sunflower is pigment deficient andhighly sensitive to light stress (Fambrini et al., 2004). There-fore, in order to minimize pleiotropic effects, the analyses of

gas exchange and chlorophyll a fluorescence were performedwith seedlings grown under dim light (3.5 �mol m−2 s−1).

The CO2 assimilation rate of the xan1 cotyledons wasstrongly depressed as compared to the wt. The variation inphotoassimilation did not correlate to stomatal closure, but

F adiativee tion ofo FD. Th

ig. 5. Response curves of non-photochemical quenching (qNP; A), non rxcitation energy trapping by PSII reaction centres (1/F ′

0 − 1/F ′m; C) in func

f sunflower seedlings grown at a photon fluence of 3.5 �mol m−2 s−1 of PP

losses in light-harvesting complexes (1/F ′m; B) and the rate constant of

irradiance in cotyledons of wild-type (open circle) and xan1 (closed circles)e measurements were carried on cotyledons 15 days after the sowing.

188 L. Guidi et al. / Environmental and Experimental Botany 56 (2006) 182–189

was attributable to an alteration in the activity of mesophyll.This is confirmed by the increase of Ci displayed by mutantcotyledons. Therefore, we can postulate that this mutant hasdefects in the primary photosynthetic process. However, thespecific activity of rubisco was not affected, which sug-gests that the “dark reactions” were presumably not alteredin xan1.

As reported by Fambrini et al. (2004), the total chloro-phyll content of xan1 cotyledons was only 56% of that of thewild-type, and the pigment deficiency clearly indicated thatmutant seedlings have a reduced number of photosyntheticcomplexes. This is supported also by the strong decrease inthe values of Fv/Fm observed in the mutant. Meurer et al.(1996) divided hcf mutants of A. thaliana into five groupswith those characterized by a value of Fv/Fm ratio lower than0.5 (Group III) reflecting PSII as being the mutational target.These genotypes were characterized by an Fv/Fm ratio of 0.5due to an increase in F0. Analogously, in the xan1 mutantcotyledons a strong decrease in Fv/Fm was observed and waslinked to a large increase in F0 values. These F0 could reflecta partially closed PSII reaction centre or a disconnection ofthe perypheral antenna system from the reaction centre. Itis important to underline that these effects on chlorophyllfluorescence parameters are detected in seedlings grown indim photon fluence (3.5 �mol m−2 s−1). Moreover, anotherimportant finding was the altered chlorophyll fluorescencepTflh

PbtdrPewacvo(

nx(epp(

g1r

dependent on the antenna size of PSII (Briantais, 1994).Apparently, there is a strong decrease in 1/F ′

m with increas-ing irradiance in the xan1 mutant and this seems to indicatea lower contribution of antenna-associated processes to themagnitude of qNP in the mutant in comparison to the wt. Theparameter 1/F ′

0 − 1/F ′m has been linked to a decrease in the

number of functional PSII units (Park et al., 1995), and it wasundetectable in the xan1 mutant suggesting, furthermore, thatthe product of the XAN1 gene is fundamental for PSII activity.

The excess energy determined as Φexc. × (1 − qP) indi-cates the excitation energy absorbed by closed PSII reactioncentres which was not dissipated as heat (Demmig-Adams etal., 1996). The accumulation of excitation energy in closedPSII may generate long-lived excited states of chlorophyll(3Chl*) and singlet oxygen (1O2). Therefore, the amountof the excess energy parameter in xan1 may lead to a pro-portional increase in the production of 1O2. The amount ofexcess energy in the mutant seems more pronounced whenphoton fluence increases. This excess energy, which is nei-ther utilized not dissipated, can cause the photoinactivationof PSII (Kato et al., 2003).

The analysis of the response of the Chl fluorescenceparameters versus increasing light intensities confirmed thatthe electron transport rate through PSII was very low at all lev-els of irradiance. The strong increase in relative excess PPFDobserved in xan1 cotyledons at low irradiance confirmed thata

oaplobfrl2rbitrilf

R

B

B

arameters of xan1 cotyledons within 7 days of germination.herefore, we can conclude that the alteration of chlorophylluorescence parameters was not the harmful consequence ofigh light stress or carbon starvation.

In the mutant, the light-adapted quantum efficiency ofSII (ΦPSII) was found to have considerably decreased underoth non-saturating and saturating irradiances as comparedo the wt. This decline in PSII was fully accounted for by theecrease in Φexc. and also by the increase in (1 − qP). It iseasonable that the over-reduction of the electron acceptors ofSII was directly related to the enhanced decay of the singletxcited Chl to triplet Chl which in turn, has potential to reactith oxygen to form toxic singlet oxygen (Demmig-Adams

nd Adams, 1993). Moreover, the strong depletion of ΦPSIIould also be connected to the observed decrease in Φexc. byirtue of pro-active and photoprotective thermal dissipationf excess excitation energy before it reaches the PSII centresDemmig-Adams et al., 1995).

Increase in thermal dissipation leads to an increase in theon-photochemical quenching coefficient, as observed in thean1 cotyledons. This coefficient has several componentsKrause and Weis, 1991) and the major one is the pH or high-nergy-dependent factor, which, in turn, down regulates thehotochemical efficiency of PSII. Its formation is thought torotect the photosynthetic apparatus against photoinhibitionBilger and Bjorkman, 1990).

As reported above, additional information on the ori-in of qNP can be derived from the parameters 1/F ′

m and/F ′

0 − 1/F ′m. The first parameter is a measure of the non-

adiative losses in light-harvesting antennae and is strictly

n excess of light energy was not utilized.In conclusion, with analogy to the results previously

btained in leaves (Fambrini et al., 2004), a high value ofbsorbed light energy cannot be utilized by cotyledonarylastids and is re-emitted as fluorescence. The defects inight energy utilization are not related to genetic alterationf the biochemical reaction of the Calvin cycle as suggestedy the results of rubisco activity. Instead, the photosyntheticunctions in the mutant are precluded probably because theeduced number of PSII units are functionally impaired. Col-ectively, the evidence previously obtained (Fambrini et al.,004) and the new data here reported demonstrate that theole of the XAN1 gene is essential for light energy utilisationoth in true leaf and cotyledon chloroplasts, and probably, its involved in the same developmental pathway. In particular,he data previously reported (Fambrini et al., 2004) and theesults here described jointly suggest that the XAN1 proteins required to organize a stable and structurally correct thy-akoid system as well as to fully express and retain the PSIIunction, in organ independent-way.

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