The Plant Journal (2011) 65, 922–935
doi: 10.1111/j.1365-313X.2010.04479.x
Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants Nicola´s E. Blanco1, Romina D. Ceccoli1, Marı´a E. Segretin2, Hugo O. Poli1, Ingo Voss3, Michael Melzer4, Fernando F. Bravo-Almonacid2, Renate Scheibe3, Mohammad-Reza Hajirezaei4 and Ne´stor Carrillo1,* 1 Divisio´n Biologı´a Molecular, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Instituto de Biologı´a Molecular y Celular de Rosario (IBR, CONICET), Universidad Nacional de Rosario, S2002LRK Rosario, Argentina, 2 Laboratorio de Virologı´a y Biotecnologı´a Vegetal, Instituto de Investigaciones en Ingenierı´a Gene´tica y Biologı´a Molecular (INGEBI, CONICET), C1428ADN Ciudad Auto´noma de Buenos Aires, Argentina, 3 Pflanzenphysiologie, Fakulta¨t Biologie/Chemie, Universita¨t Osnabru¨ck, D-49069 Osnabru¨ck, Germany, and 4 Leibniz-Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung, Corrensstraße 3, 06466 Gatersleben, Germany Received 20 October 2010; revised 7 December 2010; accepted 21 December 2010; published online 21 February 2011. * For correspondence (fax +54 341 4390465; e-mail
[email protected]).
SUMMARY Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxindeficient lines showed growth arrest, leaf chlorosis and decreased CO2 assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein. Keywords: ferredoxin, flavodoxin, complementation, photosynthesis, knock-down, transplastomic.
INTRODUCTION Optimal function of photosynthesis and assimilatory processes in the light relies on flexible distribution of photosynthetic reducing equivalents by the chloroplast electron carrier ferredoxin (Fd). Ferrodoxins are small, soluble [2Fe– 2S] proteins that participate in many oxido-reductive processes in prokaryotes, plants and animals. In chloroplasts, Fd is reduced by photosystem I (PSI) and delivers lowpotential electrons to several enzymes, including ferredoxinNADP+ reductase for NADP+ reduction, nitrite reductase and glutamate-oxoglutarate aminotransferase for nitrogen 922
assimilation and amino acid synthesis, and sulfite reductase and fatty acid desaturase (Hase et al., 2006). In addition, it acts as electron donor for ascorbate regeneration (Miyake and Asada, 1994) and for thioredoxin (Trx) reduction via Fd-Trx reductase (Schu¨rmann and Buchanan, 2008). Fd also helps to relieve the electron pressure on the photosynthetic electron transport chain (PETC) when photochemical efficiency is low due to CO2 shortage and/or excess illumination. Under such conditions, the PETC becomes overreduced, and electrons or light energy may be passed ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd
Flavodoxin complements ferredoxin deficiency 923 straight to oxygen, generating reactive oxygen species (ROS) that may damage all types of biomolecules (Apel and Hirt, 2004). Fd alleviates this situation, by returning the surplus of reducing equivalents to the PETC via cyclic electron flow (Yamamoto et al., 2006), or by delivering them to the cytosol through Trx-dependent activation of the malate valve (Scheibe, 2004). Chloroplasts contain at least two leaf-type Fd isoforms, and it has been argued that there is a division of labour between them into separate linear and cyclic electron transfer routes (Hanke et al., 2004; Voss et al., 2008; Hanke and Hase, 2008). Plastids from heterotrophic organs (e.g. roots) harbour another set of ferredoxins that function in a backward direction (Neuhaus and Emes, 2000). The levels of Fd transcripts and polypeptides decrease when plants and cyanobacteria are exposed to iron deficit or environmental conditions that cause ROS accumulation (Thimm et al., 2001; Mazouni et al., 2003; Zimmermann et al., 2004; Tognetti et al., 2006), suggesting transcriptional and/or post-transcriptional mechanisms of regulation. Several lines of evidence indicate that a decrease in Fd compromises cell survival. First, Fd is essential for the viability of cyanobacteria, as demonstrated by targeted disruption of the gene (Poncelet et al., 1998). Second, Fd down-regulation using antisense RNA technology in potato (Solanum tuberosum) indicated that its levels could not be decreased below 50% of wild-type (WT) contents without fatally affecting plant fitness (Holtgrefe et al., 2003). Third, knockout (Voss et al., 2008) and knocked-down Arabidopsis thaliana lines (Hanke and Hase, 2008), in which either the major leaf Fd or both isoforms were depleted, displayed growth arrest, reproductive handicap and partial inactivation of photosynthesis. Finally, in cyanobacteria and some algae, the decrease in Fd levels during episodes of iron starvation and environmental stress is compensated for by induced expression of flavodoxin (Fld), an isofunctional electron shuttle that contains flavin mononucleotide as the prosthetic group (Singh et al., 2004). Under these adverse growth conditions, Fld is able to replace Fd in many reactions, including photosynthesis, nitrite and Trx reduction (Sancho, 2006). Indeed, Fld up-regulation is regarded as one of the most important adaptive resources to enable successful colonization of iron-poor waters by phytoplankton (Palenik et al., 2003). Flds are not found in plants, and the adaptive advantages derived from their expression and stress-dependent induction have been lost over the course of evolution (Zurbriggen et al., 2007). However, transgenic tobacco plants (Nicotiana tabacum cv. Petit Havana) transformed with a chloroplasttargeted Fld gene from Anabaena sp. PCC7119 showed enhanced tolerance to iron starvation and to several sources of stress, such as water deficit, extreme temperatures, xenobiotics and pathogen-induced cell death (Tognetti et al., 2006, 2007; Zurbriggen et al., 2008, 2009). Fld accu-
