Photosystem II efficiency and mechanisms of energy dissipation in iron-deficient, field-grown pear trees (Pyrus communis L.)

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1 Photosynthesis Research 63: 9 21, Kluwer Academic Publishers. Printed in the Netherlands. 9 Regular paper Photosystem II efficiency and mechanisms of energy dissipation in iron-deficient, field-grown pear trees (Pyrus communis L.) Fermín Morales, Ramzi Belkhodja, Anunciación Abadía & Javier Abadía Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (C.S.I.C.), Apdo. 202, E-50080, Zaragoza, Spain; Author for correspondence Received 18 February 1999; accepted in revised form 4 November 1999 Key words: chlorophyll fluorescence, energy dissipation, field-grown pear, iron deficiency, Photosystem II efficiency, Pyrus communis Abstract The dark-adapted Photosystem II efficiency of field-grown pear leaves, estimated by the variable to maximum chlorophyll fluorescence ratio, was little affected by moderate and severe iron deficiency. Only extremely irondeficient leaves showed a decreased Photosystem II efficiency after dark adaptation. Midday depressions in Photosystem II efficiency were still found after short-term dark-adaptation in iron-deficient leaves, indicating that Photosystem II down-regulation occurred when the leaves were illuminated by excessive irradiance. The actual Photosystem II efficiency at steady-state photosynthesis was decreased by iron deficiency both early in the morning and at midday, due to closure of Photosystem II reaction centers and decreases of the intrinsic Photosystem II efficiency. Iron deficiency decreased the amount of light in excess of that which can be used in photosynthesis not only by decreasing absorptance, but also by increasing the relative amount of light dissipated thermally by the Photosystem II antenna. When compared to the controls, iron-deficient pear leaves dissipated thermally up to 20% more of the light absorbed by the Photosystem II, both early in the morning and at midday. At low light iron-deficient leaves with high violaxanthin cycle pigments to chlorophyll ratios had increases in pigment deepoxidation, non-photochemical quenching and thermal dissipation. Our data suggest that ph could be the major factor controlling thermal energy dissipation, and that large (more than 10-fold) changes in the zeaxanthin plus antheraxanthin to chlorophyll molar ratio caused by iron deficiency were associated only to moderate increases in the extent of photoprotection. Abbreviations: Chl chlorophyll; F o and F o minimal Chl fluorescence yield in the dark or during energization, respectively; F m and F m maximal Chl fluorescence yield in the dark or during energization, respectively; F p and F pl Chl fluorescence at the peak or at the plateau of the continuous fluorescence induction curve, respectively; FR far-red; F s Chl fluorescence at steady-state photosynthesis; F v (F m F o )or(f p F o ); F v (F m F o ); NPQ non-photochemical quenching; PQ plastoquinone; Q A and Q B primary and secondary quinone acceptors in PS II; qp photochemical quenching; exc. excitation capture efficiency of PS II (intrinsic PS II efficiency); PS II actual PS II efficiency; V+A+Z violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) Introduction Iron plays important roles in the structure and function of the photosynthetic apparatus of plants (Terry and Abadía 1986). The most obvious characteristic of the leaves from Fe-deficient plants is chlorosis, due to low concentrations per area of the photosynthetic pigments Chls and carotenoids (Morales et al. 1990, 1994; Abadía and Abadía 1993). However, not all photosynthetic pigments are decreased to the same extent by Fe deficiency, xanthophylls being less affected than Chls and β-carotene (Morales et al. 1990, 1994). The

2 10 relative enrichment in xanthophylls was shown to arise from relative increases in lutein and in V+A+Z cycle pigments in sugar beet (Morales et al. 1990) and pear (Morales et al. 1994). The decreases in photosynthetic pigment concentrations in Fe-deficient leaves are accompanied by decreases in photosynthetic rates (Terry 1980), which were attributed to reductions in the number of photosynthetic units per area (Spiller and Terry 1980). One of the distinctive characteristics of Fe deficiency in field crops is the lack of correlation between leaf Fe concentration and chlorosis (Morales et al. 1998b). This has been termed the chlorosis paradox (Römheld 1999). Therefore, leaf Chl concentrations are generally used to monitor Fe chlorosis. All crops growing during the summer in the Mediterranean area are exposed to high PPFDs, values as high as 2000 µmol photons m 2 s 1 occurring at midday on clear, sunny days. Therefore, these crops are potentially exposed to an excess of light, especially when a decrease in photosynthetic capacity occurs. When plants are illuminated by high PPFDs a decrease in photosynthetic activity is often found (for reviews see Powles 1984; Aro et al. 1993). Plants growing in the field may show photoinhibition or PS II down-regulation, when high PPFDs are combined with drought (Björkman and Powles 1984; Demmig et al. 1988), low temperatures (Öquist and Ögren 1985; Farage and Long 1987; Somersalo and Krause 1990; Ball et al. 1991; Farage and Long 1991; Ottander and Öquist 1991) and high temperatures (Ludlow and Björkman 1984; Gamon and Pearcy 1990). Nutrient deficiencies decreasing the concentrations of photosynthetic pigments are generally thought to promote photoinhibition (Godde and Dannehl 1994; Godde and Hefer 1994). Verhoeven et al. (1997) have reported N deficiency-mediated decreases in PS II efficiency, estimated from F v /F m Chl fluorescence ratios. These PS II efficiency decreases were ascribed to increases in thermal energy dissipation within the PS II antenna, mediated by the de-epoxidation of V into A+Z. This mechanism was proposed to protect PS II reaction centers from photoinhibitory damage which could be caused by an excess of light (Verhoeven et al. 1997). Other authors have suggested, however, that under limited N supply the reduction in Chl concentration and hence light absorption may be an important mechanism for avoiding high-irradiance damage to the photosynthetic apparatus (Bungard et al. 1997). Iron deficiency can be a useful tool to investigate some aspects of the protective functions attributed to the V+A+Z cycle. This is because in Fe-deficient leaves the molar (V+A+Z) pigments/chl ratio varies within a wide range (Morales et al. 1990, 1994), conversely to what happens in most plant materials (Demmig-Adams 1990; Demmig-Adams et al. 1995; Verhoeven et al. 1996, 1997). In most Fe-deficient leaves V is converted into Z+A in response to increases in PPFD (Morales