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Stress response of transgenic tobacco plants expressing a cyanobacterial ferredoxin in chloroplasts

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Stress response of transgenic tobacco plants expressing a cyanobacterial ferredoxin in chloroplasts
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  Stress response of transgenic tobacco plants expressinga cyanobacterial ferredoxin in chloroplasts Romina D. Ceccoli  • Nicola´s E. Blanco  • Milagros Medina  • Ne´stor Carrillo Received: 14 December 2010/Accepted: 5 May 2011/Published online: 17 May 2011   Springer Science+Business Media B.V. 2011 Abstract  Expression of the chloroplast electron shuttleferredoxin is induced by light through mechanisms thatpartially depend on sequences lying in the coding region of the gene, complicating its manipulation by promoterengineering. Ferredoxin expression is also down-regulatedunder virtually all stress situations, and it is unclear if light-dependent induction and stress-dependent repression pro-ceed through the same or similar mechanisms. Previousreports have shown that expression of a cyanobacterialflavodoxin in tobacco plastids results in plants withenhanced tolerance to adverse environmental conditionssuch as drought, chilling and xenobiotics (Tognetti et al. inPlant Cell 18:2035–2050, 2006). The protective effect of flavodoxin was linked to functional replacement of ferre-doxin, suggesting the possibility that tolerant phenotypesmight be obtained by simply increasing ferredoxin con-tents. To bypass endogenous regulatory constraints, wetransformed tobacco plants with a ferredoxin gene from  Anabaena sp.  PCC7120, which has only 53% identity withplant orthologs. The cyanobacterial protein was able tointeract in vitro with ferredoxin-dependent plant enzymesand to mediate NADP ? photoreduction by tobacco thy-lakoids. Expression of   Anabaena  ferredoxin was constitu-tive and light-independent. However, homozygous linesaccumulating threefold higher ferredoxin levels than thewild-type failed to show enhanced tolerance to oxidativestress and chilling temperatures. Under these adverseconditions,  Anabaena  ferredoxin was down-regulated evenfaster than the endogenous counterparts. The results indi-cate that: (1) light- and stress-dependent regulations of ferredoxin expression proceed through different pathways,and (2) overexpression of ferredoxin is not an alternative toflavodoxin expression for the development of increasedstress tolerance in plants. Keywords  Ferredoxin    Anabaena sp.    Overexpression   Stress tolerance Introduction Ferredoxins (Fd) are small, soluble [2Fe-2S] proteins thatplay a key role in electron distribution in all types of plastids (Hase et al. 2006). In green tissues, chloroplast Fdis reduced by the photosynthetic electron transport chain(PETC) at the level of photosystem (PS) I. In its reducedstate, Fd acts a mobile electron shuttle which delivers low-potential reducing equivalents to a plethora of metabolic,dissipative and regulatory pathways. A fraction of photo-reduced Fd is employed for NADP ? reduction viaFd-NADP ? reductase (FNR), generating the NADPHrequired for carbon assimilation and other biosyntheticpathways (Hase et al. 2006). In addition, Fd is the directelectron donor for nitrogen and sulfur assimilation, Electronic supplementary material  The online version of thisarticle (doi:10.1007/s11103-011-9786-9) contains supplementarymaterial, which is available to authorized users.R. D. Ceccoli    N. E. Blanco    N. Carrillo ( & )Instituto de Biologı´a Molecular y Celular de Rosario (IBR,CONICET), Divisio´n Biologı´a Molecular, Facultad de CienciasBioquı´micas y Farmace´uticas, Universidad Nacional de Rosario,Suipacha 531, S2002LRK Rosario, Argentinae-mail: carrillo@ibr.gov.arM. MedinaDepartamento de Bioquı´mica y Biologı´a Molecular y Celular,Facultad de Ciencias, and Institute of Biocomputation andPhysics of Complex Systems (BIFI), Universidad de Zaragoza,Zaragoza, Spain  1 3 Plant Mol Biol (2011) 76:535–544DOI 10.1007/s11103-011-9786-9  glutamate synthesis, fatty acid desaturation and severalenzymes of secondary metabolism (Brouquisse et al. 1989;Hase et al. 2006). As a substrate of Fd-thioredoxinreductase, Fd is also a key player in the regulation of thechloroplast metabolic network by the thioredoxin system,and contributes directly to antioxidant protection by itsinvolvement in ascorbate and peroxiredoxin regeneration(Miyake