mulation in chloroplasts resulted in restoration of productive routes of electron distribution, and suppression of ROS build-up in leaves (Tognetti et al., 2006). These features, and the observation that cyanobacterial Fld behaves in vitro as an efficient substrate for several Fd-dependent plant enzymes and reactions, including cyclic electron flow (Scheller, 1996), NADP+ and Trx reduction (Tognetti et al., 2006), led to the assumption that increased tolerance in the transgenic lines was caused solely by functional replacement of decreasing Fd by Fld, although no rigorous proof was provided. Here we address this question by expressing Anabaena Fld in Fd-deficient tobacco plants generated using both RNA antisense and RNA interference technologies. Knocked-down lines exhibited the typical symptoms of Fd deficiency, namely arrested growth, homogeneous leaf chlorosis or variegated leaf phenotype, inhibition of photosynthesis, and increased sensitivity to oxidants. Fld expression from the chloroplast or nuclear genome in these Fddeficient plants led to partial recovery of a WT phenotype and restoration of photosynthetic activities. Fd-deficient lines expressing chloroplast-located Fld were more tolerant than WT siblings to oxidative stress imposed by the redoxcycling herbicide methyl viologen (MV). The results confirm that Fld is able to complement the essential activities of endogenous Fd in vivo. RESULTS Preparation and characterization of Fd-deficient plants The full complement of Fd isoforms in tobacco is unknown. Three of the reported nucleotide sequences are very similar and most likely represent the same gene, but the fourth is significantly different (Figure S1). Comparison with Fd isoforms present in other solanaceous species indicates that all reported tobacco sequences cluster with the Fd2-type isoforms (Figure S2 and Appendix S1). The Fd sequence represented by GenBank accession number AY552781.1 was therefore used for the design of vectors in two complementary strategies to down-regulate Fd expression in tobacco: RNA interference (siFd plants) and RNA antisense (asFd plants). Transformed plants were screened for Fd decrease by immunoblot analysis. Lines with various levels of Fd were recovered after selection of primary transformants generated by the two methods. Those lines that were able to set seed were propagated up to the T3 generation and used for further studies. In agreement with previous observations (Holtgrefe et al., 2003), asFd plants showed leaf chlorosis and growth retardation to various degrees, depending on the levels of Fd remaining (Figure 1a and Table 1). The chlorophyll and carotenoid contents decreased to approximately 20 and 40% of WT levels, respectively, in antisense lines retaining approximately 60% of WT Fd, with only marginal modifications in the chlorophyll a/b ratio (Table 1). CO2 assimilation
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
924 Nicola´s E. Blanco et al. Figure 1. Impact of reduced ferredoxin levels on plant growth, leaf morphology and photosynthetic activities. (a) Stunted growth of 8-week-old antisense lines. From top to bottom: untransformed WT plants, and asFd plants (T3 generation) containing 80–85%, 70–75% and 55–60% of the Fd levels present in WT controls. The second youngest fully expanded leaves are shown. (b) Net CO2 uptake rates (ACO2 ) measured on the second youngest fully expanded leaves. Gas exchange determinations were performed using leaves from the plants shown in (a). Values are means SE of four measurements.
(Figure 1b) and linear electron flow rates estimated by chlorophyll fluorescence measurements (see below) showed a similar decrease. Antisense lines with less than 55–60% of WT Fd levels could not be recovered by this procedure. Silenced plants displayed phenotypes that generally resembled those of asFd lines at similar Fd levels (Table 1), although siFd leaves showed mostly variegated phenotype rather than homogeneous chlorosis (see below). The lower limit of leaf Fd content that allowed rescue of viable siFd
plants was approximately 45% in a number of attempts. The results indicate that both asFd and siFd lines displayed typical symptoms of Fd deficiency (Holtgrefe et al., 2003; Voss et al., 2008; Hanke and Hase, 2008), and are therefore suitable to investigate Fld complementation. Functional complementation of Fd-deficient plants using Anabaena Fld Two complementary approaches were again used to investigate functional complementation of Fd deficiency by Fld
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
Flavodoxin complements ferredoxin deficiency 925 Table 1 Phenotypic analysis of wild-type and ferredoxin-deficient transgenic tobacco plants expressing or not expressing flavodoxin in chloroplasts WT )1
Fd (pmol g FW) Fld (pmol g)1 FW) Number of nodes Height (cm) Internodal distance (cm) Aerial fresh weight (g) Aerial dry weight (g) % H2O Chlorophyll a (mg cm)2) Chlorophyll b (mg cm)2) Chlorophyll a + b (mg cm)2) Chlorophyll a/b Carotenoids (mg cm)2)
80 – 10.67 55.10 4.38 56.32 6.24 88.94 24.99 9.93 34.92 2.41 4.73
asFd x
7
0.52x 6.58x 0.15x 5.12x 1.09x 1.40y 2.54x 0.49x 1.66x 0.20xy 0.23x
47 – 7.67 12.55 1.52 21.49 1.21 94.45 4.92 2.37 7.29 2.06 1.95
asFd/pfld y
5
0.82z 8.30z 1.00y 11.47z 1.15z 1.25x 0.84z 0.24z 1.04z 0.17y 0.16z
10 70 9.38 39.79 3.44 39.60 3.64 90.85 14.31 4.84 19.15 2.95 4.24
WT z
2 8 1.19y 11.98y 0.74xy 7.86y 1.58y 2.05y 1.55y 0.49y 2.03y 0.17x 0.27y
80 – 10.83 57.18 4.47 55.14 6.06 89.10 24.35 9.89 34.24 2.34 4.68
siFd 7
x
0.75x 4.19x 0.05x 6.90x 1.38x 1.49y 1.35x 0.51x 1.83x 0.14x 0.18x
36 – 8.00 25.70 3.04 26.52 2.02 92.44 5.34 2.45 7.79 2.20 2.01
siFd/tfld y
3
0.00y 7.65y 0.95y 5.81z 0.73y 1.90x 0.42z 0.42z 0.68z 0.12xy 0.12z
15 269 9.00 27.76 2.81 42.61 3.35 92.15 12.95 6.32 19.27 2.05 2.91
2z 22 1.22xy 6.27y 0.65y 11.32y 1.03y 1.13x 1.31y 0.76y 2.02y 0.18y 0.27y
Plants were grown in soil for 8 weeks in a growth chamber under the conditions described in Experimental procedures. The methods used to determine the levels of chlorophylls, carotenoids, Fd and Fld are also given in Experimental procedures. Values are means SE of seven individual plants. Statistical differences between plant lines (P £ 0.05) are indicated by different letters (x, y, z).