et al. 1990, 1994), indicating that Fe-deficient leaves have an excess of light. Preliminary works suggested that the combination of Fe deficiency with high PPFDs at midday may induce some type of photoinhibition in pear leaves, as indicated by the low F v /F p ratios (Morales et al. 1992). However, these data should be re-examined because it has been recently reported that the F v /F p ratios from dark-adapted, Fe-deficient leaves could underestimate the true PS II efficiency (Belkhodja et al. 1998). Iron deficiency has been recently shown to decrease the PS II efficiency at steady-state photosynthesis in sugar beet grown in hydroponics and under a relatively low PPFD (Morales et al. 1998a). The aim of this work was to characterize the combined effects of Fe deficiency and the high PPFDs that occur during the summer in the Mediterranean area on the PS II efficiency of field-grown pear trees (Pyrus communis L.). Pear is one of the fruit tree crops of high value that is severely affected by Fe deficiency in this area. More than 70% of the pear orchards are affected and this causes large economic losses to farmers (Sanz et al. 1992). We have investigated the changes in PS II efficiency, both after dark-adaptation and at steady-state photosynthesis, in a range of Chl concentrations induced by Fe deficiency. We have examined the effects of Fe deficiency on the degree of PS II reaction center closure and the intrinsic PS II efficiency, two factors that can induce changes in the actual PS II efficiency at steady-state photosynthesis. Evidence is presented for PS II down-regulation in Fe-deficient leaves when illuminated by high PPFDs. Iron-deficient leaves dissipated thermally a larger fraction of the light absorbed by the PS II antenna than that dissipated by control leaves. The possible relationships of the increase in thermal dissipation with the (V+A+Z) /Chl ratios, the epoxidation state of the V+A+Z cycle and NPQ are discussed. Materials and methods Plant material Pear trees (Pyrus communis L., cv. Blanquilla ) were

3 11 grown in an orchard in the experimental farm of the Servicio de Investigación Agroalimentaria of the Diputación General de Aragón in the Aula Dei Campus, located close to the Gallego river, in the Ebro basin in north-eastern Spain. Trees were grown on a calcareous soil (Typic xerofluvent, clay-loamy texture, with 31% total calcium carbonate, 9.9% active lime, 2.86% organic mater and ph in water 8.0). Calcareous soils are known to induce Fe chlorosis in pear and other fruit tree crops. In this orchard Fe deficiency induced a range of leaf pigment concentrations similar to that found in a different pear cultivar previously (Morales et al. 1994). Some trees were not affected by chlorosis, with leaf Chl concentrations of 800 to 300 µmol Chl m 2. Iron-deficient trees had leaf Chl concentrations of 300 to 20 µmol Chl m 2. That chlorosis was due to Fe deficiency was confirmed by the shortterm leaf re-greening obtained after application of Fe sulfate or Fe-chelate (Fe-DTPA, Sequestrene 330 from Ciba-Geigy) to the leaves. Applying N or other nutrients to the leaves did not cause re-greening. Young, fully developed (3rd 6th from the top of the current year s growth) pear leaves from the external part of the tree crown, showing no interveinal chlorosis and an almost homogeneous color throughout the leaf, were used. During the experimental period (June August in 1996 and 1997) the daily maximum PPFD at the leaf level, on clear, sunny days, was 2000 µmol m 2 s 1. Pigment analysis Leaf disks were taken from the same area of the leaves in which modulated Chl fluorescence was measured (see below), both early in the morning and at midday. Disks were cut with a calibrated cork borer, wrapped in aluminum foil, dropped in liquid-n 2 and stored (still wrapped in foil) at 20 C. Leaf extracts were prepared and stored as described previously (Abadía and Abadía 1993). Pigment extracts were thawed on ice, filtered through a 0.45 µm filter and analyzed by HPLC (de Las Rivas et al. 1989). The Chl concentration per area was estimated nondestructively by using an SPAD-502 Minolta device. For calibration of the apparatus, 40 leaf disks across all the Chl range used were first measured with the SPAD, then frozen in liquid N 2, extracted as described previously (Abadía and Abadía 1993) and the extracts analyzed spectrophotometrically according to Lichtenthaler (1987). Chlorophyll fluorescence measurements in the ms-s time domain Room-temperature continuous Chl fluorescence was measured in excised leaves in a darkroom as described previously (Morales et al. 1991). Blue light (light from a 150 W tungsten lamp powered with a stabilized power supply and passing through 1 KG1 and 3 KG3 Schott infrared filters plus a 620 n m cut-off filter) was passed through a Copal photographic shutter (opening time 2 ms) and a Schölly fiber optic guide. Light intensity was 150 µmol photons m 2 s 1 at the leaf level. Chlorophyll fluorescence was detected through a 3 mm Schott RG665 filter and a 680 nm interference filter (10 n m bandpass) with a Hansatech photodiode, and the signal fed to a digital storage oscilloscope. Measurements were made on leaves collected early in the morning (6 h solar time) and at midday (12 h solar time) when illuminated by full sunlight. For the time-course experiments, measurements were made on leaves sampled at 6, 8, 10, 12, 16 and 20 h solar time. Pear leaves were excised, transported rapidly to the laboratory with the petioles immersed in water and kept in the dark for 30 min before Chl fluorescence measurements were made. In most experiments, 30 min dark-adapted leaves were pre-illuminated with 1 min of far-red (FR) light (7 µmol photons m 2 s 1 obtained with a RG715 Schott filter) to fully oxidize the PS II acceptor side before measuring Chl fluorescence (Belkhodja et al. 1998). Chlorophyll fluorescence transients in the µs-ms time domain Re-oxidation kinetics of Q A was measured in excised leaves using a laboratory PAM fluorometer (Walz, Effeltrich, Germany). Actinic high-intensity singleturnover flashes (8 µs half-width) were obtained from a XST 103 xenon discharge lamp (Walz, Effeltrich, Germany) connected to the PAM 103 unit. The Chl fluorescence decay was recorded in the following dark period after the flash by means of a weak modulated measuring light. Due to the PAM detector internal gating, measurements started 120 µs after the flash. The variable Chl fluorescence decays were detected at 17 µs resolution and stored in a computer. Measurements were made after 30 min of dark adaptation. In some cases, leaves were pre-illuminated with 1 min of FR light (7 µmol photons m 2 s 1 obtained with a RG715 Schott filter) after the dark period (Belkhodja et al. 1998).