and Asada 1994; Dietz et al. 2006; Schu ¨rmannand Buchanan 2008). Finally, Fd helps to relieve theelectron pressure on the PETC when photosyntheticefficiency is low due to CO 2  shortage and/or excess illu-mination. Under such conditions, the chain becomesoverreduced, there is a deficiency of oxidized electronacceptors (acceptor side limitation), and electrons or lightenergy may be passed straight to oxygen, generating reac-tive oxygen species (ROS) that could damage all types of biomolecules (Apel and Hirt 2004). Fd alleviates this situ-ation, either by returning the surplus of reducing equiva-lents to the PETC via cyclic electron flow (Yamamoto et al.2006), or to the cell cytosol through the activation of themalate valve (Scheibe 2004).Plant leaves usually contain at least two different Fdisoforms, which in some cases show higher intraspecificthan interspecific similarity (Hanke and Hase 2008). Thevarious leaf-type Fd forms accumulate to different levelsin many plant species, and it has been argued that theycould act preferentially in separate electron transferroutes (Kimata-Ariga et al. 2000; Hanke et al. 2004; Hanke and Hase 2008; Voss et al. 2008). The pivotal role played by leaf Fd in chloroplast oxido-reductive metab-olism is reflected by the pleiotropic phenotypes of bothknocked-down plants (Holtgrefe et al. 2003; Hanke andHase 2008; Blanco et al. 2011) and isoform-specific loss- of-function mutants (Voss et al. 2008), which exhibitgrowth arrest, reproductive handicaps and inactivation of photosynthesis. The severity of the symptoms correlatesinversely with the levels of remaining Fd (Holtgrefe et al.2003; Hanke and Hase 2008; Voss et al. 2008). Leaf Fd expression is induced by light through a regulatorymechanism which requires active photosynthesis andapparently responds to the redox state of the interchainsystem and the stroma. It has been shown to depend onsequences that lie within the transcribed region of thegene (Petracek et al. 1998), therefore complicatinggenetic manipulation of its expression. In addition, levelsof Fd transcripts (Thimm et al. 2001; Zimmermann et al.2004) and protein (Tognetti et al. 2006, 2007) are down- regulated by iron deficit and virtually all kinds of abioticstress situations, including drought, chilling and salinity,a decline which contributes decisively to the damageundergone by plants under such conditions. Althoughthe molecular mechanisms underlying this effect areunknown, any source of environmental stress is expectedto affect the redox status of the stroma and the PETC. Itis therefore conceivable that both processes, light induc-tion and stress repression, could share common pathwaysof sensing and regulation.A similar repression of Fd expression by adverseenvironments has been observed in cyanobacteria, theclosest living relatives of the srcinal endosymbiont thatgave origin to modern-day chloroplasts (Mazouni et al.2003; Singh et al. 2004). In these photosynthetic pro- karyotes, stress-dependent Fd decline is compensated byinduction of the isofunctional electron carrier flavodoxin(Singh et al. 2004). Flavodoxins (Fld) contain flavinmononucleotide as prosthetic group and their propertiesas redox shuttles largely match those of Fd (Sancho2006; Medina 2009). They are found in cyanobacteria and some marine algae, but not in plants (Zurbriggenet al. 2007). However, introduction of a cyanobacterialFld in the chloroplasts of transgenic tobacco resulted inremarkably enhanced tolerance to iron starvation andvarious sources of abiotic stress, including water deficit,extreme temperatures and xenobiotics (Tognetti et al.2006, 2007; Zurbriggen et al. 2008). Fld accumulation in chloroplasts led to restoration of productive routes of electron distribution and suppression of ROS build-up(Tognetti et al. 2006). Its protective role appears to belargely dependent on functional interactions withFd-based endogenous pathways and hence, on successfulFd replacement (Tognetti et al. 2006; Blanco et al. 2011), suggesting that a similar effect could be achieved bysimply overexpressing Fd. Transformed lines accumulat-ing Fd to greater levels than wild type (WT) havealready been prepared by both nuclear (Elliott et al.1989) and chloroplast transformation (Yamamoto et al.2006), but the performance under stress of the resultingtransgenic lines has not been assayed.We report herein the characterization of transgenictobacco plants expressing an active Fd from  Anabaena vegetative cells. Chloroplasts