expression (Figure 2). One of the strategies used transgenic pfld5-8 plants that expressed a plastid-targeted Anabaena Fld from a nuclear-inserted gene (Tognetti et al., 2006). They were partially depleted of Fd by using antisense RNA technology, to yield asFd/pfld lines. The aim of this strategy was to determine whether the lower limit of 45–55% of WT Fd levels observed by us and by Holtgrefe et al. (2003) could be reduced if Fd was knocked-down in a pfld5-8 background where chloroplast Fld was already present at approximately 70 pmol g)1 fresh weight (FW), in the same range of WT Fd contents (Tognetti et al., 2006). Previous results suggested that chloroplast accumulation of Fld was limited by organellar protein import efficiency (Tognetti et al., 2006). To overcome this limitation and increase leaf Fld dosage, a second approach was used, involving siFd plants into which the Fld gene was subsequently introduced (Figure 2) by direct chloroplast transformation. In this strategy, the transgene was inserted by homologous recombination into a single site of the plastidic genome (Maliga, 2004). Silenced siFd plants were trans-
Figure 2. Schematic representation of the strategies used to generate plants with decreased ferredoxin levels and/or flavodoxin expression.
formed with plasmid pBSWUTR-fld (see Experimental procedures), and homoplasmic lines (called siFd/tfld, for transplastomic fld) were isolated by successive rounds of growth under selection. In this case, independent transformants did not show position effects and yielded essentially the same leaf Fld contents (269 22 pmol Fld g)1 FW) in homoplasmic lines. Plants containing the lowest amounts of Fd in the presence of Fld were selected for further studies (Figure 3a,b). As anticipated, Fld expression allowed recovery of viable plants with very low Fd contents (Figure 3a,b and Tables 1 and 2), well below those observed in asFd and siFd siblings. The levels of Fd remaining and of Fld expressed in the various lines analyzed are indicated in the table and figure legends. Fld-expressing plants showed recovery of many features of the WT phenotypes (Figure 3c and Table 1). Antisense asFd lines retaining 60–70% Fd (Figure 3a) displayed stunted growth and severe leaf chlorosis relative to their WT siblings, and Fld expression in these plants led to significant recovery of growth rates and chlorophyll and carotenoid contents (Figure 3c and Table 1). Growth arrest was also seen in siFd plants (Figure 3c). Interestingly enough, patched leaf chlorosis, rather than the bleaching typical of asFd leaves, was frequent in siFd plants. Expression of Fld from a chloroplast-encoded gene restored growth rates and normal content and distribution of pigments in leaves (Figure 3c and Table 1). Statistical analysis indicated that recovery was partial for most phenotypic traits (Table 1). As previously reported (Tognetti et al., 2006), pfld5-8 plants, which contained similar levels of Fd and Fld (70–80 pmol g)1 FW) in their chloroplasts and were used as additional controls, did not differ significantly from WT siblings with respect to growth and pigment contents (Figure 3c).
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
926 Nicola´s E. Blanco et al.
Figure 3. Flavodoxin accumulation in chloroplasts complements the phenotypes of ferredoxin-deficient transgenic tobacco plants. Fd and Fld levels in leaves of WT plants and asFd (a) and siFd (b) plant, expressing Fld (pfld, tfld) or not, were determined in leaf extracts from 6-week-old plants grown under growth chamber conditions, using SDS–PAGE and immunoblotting. Lane pfld corresponds to pfld5-8 extracts. Cleared leaf extracts corresponding to 5 lg (a) or 10 lg (b) of total soluble protein were loaded onto each lane for Fd and Fld detection. The electrophoretic mobilities of both electron carriers are indicated by arrows. The slower-migrating band reacting with Fld antiserum that could be visualized in some pfld lanes corresponds to unprocessed Fld precursor, as reported previously (Tognetti et al., 2006). (c) Development of WT and transgenic plants grown under autotrophic conditions. Tissue culture-derived seedlings were transferred to soil, and grown for 6 weeks as indicated in Experimental procedures. Several plants were sampled, and representative results are shown (second youngest fully expanded leaf and corresponding whole plant). In the examples provided, the Fd contents remaining, relative to wild-type, were 58% (asFd), 12% (asFd/pfld), 45% (siFd) and 17% (siFd/tfld). Fld levels were 70 pmol g)1 FW in all cases except for siFd/tfld plants, which contained approximately 270 pmol g)1 FW.
To further characterize the effect of Fd deficiency and Fld expression at the cellular level, we analyzed leaf tissue from the various lines by optical and transmission electron microscopy (TEM). The micrographs in Figure 4(a) indicate that WT cells in the palisade parenchyma were rich in ellipsoid chloroplasts, which were pressed against the plasmalemma by a large vacuole. In contrast, tissues from asFd and siFd leaves displayed several abnormal features. Cell organization was irregular: there were fewer chloroplasts per cell and these chloroplasts were round. Fld complementation in asFd/pfld and siFd/tfld lines resulted in recovery of chloroplast number and geometry. Plastid ultrastructure, as determined by TEM, was largely disrupted in Fd-deficient plants, with loss of granal stacking and profusion of plastoglobules (Figure 4), indicative of ongoing thylakoid disassembly (Brehelin et al., 2007). Except for a small number of plastoglobules in the stroma, asFd/pfld and siFd/tfld lines did not show any significant differences from WT plants. Chloroplasts from pfld5-8 lines were also very similar to those of their WT counterparts. The photosynthetic performance of silenced, antisense and complemented lines was assayed by measuring CO2 assimilation (Figure 5). WT plants displayed maximal rates in the range 15–16 lmol CO2 m)2 sec)1, whereas pfld5-8 siblings did not show improved photosynthetic activity at any measured light intensity (Figure 5a), in agreement with previous reports (Tognetti et al., 2006). Silenced siFd plants retaining 45% of WT Fd levels reached only 20% of the maximal CO2 fixation rates displayed by WT siblings (Figure 5b), and asFd lines (60% of Fd remaining) performed even worse (Figure 5a). Fld expression increased maximal photosynthetic rates to approximately 12 lmol CO2 m)2 sec)1 in both Fd-deficient backgrounds, with saturation occurring at lower light intensities (Figure 5). It is worth emphasizing that Fld-expressing lines contained significantly lower Fd levels than their corresponding controls (Figures 3 and 5), suggesting that photosynthesis was largely dependent on Fld-driven electron transport in these plants. Measurements of chlorophyll fluorescence in darkadapted leaves of siFd and asFd plants showed significant decreases of the Fv/Fm value, which reflects photodamage to photosystem II (PSII) (Table 2). Values for other photosynthetic parameters were obtained after adaptation of the same leaves to 200 lmol quanta m)2 sec)1 of actinic light (Baker, 2008). The quantum yield of PSII (/PSII) and the F¢v/F¢m ratio were very low in both types of Fd-deficient plants compared to wild-type (Table 2). Decreases in Fv/Fm, F’v/F’m and /PSII values were prevented by Fld expression in the complemented lines (Table 2). The capacity for non-photochemical quenching (NPQ) was estimated under steady-state conditions, i.e. when the Calvin cycle is active after several minutes of illumination (Baker, 2008; Cardol et al., 2010). NPQ values increased