4 12 Table 1. Effect of FR pre-illumination on F v /F p and (F pl F o )/F v Chl fluorescence ratios in dark-adapted control (500 µmol Chl m 2 ), moderately Fe-deficient (150 µmol Chl m 2 )andseverely Fe-deficient (50 µmol Chl m 2 ) pear leaves sampled at midday (12 h, solar time). Measurements were made after a 30-min dark adaptation period. Data are mean ± SE of 3 replicates Chl F v /F p (F pl F o )/F v Dark-adapted +FR Dark-adapted +FR ± ± ± ± ± ± ± ± ± ± ± ± 0.08 Modulated chlorophyll fluorescence Modulated Chl fluorescence measurements were made in attached pear leaves in the field with a PAM 2000 fluorometer (Walz, Effeltrich, Germany). F o was measured by switching on the modulated light at 0.6 khz; PPFD was less than 0.1 µmol m 2 s 1 at the leaf surface. F m and F m were measured at 20 khz with a 1 s pulse of 6000 µmol photons m 2 s 1 of white light. F m was measured after 30 min of dark adaptation at 6 h solar time, when PPFD was approximately 200 µmol m 2 s 1 PAR. F m and F s were measured when morning and midday PPFDs were approximately 200 and 2000 µmol m 2 s 1 PAR, respectively. The experimental protocol for the analysis of the Chl fluorescence quenching was essentially as described by Genty et al. (1989) with some modifications. These involved the measurements of F o and F o,whichwere measured in presence of far-red light (7 µmol photons m 2 s 1 ) in order to fully oxidize the PS II acceptor side (Belkhodja et al. 1998). The actual ( PS II )and intrinsic PS II efficiency ( exc. ) were calculated as (F m F s )/F m and F v /F m, respectively (Genty et al. 1989; Harbinson et al. 1989). Photochemical quenching (qp) was calculated as (F m F s )/F v according to van Kooten and Snel (1990). Non-photochemical quenching (NPQ) was calculated as (F m /F m ) 1, according to Bilger and Björkman (1990). The fractions of light absorbed by PS II that are dissipated in the antennae (D, identical to 1- exc. ) and used in photochemistry (P, identical to PS II ) were estimated as in Demmig-Adams et al. (1996). The fraction not used in photochemistry nor dissipated in the PS II antenna (X), that may reflect de-excitation of singlet excited Chl via the triplet pathway, was estimated as (F v /F m ) (1-qp) (Demmig-Adams et al. 1996). Results The apparent PS II efficiency in Fe-deficient, dark-adapted pear leaves is improved by FR light We have reported elsewhere that the apparent PS II photochemical efficiency in Fe-deficient, dark-adapted sugar beet leaves is improved by a pre-illumination with FR light (Belkhodja et al. 1998). This effect is caused by a dark-induced accumulation of electrons in the PS II acceptor side, which is subsequently removed by FR light exciting preferentially PS I. This phenomenon also occurred in Fe-deficient pear leaves. A pre-treatment with FR light caused large decreases in the F o and F pl levels and small decreases in the F p levels in the Chl fluorescence induction curves (data not shown). Therefore, the consequences of these changes were an increase in the F v /F p ratio and a decrease in the (F pl F o )/F v ratio (Table 1). The magnitude of the changes in Chl fluorescence induced by FR depended on the time of the day and on the leaf Chl concentration, being maximal at midday in low Chl leaves (data not shown). Because of the existence of this effect we have used a FR pre-illumination step for the F v /F p measurements with Fe-deficient leaves (<300 µmol Chl m 2 ). In control pear leaves FR illumination was not used, because it caused slight reductions in the F v /F p ratio (Table 1). This effect has been also found in other plants and is possibly due to a small, steady state population of Q A formed during the FR-illumination period (Belkhodja et al. 1998; Feild et al. 1998). Effects of Fe deficiency on the PS II efficiency of dark-adapted leaves In excised pear leaves sampled early in the morning the PS II efficiency, estimated by the F v /F p ratio after dark adaptation and treatment with FR, was high even when the leaf Chl concentration decreased by 80%, from 600 to 100 µmol Chl m 2 (Figure 1A). All F v /F p ratios measured in this Chl range were close to Severely chlorotic leaves, however, had significantly lower F v /F p ratios. The most chlorotic leaves sampled (approximately 30 µmol Chl m 2 )hadf v /F p ratios as low as The decreases in PS II efficiency found early in the morning in dark-adapted, severely Fe-deficient leaves appeared to be the result of decreases in F v (Figure 1D) caused mainly by decreases in F p (Figure 1C). The changes in Chl fluorescence observed were not related to leaf excision,