of the transformed linesaccumulated total Fd contents that were * 3-fold over theWT stock. These levels were maintained through severalgenerations and, unlike the chloroplast Fd forms, were notaffected by incubation of the seedlings in the dark, indi-cating that expression of the foreign protein was indeedindependent of light-driven endogenous regulation.Surprisingly, higher Fd levels failed to confer increasedtolerance to oxidative or chilling stress. Furthermore,steady-state levels of   Anabaena  Fd protein and transcriptdeclined on exposure of the transgenic plants to theseadverse conditions. The results indicate that light- andstress-dependent regulations of plant Fd expression operatethrough different pathways, and that Fd overexpression isnot an alternative to Fld expression for the development of stress tolerance in plants. 536 Plant Mol Biol (2011) 76:535–544  1 3  Results and discussion Preparation and characterization of transgenic tobaccoplants expressing  Anabaena  FdWe generated tobacco lines with increased levels of func-tional Fd by using the  Anabaena sp.  PCC7120  petF   gene,whose product is preferentially expressed in vegetativecells and participates in photosynthesis (Bo¨hme and Sch-rautemeier 1986). The coding region of this gene hasdiverged significantly from orthologs present in plantgenomes, displaying only 53% identity at the nucleotidelevel (Supplementary Fig. 1). We argued that this lowdegree of sequence conservation made it unlikely that thecyanobacterial  petF   gene could be recognized by the plantregulatory networks once introduced into the tobaccogenome. Moreover, Mazouni et al. (2003) have shown that,unlike plant Fds, regulation of the Fd gene in the cyano-bacterium  Synechocystis  occurs through a purely tran-scriptional mechanism involving sequences locatedupstream of the transcription start site. Finally, the  Ana-baena  protein has been shown to productively interact invitro with plant PSI with efficiencies similar to those of itseukaryotic counterparts (Navarro et al. 1995).The coding region of the  petF   gene was fused in-frameto an N-terminal extension encoding the chloroplast transitpeptide of pea FNR, which has been previously employedto efficiently direct plastid targeting of a cyanobacterial Fldin transgenic tobacco (Tognetti et al. 2006). The fused genewas placed under control of the constitutive cauliflowermosaic virus (CaMV) 35S promoter (Fig. 1a) and intro-duced into tobacco (  Nicotiana tabacum  cv Petit Havana)by  Agrobacterium -mediated leaf disc transformation, toyield  pAnfd   lines (for  p lastidic  An abaena  Fd  ). The pres-ence of   Anabaena  Fd in foliar tissue was evaluated bysodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot (Fig. 1b). Antisera raisedagainst the cyanobacterial protein did not cross-react withendogenous Fds.  Anabaena  Fd was predominantly recov-ered as a mature-sized species in both leaf and chloroplastextracts (Fig. 1b), suggesting that the precursor wasimported and the transit peptide cleaved in vivo. In somepreparations, slower migrating bands could also be recog-nized by the antiserum (see below), presumably resultingfrom incomplete cleavage of the pre-sequence, as reportedfor other plastid-targeted transgenic proteins (Tognettiet al. 2006). Leaf contents of the introduced prokaryotic Fdshowed ample variations among transformed  pAnfd   lines,reflecting position effects during T-DNA integration(Fischer et al. 2008). Primary transformants displayingvarious levels of Fd expression were self-pollinated, and T 2 seedlings were tested for segregation of the kanamycinresistance trait to confirm a single transgene insertion  locus per genome (Supplementary Table 1). Homozygous lineswere identified by segregation analysis of the selectionmarker after back-crosses into the WT. Homozygosity wasconfirmed in all cases by proportional increases in Fdcontents.Lines  pAnfd2  and  pAnfd4  (Fig. 1b) were singled out forfurther studies. They accumulated 171  ±  14 and154  ±  12 pmol  Anabaena  Fd per g of leaf fresh weight(fw), respectively, as estimated by integration of immu-noblot signals and comparison with pure standards. Thisrepresents about twice the amount of endogenous Fd,indicating that these transgenic lines contained  * 3-foldmore Fd (of any source) than their WT siblings. The levelsof   Anabaena  Fd were maintained along all stages of plantdevelopment and through several generations, and did notdecline when plants were incubated for several hours in the Fig. 1  Expression of   Anabaena  Fd in transgenic tobacco plants. a  Schematic representation of the chimeric gene used to generatetransgenic plants expressing Fd. The coding sequence of Fd (  petF  ),ligated in-frame to the sequence encoding the chloroplast-targetingtransit peptide (TP) of pea FNR, was cloned between the CaMV 35Spromoter and polyadenylation regions (T). LB and RB, left and rightborders, respectively.  