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
Flavodoxin complements ferredoxin deficiency 927 Table 2 Determination of chlorophyll fluorescence parameters in ferredoxin-deficient and flavodoxin-complemented tobacco plants
Fd (pmol g)1 FW) Fld (pmol g)1 FW) Fv/Fm F’v/F’m /PSII NPQ 1 – qP
WT
asFd
asFd/pfld
80 7x – 0.724 0.015x 0.615 0.017x 0.546 0.024x 0.170 0.042y 0.111 0.016z
48 5y – 0.513 0.029y 0.142 0.028z 0.056 0.020y 0.822 0.092x 0.615 0.061x
10 70 0.697 0.446 0.318 0.246 0.296
2z 8 0.037x 0.069y 0.087x 0.044y 0.085y
WT
siFd
siFd/tfld
80 7x – 0.721 0.028x 0.659 0.020x 0.634 0.020x 0.186 0.032y 0.039 0.018y
36 3y – 0.611 0.064y 0.137 0.050y 0.063 0.016z 0.881 0.033x 0.530 0.063x
15 269 0.697 0.609 0.563 0.195 0.075
2z 22 0.022x 0.021x 0.028y 0.045y 0.025y
Eight week-old plants were sampled 3–4 h into the light period, and dark-incubated for 30 min prior to dark-adapted measurements (Fv/Fm). Lightadapted determinations were performed after 25 min of exposure to 200 lmol quanta m)2 sec)1. Values are means SE of at least six measurements on independent plants. Statistical differences between plant lines (P £ 0.05) are indicated by different letters (x, y, z).
dramatically in the antisense and silenced plants relative to WT controls (Table 2). The value of the 1 – qP parameter was also greater in the two Fd-deficient lines, reflecting overreduction of the QA acceptor of PSII (Krause and Weis, 1991). The increase in both NPQ and 1 – qP was prevented by Fld expression (Table 2). Statistical analysis indicates that recovery was complete for most photosynthetic parameters (Table 2). When plants are exposed to actinic light, they undergo a transient increase in NPQ, which reaches a maximum and subsequently decreases due to photochemical quenching (as the Calvin cycle becomes active), to reach steady-state values as reported in Table 2. Under the experimental conditions used (see Discussion), the increase in NPQ stems mostly from two components with characteristic kinetics: a rapid phase of NPQ induction (rise time of approximately 1 min in Arabidopsis) that involves generation of a pH gradient across thylakoid membranes (originated by photosynthetic electron transport), followed by a slower phase that is dependent on zeaxanthin turnover (Nilkens et al., 2010). If the light is turned off when NPQ is at its maximum, NPQ relaxation proceeds through the same fast and slower components. In cases in which a photoinhibitory process takes place (i.e. when plants are illuminated at high intensity for extended periods), a third relaxation phase can be detected over longer time scales, representing the photoinhibitory (qI) quenching component (Krause and Weis, 1991; Nilkens et al., 2010). Analysis of the time courses of both light induction and dark relaxation of NPQ provides valuable data on the status of the PETC and the thermal dissipation of excess excitation energy in PSII. Figure 6 shows the NPQ induction/relaxation kinetics obtained for WT, siFd and siFd/tfld lines grown under growth chamber conditions (200 lmol quanta m)2 sec)1), and assayed under actinic light of the same intensity after a dark-adaptation period of 30 min. The /PSII parameter, which provides an estimation of linear electron flow through PSII (Baker, 2008), was determined simultaneously on the same leaves and under the same conditions (Figure 6, insets). All lines showed NPQ induction curves with a similar general shape (Figure 6), with the rapid DpH-dependent
phase being completed within the time frame of the first data point recorded (30 sec). However, there were significant differences in signal amplitudes among the various lines. Compared to WT plants (Figure 6a), siFd siblings showed a 39% increase in the signal amplitude of NPQ induction (Figure 6b), accompanied by a 35% decrease in /PSII (insets to Figure 6a,b). Relaxation time courses showed a fast decrease within the first 10 sec, followed by a second phase that was 25% slower in siFd plants relative to the wildtype (Figure 6a,b). The results suggest that Fd deficiency partially impairs the ability of the silenced plants to perform photochemistry at the level of PSII, as reflected by a lower /PSII value during measurements under actinic light (Figure 6b, inset), but the PETC and the photosystems are still able to deploy an enhanced NPQ response. When Fld was expressed in the siFd background, the induction and relaxation kinetics were intermediate between those of the WT and siFd plants, but with amplitudes closer to the WT values (Figure 6c). However, the value of the /PSII parameter was restored to WT levels by the presence of the cyanobacterial flavoprotein (Figure 6c, inset). Sub-optimal performance of PSII and the PETC as observed in siFd lines might affect the response of these plants to prolonged exposure to higher light intensities. To evaluate this possibility, WT, siFd and siFd/tfld plants were exposed to 600 lmol quanta m)2 sec)1 for 6 h, dark-adapted for 30 min, and assayed at 200 lmol quanta m)2 sec)1 of actinic light as described above. As anticipated, these moderate light intensities had little effect on the NPQ induction and relaxation kinetics of WT plants (Figure 6a), or their photochemical capacity, as reflected by the /PSII values (Figure 6a, inset). In contrast, light treatment severely impaired the ability of Fd-deficient lines to induce NPQ, decreasing the signal amplitude by 60% relative to untreated siFd plants (Figure 6b). This effect was correlated with protracted NPQ relaxation after turning off the actinic light (Figure 6b), and an approximately 42% inhibition of /PSII relative to untreated silenced controls (Figure 6b, inset). The /PSII values recovered in Fld-expressing siFd/tfld plants to approximately 85% of those of untreated controls (Figure 6c, inset). However, the signal amplitudes of NPQ induction