5 13 Figure 2. Time-course of the daily changes in the ratio of variable to maximum Chl fluorescence (F v /F p ) in control (500 µmol Chl m 2 solid triangles), moderately Fe-deficient (150 µmol Chl m 2 open triangles) and severely Fe-deficient (50 µmol Chl m 2 open squares) pear leaves. Data are the mean ± SE of 4 measurements. Daily changes in PPFD are also shown. The dashed line was the PPFD obtained by placing the PAR sensor, outside the tree crown, in the sun direction, and the continuous line was the mean PPFD obtained at the leaf surface level. because similar PS II efficiency estimates were obtained in some attached leaves in the field by using the F v /F m modulated Chl fluorescence ratio (grey squares in Figure 1A). At midday, after being illuminated by full sunlight, pear leaves showed decreases in the F v /F p ratios after dark-adaptation from approximately 0.80 in control leaves ( µmol Chl m 2 )downto0.65 in leaves with 100 µmol Chl m 2. Severely chlorotic leaves (30 40 µmol Chl m 2 )showedverylowf v /F p ratios of approximately The midday decreases in PS II efficiency after dark-adaptation were due to decreases in F v (Figure 1D) caused by increases in F o (Figure 1B) and decreases in F p (Figure 1C), both in moderately and severely Fe-deficient leaves. The values for F o,f p and F v were always lower at midday than in the early morning. Figure 1. Ratio of variable to maximum Chl fluorescence (F v /F p ) (A), Chl fluorescence at F o (mv, B) and F p (mv, C) and variable part of Chl fluorescence, F v (mv, D). All data are plotted versus total leaf Chl. Measurements were made after a 30-min dark adaptation period. F v /F p ratios were obtained from continuous Chl fluorescence measurements in dark-adapted, excised leaves. In the figure are also shown (large grey squares) the F v /F m ratios obtained from modulated Chl fluorescence measurements in dark-adapted, attached leaves. Measurements were made in leaves sampled early in the morning (6 h solar time; solid circles) or at midday (12 h solar time; open circles). Morning and midday PPFDs were approximately 200 and 2000 µmol photons m 2 s 1 PAR. A dashed line separates Fe-sufficient and deficient leaves. Data are the mean ± SE of 4 measurements. Diurnal course of changes in PS II efficiency in dark-adapted pear leaves Since the PS II efficiency of dark-adapted, Fe-deficient pear leaves was different in the early morning and at midday, we investigated the daily course of the changes in this parameter, along with the changes in incident PPFD (Figure 2). The F v /F p ratio in control pear leaves was approximately from early morning to sunset (Figure 2). However, the PS II efficiency of moderately and severely Fe-deficient leaves changed significantly throughout the day. Progressive decreases of the F v /F p ratios occurred from morning

6 14 vivo (Chylla and Whitmarsh 1989). When leaves were sampled early in the morning, control (Figure 3A) and moderately Fe-deficient leaves (Figure 3B) had similar Q A re-oxidation kinetics, whereas severely Fe-deficient leaves (Figure 3C) had slightly slower kinetics. When similar leaves were sampled at midday, the Q A re-oxidation was slowed down in all leaves, severely Fe-deficient leaves having the slowest kinetics (Figures 3A C). Far-red (FR) light, exciting preferentially PS I, was used to remove any electron accumulation in the PS II acceptor side. Far-red pre-illumination did not affect significantly the Chl fluorescence decays from control leaves at midday (Figure 3A) and from control, moderately and severely Fe-deficient leaves sampled early in the morning (data not shown). However, decays from moderately and severely Fe-deficient leaves sampled at midday became much faster after FR preillumination (Figures 3B and C). Effects of Fe deficiency on Chl fluorescence quenching parameters Figure 3. Decays of the variable Chl fluorescence following an actinic flash measured in control (500 µmol Chl m 2 A), moderately Fe-deficient (150 µmol Chl m 2 B) and severely Fe-deficient (50 µmol Chl m 2 C) pear leaves sampled early in the morning (6 h solar time; solid circles) or at midday (12 h solar time; open symbols). Measurements were made after 30 min dark adaptation (circles) or in 30 min dark-adapted leaves, 2 s after the end of a treatment with FR light (triangles). Curves were normalized to the same F m. For clarity, only 1 out of 5 points were drawn in each curve. to midday, and opposite increases occurred from midday to sunset. Midday F v /F p ratios were found to be lower in severely (50 µmol Chl m 2 ) than in moderately (150 µmol Chl m 2 ) Fe-deficient leaves (0.58 and 0.67, respectively; Figure 2). Effects of Fe deficiency on the kinetics of Q A re-oxidation The decay of variable Chl fluorescence after a saturating flash can be used to evaluate the characteristics of Q A re-oxidation in intact, dark-adapted leaves in When analyzed early in the morning, moderately (200 µmol Chl m 2 ) and severely (50 µmol Chl m 2 ) Fe-deficient leaves had lower PS II (actual PS II efficiency at steady-state photosynthesis) values than those found in control leaves (Figure 4A). These decreases in PS II were accompanied by decreases in intrinsic PS II efficiency ( exc. ) (Figure 4B) and by decreases in the proportion of open PS II reaction centers, estimated by qp (Figure 4C). At midday, PS II, exc. and qp decreased both in control and in Fe-deficient pear leaves (Figures 4A C), reflecting the excess excitation pressure on the photosynthetic apparatus occurring at full sunlight. Iron-deficient leaves had lower PS II values than those found in control leaves (Figure 4A). These decreases in PS II were accompanied by decreases in intrinsic PS II efficiency ( exc. ) (Figure 4B). Photochemical quenching (qp) was in control leaves and 0.67 and 0.50 in moderately and severely Fe-deficient leaves respectively (Figure 4C). In the morning, control leaves dissipated approximately 20% of the absorbed light (Figure 5A). This value was close to the constitutive level of dissipation in the PS II antenna which is unrelated to the xanthophyll cycle and results in dark-adapted F v /F p ratios of 0.80 (Demmig-Adams et al. 1996). Early in the morning, Fe-deficient leaves dissipated, when compared to the controls, up to 20% more of the light absorbed by