b  Anabaena  and endogenous Fd accumulationin leaves of WT and two independent  pAnfd   primary transformants.Leaf (LE) and intact chloroplasts (CE) extracts from 8-week-oldplants corresponding to 0.1 mg fw and 5  l g chlorophyll, respectively,were fractionated by SDS-PAGE and blotted onto nitrocellulosemembranes for immunodetection of Fd. The levels of the smallsubunit of Rubisco (RbcS) are shown as loading controls in CE. c  Comparative amounts of Fds in leaf extracts of transgenic and WTtobacco plants incubated for 24 or 48 h in the  dark   under growthchamber conditions. The srcins of the Fd, as determined by thespecific antisera, are indicated as  subscripts : An,  Anabaena ; Nt,tobaccoPlant Mol Biol (2011) 76:535–544 537  1 3  dark (Fig. 1c), indicating that expression of the cyano-bacterial iron-sulfur protein was not affected by the light-dependent plant regulatory system. When cultured undergrowth chamber conditions, transformed plants displayedWT phenotypes with respect to biomass accumulation andphotosynthetic pigment contents (Supplementary Table 2),in good agreement with previous reports (Yamamoto et al.2006). Linear electron flow and CO 2  assimilation rates alsofailed to reveal significant differences between transgenicplants and non-transformed siblings (SupplementaryTable 2).To confirm that  Anabaena  Fd was expressed in tobaccoas an active holoprotein, the electron carrier was isolatedfrom leaves of the transgenic plants (Fig. 2a). Purified  Anabaena  Fd displayed the normal absorption spectrumcorresponding to the iron-sulfur cluster, very similar to thatof a plant Fd (Fig. 2b). It was able to mediate NADP ? photoreduction by isolated tobacco thylakoids at1.82  ±  0.13  l mol NADPH mg - 1 chlorophyll min - 1 , andto accept electrons from pea FNR in the cytochrome c  reductase assay (Fig. 2c). Specific activities at 10  l M Fd(198  ±  21  l mol cytochrome  c  l mol - 1 FNR s - 1 ) weresimilar to those reported for plant Fd (Carrillo and Cec-carelli 2003). The results indicate that  Anabaena  Fdproperly assembled the iron-sulfur cluster when expressedin chloroplasts, and that it was able to interact with chlo-roplast Fd-dependent systems as efficiently as the endog-enous Fd.Expression of   Anabaena  Fd in tobacco chloroplastsdoes not increase the stress tolerance of transgenic linesTo evaluate the stress tolerance of   pAnfd   plants, leaf discsfrom WT and transgenic lines were illuminated in thepresence of 10  l M methyl viologen (MV), a redox-cyclingherbicide which generates oxidative stress in chloroplasts(Babbs et al. 1989). The degree of cell damage was esti-mated by measuring electrolyte leakage (Fig. 3a), whilechlorophyll contents were determined after alcoholicextraction (Fig. 3b). Surprisingly, expression of thecyanobacterial ferrosulfoprotein did not ameliorate mem-brane deterioration and degradation of photosyntheticpigments caused by the herbicide (Fig. 3a, b). Whenincubated in the absence of MV, electrolyte (Fig. 1a) andchlorophyll (data not shown) losses were less than 10% forall lines in the timeframe of the experiment, indicating thatdisc excision did not impose any significant stress on theleaf tissue.The  F  v  /  F  m  parameter, which is calculated from chloro-phyll fluorescence data, reflects accumulated photooxida-tive damage to PSII (Baker 2008). As expected, exposureof WT leaf discs to chilling temperatures and relativelyhigh light intensities (LT/HL conditions) led to a steadydecline of   F  v  /  F  m  values (Fig. 3c). Once again,  pAnfd   plantsdid not exhibit significant differences in stress tolerancerelative to their WT siblings (Fig. 3c), although theyexpressed a fully functional  Anabaena  Fd.Levels of   Anabaena  Fd protein and transcript declinein transgenic tobacco plants exposed to stressconditionsFailure of   Anabaena  Fd to confer increased toleranceto transgenic tobacco plants contrasts sharply with the Fig. 2  Anabaena  Fd purified from transgenic tobacco leaf extracts isan active holoprotein.  