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
928 Nicola´s E. Blanco et al.
Figure 4. Light and transmission electron microscopy of leaf cross-sections. Histological and ultrastructural analysis of leaf cross-sections of WT, asFd, asFd/pfld, pfld5-8, siFd and siFd/tfld plants. The cell organization of leaves of asFd and siFd appeared irregular: there were fewer chloroplasts per cell and these chloroplasts were round. The magnified TEM images highlight the differences in thylakoid stacking and plastoglobule formation of these lines compared to the wild-type. Due to Fld complementation, asFd/pfld and siFd/tfld lines did not show any significant structural differences compared to the wild-type. Fd and Fld levels are indicated in Table 1. In pfld5-8 plants Fd and Fld contents were 80 and 70 pmol g)1 FW, respectively. Scale bars = 30 lm (light microscopy images), 1 lm (TEM left panel) and 0.2 lm (TEM right panel).
were lower than those of siFd/tfld plants kept under growth chamber conditions (Figure 6c), indicating some degree of damage. These results indicate that, after 6 h of moderate light treatment, siFd plants undergo severe deterioration in the PETC and/or PSII, and are no longer able to establish a fully functional NPQ response. For both light-exposed and
untreated plants, incorporation of Fld restored NPQ and /PSII values to levels close to those of wild-type. Electron flow from PSI, estimated by the oxidation of P700 under near infra-red light, decreased significantly in the antisense (Figure 7a) and silenced (Figure 7b) Fd-deficient lines, affecting both the maximum amplitude and the half-time of oxidation. The decrease in maximum amplitude
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
Flavodoxin complements ferredoxin deficiency 929
Figure 5. Flavodoxin expression restores CO2 assimilation activities in ferredoxin-deficient tobacco plants. (a) Light-response curves of net photosynthetic rates corresponding to 8-week-old WT (closed circles), asFd (open circles), asFd/pfld (grey circles) and pfld5-8 (closed diamonds) plants. (b) Light-response curves for CO2 assimilation rates corresponding to WT (closed squares), siFd (open squares) and siFd/tfld (grey squares) plants. Determinations were performed using the second youngest fully expanded leaves. Fd and Fld levels are indicated in Table 1. Values are means SE of triplicate assays on three independent plants (P £ 0.05).
indicates lower efficiency of P700 oxidation and/or a lower availability of this reaction centre pigment for oxidation. The effect was prevented in Fld-expressing plants, which showed nearly WT values for these parameters (Figure 7). Fld expression restores stress tolerance to Fd-deficient lines As indicated, exposure to hostile environments leads to down-regulation of Fd expression in both plants and cyanobacteria, contributing to the damage experienced by the stressed organism (Mazouni et al., 2003; Zimmermann et al., 2004; Singh et al., 2004; Tognetti et al., 2006). We therefore evaluated the effect of Fd depletion on stress tolerance by exposing leaves from antisense, silenced and complemented plants to methyl viologen (MV), a contact herbicide that generates superoxide radicals, mostly in chloroplasts, through a redox-cycling reaction of the reduced form with oxygen (Babbs et al., 1989). Typical results obtained with siFd and siFd/tfld lines are shown in Figure 8(a). Fd-deficient siFd specimens were abnormally sensitive to MV relative to the wild-type, undergoing progressive leaf bleaching that eventually led to necrosis over the entire leaf (Figure 8a). Similar symptoms were observed in the WT controls, but the number and severity of the lesions were significantly lower (Figure 8a). Fld expression in chloroplasts of these silenced plants resulted in increased tolerance to MV toxicity, comparable to that of the wild-type with respect to the frequency of chlorotic leaf spots. Moreover, unlike WT plants, these lesions did not become necrotic at the end of the 7-day assay (Figure 8a). MV-dependent damage was also evaluated in short-term exposure experiments by measuring Fv/Fm (Figure 8b) and electrolyte leakage (Figure 8c) in leaf discs. Two hours after MV treatment, WT discs showed a 60% decrease in the Fv/Fm ratio, indicating substantial photodamage to PSII. Deterioration was significantly higher in siFd discs and lower in
Figure 6. Induction/relaxation curves of non-photochemical quenching and /PSII in plants containing various amounts of ferredoxin and flavodoxin. NPQ and /PSII (inset) measurements were carried out using induction and relaxation periods of 6 and 18 min, respectively, as indicated by the boxes at the top. Plants had been kept under growth chamber light conditions (200 lmol quanta m)2 sec)1, closed circles), or incubated for 6 h at 600 lmol quanta m)2 sec)1 (open circles). Values are means SE of three assays on independent plants.
siFd/tfld tissue (Figure 8b). Partial protection against membrane damage by Fld expression in siFd plants was evident, as measured by the extent of ion leakage (Figure 8c). The tolerance of siFd/tfld plants to MV toxicity was intermediate
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930 Nicola´s E. Blanco et al.
Figure 7. P700 absorbance kinetics under near infra-red light. P700 oxidation by near infra-red light was monitored on discs excised from the second youngest fully expanded leaves of 8-week-old WT, asFd (a) and siFd (b) plants and their Fld-complemented siblings. The upper panel shows two typical time courses for each line, with upward arrows indicating application of the light pulses. The middle and lower panels show maximal amplitudes and half-times of P700 oxidation. Fd and Fld levels are indicated in Table 1. Values are means SE of five replicate experiments. Letters above each bar indicate the statistical significance of the observed differences among lines: values with the same letter are not significantly different at P £ 0.05; those with different letters are significantly different at P £ 0.05.