7 15 Figure 4. Actual PS II efficiency, PS II (A), intrinsic PS II efficiency, exc. (B) and photochemical quenching, qp (C) versus total Chl in pear leaves affected by Fe deficiency. See the legend of Figure 1 for further details. Data are the mean ± SE of 4 measurements. PS II (Figure 5A). This could be ascribed to increases in xanthophyll cycle-related thermal dissipation, and resulted in a 2-fold increase in total D when compared to the controls (Figure 5A). At this time Fe-deficient leaves used in photochemistry a lower proportion of the absorbed light than the controls (Figure 5A). At midday, all leaves increased markedly the amount of light dissipated thermally. Severely and moderately Fe-deficient leaves dissipated a larger proportion (20% more) of absorbed light than control leaves (Figure 5B). At this time of the day Fe-deficient leaves used a lower proportion of the absorbed light in photochemistry than the controls (Figure 5B). Early in the morning the NPQ values for control leaves were approximately 0.10, and increased to 0.25 and 0.35 in moderately and severely Fe-deficient Figure 5. Fractions of light absorbed by the PS II antenna that are used in photochemistry (P), thermally dissipated (D) and not used in photochemistry nor dissipated in the antenna (X) (A and B) and nonphotochemical quenching, NPQ, (C) versus total Chl in pear leaves affected by Fe deficiency. In A and B, the lower series of points represents D and the middle series of points represents D + X. In C, solid circles represents data obtained early in the morning and open circles those obtained at midday. The horizontal line of points in A and B corresponds to the level of dissipation independent of the xanthophyll cycle, as estimated from a dark-adapted F v /F p ratio of See the legend of Figure 1 for further details. Data are the mean ± SE of 4 measurements. A dashed line separates control and Fe-deficient leaves. leaves, respectively (Figure 5C). At midday, the NPQ values were not significantly different in control, moderately and severely Fe-deficient leaves (Figure 5C). Effects of Fe deficiency on photosynthetic pigment composition Changes induced by Fe deficiency in the photosynthetic pigment composition of field-grown leaves of

8 16 Figure 6. Chl a/chl b ratio (A) and neoxanthin/chl ratio (B) plotted versus total Chl in pear leaves. Samples were harvested early in the morning (6 h solar time; solid circles) or at midday (12 h solar time; open circles). See the legend of Figure 1 for further details. Data are the mean ± SE of 4 measurements. pear were very similar to those reported in a previous work (Morales et al. 1994). Iron deficiency increased the Chl a/chl b ratio from in control leaves to 4.0 and 4.8 in moderately and severely Fe-deficient leaves, respectively (Figure 6A). No significant differences in the Chl a/chl b ratio were found when sampling was made in the morning or at midday (Figure 6A). The molar ratio neoxanthin/chl was unchanged by Fe deficiency, being both in the morning and at midday (Figure 6B). Relationships between quenching parameters and V+A+Z cycle pigments In the pear leaves used in this study the molar (V+A+Z)/Chl ratio varied between in controls to 0.30 in severely Fe-deficient leaves (Figure 7A). This was caused by the fact that the decreases in leaf Chl concentration are much larger than the decreases in the concentration of the V+A+Z pigments. (V+A+Z)/Chl ratios as high as 0.9 have been reported before in extremely Fe-deficient leaves of a different pear cultivar (Morales et al. 1994). The V+A+Z cycle responded to the daily changes in PPFDs, and in all leaves the molar (Z+A)/Chl ratios (Figure 7B) as well Figure 7. Number of (V+A+Z)/1000 Chl (A), (Z+A)/1000 Chl (B) and (Z+A)/(V+A+Z) ratio (C) plotted versus total Chl in pear leaves. Samples were harvested early in the morning (6 h solar time; solid circles) or at midday (12 h solar time; open circles). See the legend of Figure 1 for further details. Data are the mean ± SE of 4 measurements. as the (Z+A)/(V+A+Z) ratios (Figure 7C) increased from morning to midday. We have plotted the D and NPQ levels in the morning and at midday versus the (Z+A)/Chl and (Z+A)/(V+A+Z) molar ratios. The additional 20% increase in D found in Fe-deficient leaves when compared to the controls was associated to 21- and 10-fold increases in the (Z+A)/Chl ratio (Figure 8A) and 4- and 2-fold increases in the (Z+A)/(V+A+Z) ratio (Figure 8C) in the morning and at midday, respectively. These changes in pigment ratios were accompanied by large increases in NPQ in the morning but not at midday (Figures 8B and D).