a  The presence of   Anabaena  (An) andendogenous (Nt) Fd in crude ( - ) and purified ( ? ) tobacco leaf extracts was verified by SDS–PAGE and immunodetection assaysusing specific antibodies.  b  Visible absorption spectra of   Anabaena Fd purified from tobacco leaf extracts ( dashed line ), purified  Anabaena  Fd ( solid line ), and purified pea Fd ( dotted line ).  c  Kineticsof cytochrome  c  reductase activity as determined by the increase inabsorbance at 550 nm with 0, 2 and 10  l M of purified  Anabaena  Fd.Other experimental details are given in ‘‘Materials and Methods’’538 Plant Mol Biol (2011) 76:535–544  1 3  wide-range protective effect of Fld, even though the iso-functional flavoprotein accumulated to lower amounts andwas less efficient for electron transfer in vitro (Tognettiet al. 2006). Even though these observations raises ques-tions on the proposal that the protective effect of Fld stemsfrom functional replacement of Fd, other possibilitiesremain open to explain the lack of stress tolerance of   pAnfd  plants. Previous results have indicated that accumulation of plant Fd protein (Tognetti et al. 2006) and transcripts(Zimmermann et al. 2004) was down-regulated undervirtually all forms of abiotic stress. To determine if thelevels of   Anabaena  Fd were also affected by the stresstreatments, we measured Fd contents at various times  after  imposition of the adverse situation. Immunoblot analysesshow that  Anabaena  Fd indeed declined significantly, in atime-dependent manner, when  pAnfd2  plants were exposedto MV (Fig. 4a) or LT/HL conditions (Fig. 4b). Similar results were obtained with  pAnfd4  plants (data not shown).Stress-dependent decrease in the contents of the foreignprotein was even sharper than that of the endogenousorthologs (Fig. 4a), and could be largely accounted for byequivalent declines in the corresponding transcripts, asrevealed by real-time reverse-transcription (RT) PCR(Fig. 4c). As anticipated, transcripts encoding the endog-enous Fd(s) were down-regulated by MV and LT/HLexposure (data not shown), in good agreement with pre-vious observations made on a genome-wide scale (Zim-mermann et al. 2004). Then, and although the possibilitythat Fld has additional effects besides Fd replacement whenexpressed in plants cannot be ruled out by these results andthose of Blanco et al. (2011), the failure of   pAnfd   lines todisplay enhanced stress tolerance can be explained by thestress-dependent decline of the foreign cyanobacterial Fd.Expression of leaf-type Fd in higher plants is regulatedin a complex manner, responding to both developmentaland environmental cues. Among them, light-dependentinduction is the most thoroughly studied.  Cis -acting ele-ments located upstream of the transcription initiation sitedetermine tissue specificity and provide for moderate lightresponsiveness (Gallo-Meagher et al. 1992; Vorst et al.1993). However, replacement of these regions by a con-stitutive promoter does not abolish light induction (Elliottet al. 1989), indicating that Fd expression depends onsequences located in the transcribed portion of the gene andis therefore subject to post-transcriptional regulation.Proper Fd expression and accumulation was shown torequire active photosynthesis (Petracek et al. 1998), andtranslation of the gene (Dickey et al. 1994). Using chimeric Fig. 3  Effect of stress on WT and transgenic tobacco plantsexpressing  Anabaena  Fd.  a  MV-induced membrane damage. Ionleakage was estimated by measuring the increase in conductivity of the medium after MV treatment of leaf discs from 8-week-old WT( closed circles ),  pAnfd2  ( closed triangles ), and  pAnfd4  ( closed squares ) plants. Control leaf discs ( open symbols ) were incubatedin water for the same time.  b  MV-induced degradation of photosyn-thetic pigments. Chlorophyll contents were measured in leaf discsfrom WT and transformed plants after incubation with 10  l M MV for0 ( black bars ), 3 ( grey bars ) and 6 h ( white bars ), at 600  l mol quantam - 2 s - 1 . Values are means  ±  SD of 6 individual plants.  c  Chilling-and light-dependent damage to PSII as estimated by the decrease in F  v  /F  m . Leaf discs from WT ( closed circles ),  pAnfd2  ( closed triangles ), and  pAnfd4  ( closed squares ) plants were irradiated with600  l mol quanta m - 2 s - 1 at 4  C during the indicated times.  Opensymbols  correspond to control leaf discs b Plant Mol Biol (2011) 76:535–544 539  1 3
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