between that of WT and pfld5-8 siblings in both assays (Figure 8b,c). DISCUSSION We tested the hypothesis that the stress protection conferred by cyanobacterial Fld when expressed in plant chloroplasts was caused by functional replacement of endogenous Fd (Tognetti et al., 2006). To address this question, we produced Fd-deficient plants by two complementary approaches, RNA antisense and RNA interference, followed by complementation with an Fld gene expressed from either the nuclear or the plastidic genome (Figure 2). A scheme summarizing the results obtained is shown in Figure 9. Both siFd and asFd lines showed the typical symptoms of Fd deficiency reported for potato (S. tuberosum) and Arabidopsis (Holtgrefe et al., 2003; Voss et al., 2008; Hanke and Hase, 2008), namely growth arrest, leaf chlorosis, chloroplast abnormalities, inhibition of photosynthesis and reduced electron transport (Figures 1 and 3–7 and Tables 1 and 2). Expression of Fld reversed most phenotypical
Figure 8. Flavodoxin expression in chloroplasts of ferredoxin-deficient plants increases tolerance to methyl viologen toxicity. (a) The second youngest fully expanded leaves from 8-week-old WT plants and T3 transformants were exposed to MV as described in Experimental procedures. Photographs were taken at 4 (upper panel) and 7 (lower panel) days after exposure. Fd and Fld levels are given in Table 1. (b, c) Fv/Fm (b) and electrolyte leakage (c) were measured on leaf discs from WT and T3 transformed plants after incubation with 10 lM MV for 2 and 4.5 h, respectively, at 600 lmol quanta m)2 sec)1. Results are given as the percentages of Fv/Fm and ion leakage relative to discs incubated in water under the same conditions. Values are means SE of three assays. Letters above each bar represent the statistical significance of the observed differences among lines (P £ 0.05): values with the same letter are not significantly different at P £ 0.05; those with different letters are significantly different at P £ 0.05.
symptoms of Fd deficiency, including re-establishment of photosynthetic activities to nearly WT levels (Figures 3–6 and Tables 1 and 2). Leaf Fd contents of approximately 45% (relative to the wild-type) were determined to be the lowest limit for recovery of viable Fd-deficient lines, but Fldcomplemented plants with only 12–18% of remaining Fd were able to grow autotrophically on soil and set seed,
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Flavodoxin complements ferredoxin deficiency 931
Figure 9. Cyanobacterial flavodoxin complements ferredoxin deficiency in tobacco plants. Schematic model showing typical phenotypes displayed by transformed lines after Fd depletion by antisense RNA technology (asFd) or RNA interference (siFd), and after Fld introduction by nuclear (pfld, asFd/pfld) or chloroplast (siFd/tfld) transformation. Second fully expanded leaves of 8-week-old plants were photographed. Typical immunoblots for Fd and Fld obtained using extracts from the same leaves are shown for each line.
indicating that Fld was able to take over most, if not all, Fd functions in these transgenic organisms. Accumulation of the cyanobacterial flavoprotein in a WT background did not affect expression of endogenous Fd (Tognetti et al., 2006, 2007), suggesting that the Fd depletion observed in these lines was due to better survival in the presence of Fld, rather than down-regulation of endogenous Fd expression. Dark-adapted antisense and silenced leaves showed moderate but significant decreases in the Fv/Fm ratio relative to WT siblings (Table 2), indicating that the maximum quantum yield of PSII primary photochemistry was reduced even when plants were cultured under non-stressing light intensities. The lower chlorophyll and carotenoid levels (Table 1) suggest that the decrease in the capacity to perform photochemistry might result from a reduced ability of the light-harvesting complexes (LHC) to collect excitation energy. Moreover, the decrease in F’v/F’m values indicates difficulties in transferring energy to open PSII reaction
centres (Table 2), probably as a consequence of alterations in LHC associated with PSII (Krause and Weis, 1991). Impaired photochemistry is also indicated by the lower /PSII values and increased excitation pressure 1 – qP exhibited by Fd-deficient lines (Table 2). The fluorescence analysis shows that the alterations in photosynthetic parameters in asFd and siFd plants are probably a consequence of an over-reduced plastoquinone pool resulting from lower amounts of Fd. Although decreases in Fd led to over-reduction of the PETC and probably a rearrangement in the organization of the LHC of PSII, the presence of Fld alleviated most of these symptoms. Fluorescence-derived parameters associated with PSII returned to nearly WT levels (Table 2), indicating that the excitation pressure on the acceptor side of PSII was relieved in the complemented plants. We also evaluated photochemical efficiency in the various lines by determining the level of NPQ and its induction/ relaxation kinetics. Under steady-state conditions (obtained after 25 min of illumination), and in agreement with their lower photochemical capacity, siFd and asFd plants showed elevated NPQ levels relative to the wild-type (Table 2), indicating that, even when illuminated at low intensity (200 lmol quanta m)2 sec)1), these Fd-deficient lines dissipate a significant amount of the absorbed energy by nonphotochemical mechanisms. NPQ can be split into various overlapping components, involving energy-dependent (qE), state transition (qT) and photoinhibitory (qI) dissipation processes (Krause and Weis, 1991; Horton et al., 1996; Nilkens et al., 2010). At moderate light intensities, the contribution of the two latter components in the evaluated time frame is negligible, and qE accounts for most of the NPQ (Baker, 2008; Nilkens et al., 2010). Using selected Arabidopsis mutants, Nilkens et al. (2010) were able to dissect this energy-dependent phase into two separate components: a fast process that is dependent on establishment of a proton gradient across the thylakoid membrane, its sensing by the PsbS subunit of PSII (Niyogi, 2000) and zeaxanthin synthesis (Nilkens et al., 2010), followed by a slower phase (rise time of 10–15 min) that still requires zeaxanthin but is independent of DpH and PsbS (Nilkens et al., 2010). Under our experimental set-up, only this phase could be recorded; most of the DpH-dependent NPQ increase presumably occurs prior to the first data point (Figure 6). WT tobacco plants displayed typical NPQ induction and relaxation kinetics that were mostly unaffected by previous exposure to moderate light intensities (Figure 6a). As anticipated from steady-state measurements (Table 2), untreated siFd plants showed higher NPQ induction and lower /PSII values (Figure 6b). Interestingly, after moderate light treatment, these Fd-deficient lines were no longer able to deploy exacerbated NPQ (Figure 6b), an effect that was correlated with a significant decrease in /PSII relative to untreated siFd controls (Figure 6b, inset). The results indicate that, under