9 17 Figure 8. Relationships between Chl fluorescence and pigment data in pear leaves affected by Fe deficiency. Chlorophyll fluorescence parameters used are the fraction of light absorbed by the PS II antenna that is thermally dissipated, D (cells A and C) and the non-photochemical quenching, NPQ (cells B and D). Pigment data include the (Z+A)/Chl molar ratio (cells A and B) and the (Z+A)/(V+A+Z) ratio (cells C and D). The horizontal line of points in cells A and C corresponds to the level of dissipation independent of the xanthophyll cycle, as estimated from a dark-adapted F v /F p ratio of See the legend of Figure 1 for further details. Data are the mean ± SE of 4 measurements. Dashed lines separate control and Fe-deficient leaves in each graph. Discussion The results of this paper confirm that some apparent effects of Fe deficiency on Chl fluorescence can be induced artifactually. The sustained decreases in F v /F p and increases in (F pl F o )/F p that occur in moderately Fe-deficient pear leaves arise from the marked increases in F o and F pl that develop during the darkadaptation protocol used to measure continuous Chl fluorescence. In Fe-deficient sugar beet leaves these Chl fluorescence increases have recently been shown to arise from decreases in photochemical quenching caused by a dark reduction of electron carriers in the PS II acceptor side, probably at the PQ level (Belkhodja et al. 1998). This process also slows down the electron transport between Q A and Q B. Our results indicate that normal protocols for measuring PS II efficiency and variable Chl fluorescence decays should be modified when using Fe-deficient leaves to obtain accurate F o values and Chl fluorescence decays. This can be made by including a pre-illumination with FRlight to oxidize electron carriers at the PS II acceptor side. Results obtained in preliminary works suggesting that moderate Fe deficiency could decrease F v /F p ratios in pear leaves (Morales et al. 1992) were likely caused by this effect. Sustained decreases in dark-adapted PS II efficiency associated to moderate and severe Fe deficiency in field-grown pear leaves were quite limited. In the early morning or in the evening these leaves showed a fairly good PS II efficiency, even when the Chl concentration was as low as 10 20% of control values. For instance, morning F v /F p (and F v /F m ) values were approximately 0.80, 0.78 and 0.70 in control, moderately and severely Fe-deficient pear leaves, respectively. The slightly low morning F v /F p values found in severely Fe-deficient pear leaves could be associated to a small extent of sustained NPQ, due to some A+Z molecules that are maintained overnight (Morales et al. 1994). A similar sustained NPQ has been reported recently in N-deficient spinach leaves (Verhoeven

10 18 et al. 1997) and in Fe-deficient sugar beet grown in controlled environments (Morales et al. 1998a). Significant, sustained decreases in PS II efficiency were found only in dark-adapted, extremely chlorotic pear leaves with Chl concentrations below 40 µmol m 2. These decreases in PS II efficiency were associated to decreases in F v caused by small increases in F o and major decreases in F p, and may reflect, at least in part, real PS II damage. The marked reduction in PS II efficiency in dark-adapted, extremely Fe-deficient pear leaves is not likely to be related to overnight-sustained (Z+A)-dependent NPQ, because these leaves have been reported to have a low deepoxidation state at dawn (Morales et al. 1994). It should be mentioned that in the present paper no pigment data are shown from these extremely chlorotic leaves. Iron-deficient pear leaves showed midday depressions in the efficiency of PS II, estimated from the F v /F p ratio after dark-adaptation, similar to those reported for other field-grown plant species (Demmig- Adams 1990; Demmig-Adams and Adams 1992 and references therein). The F v /F p ratios decreased from morning to midday and increased thereafter to reach in the evening values similar to those found in the morning. The midday F v /F p decreases were larger in severely than in moderately Fe-deficient leaves. In most Fe-deficient leaves the recovery of the F v /F p ratio was practically complete within a few h of low light. These easily reversible changes in PS II efficiency are usually ascribed to a down-regulation of PS II (Krause 1988; Falk and Samuelsson 1992) that may reflect protective or regulatory mechanisms to avoid severe damage to the photosynthetic apparatus. These changes could contribute to the long life of Fe-deficient leaves exposed to high PPFD in field conditions. The extent of these easily reversible, midday decreases in the F v /F p ratio was maximal on sunny days and minimal on cloudy days, and was also dependent on the relative leaf position (data not shown), possibly due to variations in the amount of absorbed light. At steady-state photosynthesis, when several types of Chl fluorescence quenchings were present (for instance, ph-related and other non-photochemical quenching processes, photochemical quenching, etc.), Fe-deficient pear leaves had a lower actual PS II efficiency ( PS II ) than control leaves. This occurred both in the early morning and at midday (i.e. at low and high PPFDs). The decreases in PS II at steadystate photosynthesis in Fe-deficient pear leaves were associated to increases in the proportion of closed PS II centers (estimated by qp) and also to decreases in the intrinsic PS II efficiency ( exc. ). Both factors have been shown to contribute to the decrease in PS II efficiency in Fe-deficient sugar beet grown in hydroponics (Morales et al. 1998a). Decreases in exc. have been associated to increases in energy dissipation in the PS II antenna (Adams et al. 1995; Demmig- Adams et al. 1995; Demmig-Adams and Adams 1996; Verhoeven et al. 1997). These data indicate that Fe deficiency modified the allocation of the light absorbed by the PS II antenna at steady-state photosynthesis, increasing the proportion of light dissipated thermally within the PS II antenna and decreasing the proportion used in photochemistry. On the other hand, midday PS II values in severely Fe-deficient pear leaves did never approach zero (they were approximately 0.10), indicating that there was always some electron transport through PS II, even in the harshest conditions Fe-deficient pear leaves must face in full sunlight. In pear tree leaves Fe deficiency decreases the concentrations of all major photosynthetic pigments on an area basis (Morales et al. 1994). The Fe deficiency-mediated reduction in photosynthetic