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932 Nicola´s E. Blanco et al. Fd deficiency, even a mild stress results in considerable damage to the PETC, and to the ability of PSII to elicit NPQ based on electron transport. Lower carotenoid contents in these plants (Table 1) may also contribute to the effect, although determination of zeaxanthin levels is required to support this suggestion. Once again, Fld expression alleviated most of these symptoms (Figure 6c), indicating that Fld allowed proper function of the PETC and electron delivery at the reducing side of PSI, preventing chronic damage to electron transfer components, and allowing sustained and efficient photochemistry even with very low levels of Fd remaining. Similar results were obtained when studying the effects of Fd depletion and Fld expression on PSI function by measuring P700 turnover. Both the maximum amplitude and the half-time of P700 oxidation were severely decreased in Fd-deficient plants (Figure 7). Although this inhibition could be due to lower levels of functional PSI, the most likely explanation is inhibition of electron output caused by lack of Fd at the acceptor side. A similar situation has been reported for plants under cold stress and in PSI mutants (Sonoike et al., 1995; Haldrup et al., 2003; Kim et al., 2005), and is the initial stage of chronic PSI photoinhibition (Tjus et al., 2001). The hypothesis of acceptor-side limitation at PSI is supported by the observation that both the amplitude and kinetics of P700 oxidation recovered in Fld-expressing plants (Figure 7). Oxidants such as MV and virtually all environmental stresses lead to down-regulation of Fd expression in plants and photosynthetic micro-organisms (Thimm et al., 2001; Mazouni et al., 2003; Tognetti et al., 2006). This decrease, with its sequel of electron transfer inhibition, over-reduction of the PETC and ROS propagation, has been proposed to contribute significantly to the damage undergone by the stressed plant. Our results agree with this proposal, as siFd lines were abnormally sensitive to MV toxicity compared to WT siblings (Figure 8a). Expression of Fld allowed recovery of stress tolerance beyond that displayed by WT plants (Figure 8a). In short-term experiments, the MV-dependent damage undergone by siFd/tfld plants at the level of PSII (Fv/Fm) and membrane integrity (ion leakage) was intermediate between that of wild-type and transgenic plants expressing Fld in a WT genetic background (Figure 8b,c). The results indicate that Fd-deficient lines have an overreduced PETC, probably due to PSI acceptor-side limitation, a situation that is corrected by Fld introduction. Photosynthetic parameters of the complemented lines showed little or no statistical differences from those of WT siblings, strongly suggesting that cyanobacterial Fld could indeed replace most functions of plant Fd in the PETC. However, recovery of CO2 assimilation rates (Figure 5) and phenotypic complementation (Table 1), although substantial, were only partial. These results may reflect a lower efficiency of the cyanobacterial electron shuttle in chloroplast Fd-dependent reac-
tions other than photosynthesis, as has been shown for in vitro Trx reduction (Tognetti et al., 2006). Fld expression re-established photosynthetic electron transport and delivery of reducing equivalents to productive assimilatory routes in plants containing as little as 12–18% of the physiological Fd content (Figures 4–6 and Table 1). In conclusion, stress tolerance conferred by Fld expression in transgenic tobacco plants appears to stem largely, if not exclusively, from the ability of this cyanobacterial electron transfer flavoprotein to replace chloroplast Fd. Conjugated iron, and especially iron–sulfur clusters, is believed to have been among the first biological catalysts (Wa¨chtersha¨user, 1992), suggesting that ferredoxins are very ancient proteins that were widely used by anaerobic organisms for a plethora of metabolic pathways. Then, approximately 2.7 billion years ago, cyanobacteria took the epochal step of evolving PSII and oxygenic photosynthesis, leading to one of the most dramatic ecological crises since the origin of life: the atmosphere became oxidative, and iron availability, on which photosynthesis relied heavily, was compromised. Many iron-containing proteins and enzymes were replaced by isofunctional counterparts, either constitutive or inducible, that used alternative prosthetic groups. Among photosynthetic components, Fld substitution of Fd appears to be particularly important, but there are other examples. Haem-containing cytochrome c6 was progressively displaced from the algal lineage by copper-containing plastocyanin, and has disappeared from land plants. Indeed, a genome-wide comparison between closely related cyanobacteria thriving in iron-replete and iron-deficient habitats revealed a number of genes that were either only present or induced in the isolates collected from iron-deficient regions (Palenik et al., 2003). The ability of cyanobacterial Fld to complement deficiency of a plant Fd in vivo indicates that some of these ancient strategies, although lost in plants, are imprinted in the biochemical blueprint of photosynthetic organisms. Chida et al. (2007) have also reported that expression of a green algal cytochrome c6 in Arabidopsis enhanced photosynthesis and growth. It is unknown how many of these responses are still functional in plants, or why genetic traits that confer such obvious advantages were not retained. Understanding the interactions and activities of Fld when expressed in plants could be a powerful tool to address these issues and to shed some light on the rich evolutionary history of photosynthetic organisms. EXPERIMENTAL PROCEDURES Construction of binary vectors and transformation of tobacco A DNA fragment encoding tobacco Fd precursor (GenBank accession number AY552781.1) was obtained by PCR amplification of
ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
Flavodoxin complements ferredoxin deficiency 933 genomic DNA using primers 5¢-TTCTGCAGAAATGGCCAGTATTTCAGGTAC-3¢ and 5¢-GACGGATCCTAAAGTGTTTTTTGAAATAATG3¢, containing PstI and BamHI recognition sites, respectively. Amplified DNA was digested using the corresponding enzymes, and cloned in the antisense orientation between the CaMV 35S promoter and polyadenylation regions of pDH51 (Pietrzak et al., 1986). This construct was excised using EcoRI, and inserted into binary vectors pCAMBIA2200 or pCAMBIA1200, which display kanamycin and hygromycin resistance, respectively (Hajdukiewicz et al., 1994), to yield pCAMBIA2200-asFd or pCAMBIA1200-asFd. Recombinant plasmids were verified by DNA sequencing and used to transform WT (pCAMBIA2200-asFd) and pfld5-8 (pCAMBIA1200-asFd) tobacco plants (Tognetti et al., 2006) by A. tumefaciens-mediated leaf disc transformation (Gallois and Marinho, 1995). Primary transformants (asFd and asFd/pfld lines) were propagated up to the T3 generation by self-pollination. To silence Fd expression, the attB1/attB2 recombination sites were added to the extremes of the tobacco Fd sequence as described by Helliwell and Waterhouse (2003). The resulting attBFd product was inserted in tandem and reverse orientations in pHELLSGATE2, which contains a kanamycin resistance gene (Wesley et al., 2001). The correct orientation and size of the two attB-Fd copies and the Pdk intron between them were confirmed by sequence determination, and the resulting plasmid was introduced into WT leaves via A. tumefaciens. Primary transformants (siFd) were propagated up to the T3 generation by self-pollination. For chloroplast transformation, a DNA fragment encoding the entire Fld gene from Anabaena sp. PCC7119 (GenBank accession number S68006) was cloned between the NdeI and XbaI restriction sites of plasmid pBSWUTR (Wirth et al., 2006), under the control of the plastidic rnn and psbA promoters, to produce pBSWUTR-fld. In vitro propagated plants of an established siFd line were subjected to plastidic transformation with pBSWUTR-fld as described by Wirth et al. (2006). After three rounds of regeneration on selective medium containing 500 lg ml)1 spectinomycin, several siFd/tfld lines with both transgenic traits were obtained.
distilled water. For the secondary fixation, samples were transferred to 1% w/v OsO4. After 1 h, they were washed three times with distilled water. Dehydration, resin embedding and thin sectioning for light and electron microscopy were performed as described previously (Tognetti et al., 2006), except that an FEI Tecnai G2 Sphera transmission electron microscope (FEI, http://www.fei.com) was used for ultrastructural analysis at 120 kV.
Determination of photosynthetic parameters Net CO2 uptake rates (ACO2 ) were determined as described previously (Hajirezaei et al., 2002). Chlorophyll fluorescence measurements were performed using a pulse-modulated fluorometer (Qubit Systems, http://qubitsystems.com) and a MINI-PAM 2000 fluorometer (Walz, http://www.walz.com). Fv and Fm were determined after dark adaptation for 30 min. Subsequently, leaves were exposed to 200 lmol quanta m)2 sec)1 of actinic light, and lightadapted values (F¢v, F¢m) were determined at the end of each actinic light period. Photosynthetic parameters (Fv/Fm, F’v/F’m, /PSII, 1 – qP and NPQ) were calculated as described previously (Baker, 2008). Induction/relaxation NPQ curves were determined using the program of the MINI-PAM 2000 fluorometer. Light induction measurements were performed on dark-adapted leaves by application of a saturating pulse to obtain the Fm values, and a second pulse 5 sec later. After 30 sec, NPQ induction was followed over 6 min at 200 lmol quanta m)2 sec)1 of actinic light, and saturating pulses were applied every 30 sec to obtain F¢m values. The actinic light was subsequently turned off, and the dark relaxation process was monitored at various times (30 sec and 1, 2, 5 and 10 min) using saturating pulses. To evaluate PSI turnover, the time courses of P700 oxidation by near infra-red light (715 nm) were determined using leaf discs (1.54 cm2) of the second youngest fully expanded leaves, using a PDA-100 system (Walz). The DAP700-max values were calculated from the changes in absorption at 830 and 860 nm (Backhausen et al., 1998; Voss et al., 2008). The apparent half-times for P700 oxidation (t½) were estimated by fitting the experimental time courses to the curve of a mono-exponential process.
Plant growth and characterization
Methyl viologen treatment
Seeds were germinated on Murashige & Skoog (MS0) agar plates supplemented with 2% w/v sucrose, and, in the case of transformants, with the required antibiotics at the following concentrations: 100 lg ml)1 kanamycin, 40 lg ml)1 hygromycin and/or 500 lg ml)1 spectinomycin. After 4 weeks, seedlings were transferred to soil, watered daily with nutrient medium (Geiger et al., 1999), and grown at 200 lmol quanta m)2 sec)1, 25C and a 16/8 h photoperiod (growth chamber conditions). Unless otherwise stated, experiments were performed using 8-week-old specimens grown in soil. To improve reproducibility and facilitate comparisons between lines, we performed side-by-side assays of the second and third youngest fully expanded leaves belonging to the same node (counting from the apex). The presence of Fld and Fd in leaf extracts of the various lines was determined by SDS–PAGE and immunoblot analysis. Reactive bands were integrated, and levels of the electron carriers were estimated by comparison with pure standards. The levels of photosynthetic pigments were determined spectrophotometrically after ethanol extraction of leaf discs (Lichtenthaler, 1987).
Attached second and third youngest fully developed leaves were dipped into 250 lM MV, 0.1% v/v Tween-20 for 30 sec, and incubated under growth chamber conditions for up to 7 days. For shortterm experiments, leaf discs (12 mm diameter) were floated topside up in 1 ml water or 10 lM MV, and illuminated at 600 lmol quanta m)2 sec)1 for 2 h (Fv/Fm) or 4.5 h (ion leakage), as described previously (Tognetti et al., 2006).
Histological and ultrastructural analysis of leaf tissue For primary fixation, 1 mm2 sections of the second youngest fully expanded leaf were incubated for 4 h at 25C in 50 mM cacodylate buffer, pH 7.2, containing 2% v/v glutaraldehyde and 2% v/v formaldehyde, followed by one wash with buffer and two washes with
Statistical analyses Data were analyzed using one-way ANOVA and Tukey multiple range tests. When the normality and/or equal variance assumptions were not met, Kruskall–Wallis ANOVA by ranks and Dunn’s multiple range tests were used. The significance refers to statistical significance at P £ 0.05.
ACKNOWLEDGEMENTS This work was supported by grant PICT01-14648 from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, Argentina, grant PID10777 from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina, and by the Deutscher Akademischer Austauschdienst. N.C. and F.F.B.A. are staff members and R.D.C. and M.E.S. are fellows of CONICET. We are indebted to Mr E. Geyer and the Leibniz-Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung gardeners for their excellent work. We
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934 Nicola´s E. Blanco et al. also wish to thank Dr A. ten Have (Instituto de Investigaciones Biolo´gicas-CONICET, Argentina) for helpful comments on sequence analysis.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Multiple alignment of nucleotide sequences of N. tabacum ferredoxins. Figure S2. Analysis of ferredoxin mature protein sequences. Appendix S1. Detailed description of tobacco Fd sequence analysis. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
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ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 922–935