pigment concentrations and hence light absorption may limit high-light damage to the photosynthetic apparatus of these leaves (Abadía et al. 1999). Similar conclusions have been reported in N-limited plants (Bungard et al. 1997). However, not all photosynthetic pigments were decreased to the same extent, Chl b being more affected than Chl a, and lutein and the V+A+Z xanthophylls being less affected than the other carotenoids (Figure 6; Morales et al. 1994). These data indicate a preferential loss of Chl b and therefore of antenna light-harvesting complexes (LHC). Taking into account the characteristic photosynthetic pigment composition of the different LHCs (Ruban et al. 1994; Verhoeven et al. 1999), our data may suggest different rates of degradation of the different LHCs. Senescence did not occur in chlorotic pear leaves, as indicated by their long life (several months). Also, the almost full interconversion of V into Z in response to light in the chlorotic leaves indicate that these pigments are located in functional LHCs (Abadía et al. 1999). The large PPFD increase from morning to midday was associated to large increases in thermal dissipation in the PS II antenna and also in NPQ, supporting that ph controls thermal dissipation. Chlorophyll fluorescence changes were associated to F m and F o quenching, a distinctive characteristic of this mechanism (Gilmore and Yamamoto 1993; Verhoeven et

11 19 al. 1997). Under the low light conditions occurring in the morning, Fe-deficient leaves had increases in the (Z+A)/Chl molar ratio, in the de-epoxidation state and in NPQ which were associated to significant (20%) increases in D, when compared to the controls. This indicates that xanthophyll cycle-related thermal dissipation is significant in Fe-deficient pear leaves already at low PPFDs, and suggests that this mechanism may be operating in a relatively large time-window during the day in these leaves. At the high PPFDs found at midday Fe-deficient leaves also showed dissipation values 20% higher than the controls. This was again associated to increases in the (Z+A) /Chl molar ratio and in the de-epoxidation state, whereas the NPQ levels were apparently unchanged by Fe deficiency. This higher level of dissipation may constitute a significant increase in photoprotection. Our data suggest that ph could be the major factor controlling thermal energy dissipation, since large (more than 10-fold) changes in (Z+A)/Chl molar ratio caused only moderate increases in the extent of photoprotection at low NPQ levels. With increasing Z+A, midday NPQ (indicating relative differences in the rate constant of thermal deactivation) apparently remains constant, whereas D (thermal deactivation) does increase. This may arise from the different conditions used to measure both parameters. To estimate D the only parameters used (F v and F m ) are obtained from illuminated leaves, whereas to estimate NPQ according to Stern-Volmer (as (F m /F m ) 1) one of the parameters used (F m )must be obtained from dark-adapted leaves. The actual NPQ values of Fe-deficient pear leaves may be higher than those estimated from the formula (F m /F m ) 1. For instance, early in the morning severely Fe-deficient pear leaves had, when compared to the controls, low F v /F p ratios after 30 min of dark adaptation, due to both increases in F o and decreases in F p (and F m ). Therefore NPQ would be lower than when referred to an unquenched F m (Adams et al. 1995; Adams and Demmig-Adams 1995; Morales et al. 1998a). Low F v /F m ratios early in the morning may arise from a certain degree of de-epoxidation in the V+A+Z cycle, and therefore an initial NPQ, at the moment of measuring F m (Verhoeven et al. 1997). In fact, severely Fedeficient pear leaves (50 µmol Chl m 2 ) had early in the morning (Z+A) / (V+A+Z) ratios of approximately 0.85, compared to control values of 0.20 (Figure 8C), thus supporting the existence of an initial NPQ. These high de-epoxidation values, that remained overnight (Morales et al. 1994) may conceal the increases in the rate constant of thermal deactivation in Fe-deficient pear leaves. In summary, only extremely Fe-deficient leaves (below 40 µmol Chl m 2 ) showed sustained decreases in PS II efficiency when measurements are carried out in dark-adapted leaves and after a FR pre-treatment. Midday depressions in PS II efficiency were still found after short-term dark-adaptation in Fe-deficient leaves, indicating that PS II down-regulation occurred when the leaves were illuminated by an excessive PPFD. The actual PS II efficiency at steady-state photosynthesis, PS II, was decreased by Fe deficiency both early in the morning and at midday, and these decreases were associated to closure of PS II reaction centers and decreases in the intrinsic PS II efficiency, exc.. Iron deficiency increased the NPQ values at low PPFDs, and increased the proportion of light dissipated by the PS II antenna, possibly through xanthophyll cycle-related mechanisms, both at low and high PPFDs. Acknowledgements This work was supported by grants AGF from the Spanish Comisión Interministerial de Ciencia y Tecnología to A.A. and PB from the Spanish Dirección General de Investigación Científica y Técnica and AIR3-CT from the Commission of European Communities to J.A. Support to F.M. and R.B. was provided by a contract from the Spanish Ministry of Science and Culture and a fellowship from the Spanish Institute of International Cooperation. Authors thank Dr. Carlos Zaragoza (SIA-DGA) for permitting the use of the experimental orchard. References Abadía J and Abadía A (1993) Iron and plant pigments. In: Barton LL and Hemming BC (eds) Iron Chelation in Plants and Soil Microorganisms, pp Academic Press, San Diego, California Abadía J, Morales F and Abadía A (1999) Photosystem II efficiency in low chlorophyll, iron-deficient leaves. Plant Soil (in press) Adams III WW and Demmig-Adams B (1995) The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter. Plant Cell Environ 18: Adams III WW, Demmig-Adams, B, Verhoeven AS and Barker DH (1995) Photoinhibition during winter stress: Involvement of sustained xanthophyll cycle-dependent energy dissipation. Aust J Plant Physiol 22:

12 20 Aro E-M, Virgin I and Andersson B (1993) Photoinhibition of Photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143: Ball, MC, Hodges VS and Laughlin GP (1991) Cold-induced photoinhibition limits regeneration of snow gum at tree-line. Funct Ecol 5: Belkhodja, R, Morales F, Quílez R, López-Millán A-F, Abadía A and Abadía J (1998) Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the Photosystem II acceptor side. Photosynth Res 56: Bilger W and Björkman O (1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth Res 25: Björkman O and Powles SB (1984) Inhibition of photosynthetic reactions under water stress: Interaction with light level. Planta 161: Bungard RA, McNeil D and Morton JD (1997) Effects of nitrogen on the photosynthetic apparatus of Clematis vitalba grown at several irradiances. Aust J Plant Physiol 24: Chylla RA and Whitmarsh J (1989) Inactive Photosystem II complexes in leaves. Turnover rate and quantification. Plant Physiol 90: de las Rivas J, Abadía A and Abadía J (1989) A new reversed phase HPLC method resolving all major higher plant photosynthetic pigments. Plant Physiol 91: Demmig B, Winter K, Krüger A and Czygan F-C (1988) Zeaxanthin and the heat dissipation of excess light energy in Nerium oleander exposed to a combination of high light and water stress. Plant Physiol 87: Demmig-Adams B (1990) Carotenoids and photoprotection: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1 24 Demmig-Adams B and Adams III WW (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: Demmig-Adams B and Adams III WW (1996) Xanthophyll cycle and light stress in nature: Uniform response to excess direct sunlight among higher plant species. Planta 198: Demmig-Adams B, Adams III WW, Logan BA and Verhoeven AS (1995) Xanthophyll cycle-dependent energy dissipation and flexible PS II efficiency in plants acclimated to light stress. Aust J Plant Physiol 22: Demmig-Adams B, Adams III WW, Barker DH, Logan BA, Bowling DR and Verhoeven AS (1996) Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiol Plant 98: Falk S and Samuelsson G (1992) Recovery of photosynthesis and Photosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light. Physiol Plant 85: Farage PK and Long SP (1987) Damage to maize photosynthesis in the field during periods when chilling is combined with high photon fluxes. In: Biggins J (ed) Progress in Photosynthesis Research, Vol 4, pp Martinus Nijhoff Publishers, Dordrecht, The Netherlands Farage PK and Long SP (1991) The occurrence of photoinhibition in an over-wintering crop of oil-seed rape (Brassica napus L.) and its correlation with changes in crop growth. Planta 185: Feild TS, Nedbal L and Ort DR (1998) Nonphotochemical reduction of the plastoquinone pool in sunflower leaves originates from chlororespiration. Plant Physiol 116: Gamon JA and Pearcy RW (1990) Photoinhibition in Vitis californica. The role of temperature during high-light treatment. Plant Physiol 92: Genty B, Briantais J-M and Baker NR (1989) The relationship between quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: Gilmore AM and Yamamoto HY (1993) Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching. Photosynth Res 35: Godde D and Dannehi H (1994) Stress-induced chlorosis and increase in D1-protein turnover precede photoinhibition in spinach suffering under magnesium/sulphur deficiency. Planta 195: Godde D and Hefer M (1994) Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of Photosystem II are enhanced by mineral-stress conditions. Planta 193: Harbinson J, Genty B and Baker NR (1989) Relationship between the quantum efficiencies of Photosystems I and II in pea leaves. Plant Physiol 90: Krause GH (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74: Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth Enzymol 148: Ludlow MM and Björkman O (1984) Paraheliotropic leaf movement in Siratro as a protective mechanism against droughtinduced damage to primary photosynthetic reactions: Damage by excessive light and heat. Planta 161: Morales F, Abadía A and Abadía J (1990) Characterization of the xanthophyll cycle and other photosynthetic pigment changes induced by iron deficiency in sugar beet (Beta vulgaris L.). Plant Physiol 94: Morales F, Abadía A and Abadía J (1991) Chlorophyll fluorescence and photon yield of oxygen evolution in iron-deficient sugar beet (Beta vulgaris L.) leaves. Plant Physiol 97: Morales F, Susín S, Abadía A, Carrera M and Abadía J (1992) Photosynthetic characteristics of iron chlorotic pear (Pyrus communis L.). J Plant Nutr 15: Morales F, Abadía A, Belkhodja R and Abadía J (1994) Iron deficiency-induced changes in the photosynthetic pigment composition of field-grown pear (Pyrus communis L.) leaves. Plant Cell Environ 17: Morales F, Abadía A and Abadía J (1998a) Photosynthesis, quenching of chlorophyll fluorescence and thermal energy dissipation in iron-deficient sugar beet leaves. Aust J Plant Physiol 25: Morales F, Grasa R, Abadía A and Abadía J (1998b) Iron chlorosis paradox in fruit trees. J Plant Nutr 21: Öquist G and Ogren E (1985) Effects of winter stress on photosynthetic electron transport and energy distribution between the two photosystems of pine as assayed by chlorophyll fluorescence kinetics. Photosynth Res 7: Ottander C and Öquist G (1991) Recovery of photosynthesis in winter-stressed Scots pine. Plant Cell Environ 14: Powles SB (1984) Photoinhibition of photosynthesis induced by visible light. Ann Rev Plant Physiol 35: Römheld V (1999) The chlorosis paradox: Fe inactivation in leaves as a secondary event in Fe deficiency chlorosis. J Plant Nutr 22: in press Ruban AV, Young AJ, Pascal AA and Horton P (1994) The effects of illumination on the xanthophyll composition of the Photosystem II light-harvesting complexes of spinach thylakoid membranes. Plant Physiol 104: Sanz M, Cavero J and Abadía J (1992) Iron chlorosis in the Ebro river basin, Spain. J Plant Nutr 15:

13 21 Somersalo S and Krause GH (1990) Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures. Planta 180: Spiller S and Terry N (1980) Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units. Plant Physiol 65: Terry N (1980) Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant Physiol 65: Terry N and Abadía J (1986) Function of iron in chloroplasts. J Plant Nutr 9: van Kooten O and Snel JFH (1990) The use of chlorophyll fluorescence in plant stress physiology. Photosynth Res 25: Verhoeven AS, Adams III WW and Demmig-Adams B (1996) Close relationship between the state of the xanthophyll cycle pigments and Photosystem II efficiency during recovery from winter stress. Physiol Plant 96: Verhoeven AS, Demmig-Adams B and Adams III WW (1997) Enhanced employment of the xanthophyll cycle and thermal energy dissipation in spinach exposed to high light and N stress. Plant Physiol 113: Verhoeven AS, Adams III WW, Demmig-Adams B, Croce R and Bassi R (1999) Xanthophyll cycle pigment localization and dynamics during exposure to low temperatures and light stress in Vinca major. Plant Physiol 120: