gene expression analysis in the green macroalga acrosiphonia arcta (dillw.) j. ag.: method...

Gene expression analysis in the green macroalgaAcrosiphonia arcta (Dillw.) J. Ag.: Method optimization andinfluence of ultraviolet radiationpre_647 1..10

Stephan Kremb,1,2,* Dieter Ernst,1 Werner Heller1 and Christian Wiencke2

1Helmholtz Zentrum München – German Research Center for Environmental Health, Institute of Biochemical PlantPathology, Neuherberg, and 2Alfred-Wegener-Institute for Polar and Marine Research, Section Seaweed Biology,Bremerhaven, Germany

SUMMARYMarine macroalgae represent an important part ofcoastal ecosystems in temperate and polar areas.Increasing surface ultraviolet radiation (UVR) levels dueto stratospheric ozone thinning may lead to deleteriouseffects on the physiology of marine macroalgae. Onlyfew studies concentrate on the response of macroalgalgene expression towards elevated UVR. Analyzinggene expression in marine algae is challenging regard-ing RNA extraction, enrichment of differentiallyexpressed transcripts and identification of resultingsequences. Thus, we focused on the optimization of theappropriate techniques using the widely distributedgreen marine macroalga Acrosiphonia arcta. We suc-cessfully used a combination of suppression subtractivehybridization with microarray-based screening of size-selected cDNA libraries and were able to substantiallyimprove the outcome compared with standard tech-niques. Analysis of differential gene expressionrevealed a distinct pattern of reactions towards ecologi-cally relevant UVR levels pointing to specific mecha-nisms that include modulation of the photosyntheticapparatus, induction of glutathione metabolism,removal of toxic photoproducts and use of the malatevalve to dissipate excess energy. The results of thisstudy clearly correlate with previous physiological find-ings on photosynthesis and the antioxidative capacity ofthis alga.

Key words: Acrosiphonia arcta, gene expression,microarray, subtractive suppression hybridization,ultraviolet radiation.

INTRODUCTION

High levels of ultraviolet radiation (UVR) reaching thesurface of the earth due to a thinning of the ozone layerare a serious problem for the biosphere. Most notably inpolar regions, surface UVR levels have been found to be

considerably enhanced (Rex et al. 1997; Madronichet al. 1998; Langer 1999; Mackerness & Jordan 1999;McKenzie et al. 2004) with a very recent major ozoneloss in 2011 (Manney et al. 2011).

The reactions of marine macroalgae towards UVRhave been studied in detail with regard to photosynthe-sis, growth, reproduction, formation of UV protectivesubstances, biochemical detoxification of reactiveoxygen intermediates and repair of DNA damage(Roleda et al. 2009, 2010; Karsten et al. 2011).Overall, many macroalgae seem to be well equippedto acclimate to increasing UVR levels (Bischof et al.2002, 2006; Karsten et al. 2011). Regarding theadjustment of photosynthesis to high radiation intensi-ties, the accumulation of UV absorbing compounds andthe conversion of harmful reactive oxygen species havebeen shown to be important mechanisms in stress tol-erance of macroalgae (Perez-Rodriguez et al. 2001,2003; Aguilera et al. 2002; Bischof et al. 2006). Thepotential for biochemical acclimation is strongly depen-dent on the species and the developmental stage andmostly correlates with the natural vertical distributionof the particular algal species (Bischof et al. 1998,2006; Collen & Davison 1999; Aguilera et al. 2002;Wiencke et al. 2006).

The reactions of plants towards increased UVR levelsoften correlate with pronounced changes of geneexpression patterns and are mediated by nonspecific aswell as UV-specific pathways (Agrawal et al. 2009;Jenkins 2009). In higher plants a clear correlation inthe expression status of several key genes of photosyn-thesis and protective mechanisms and the reaction ofthe plant towards elevated UVR levels has been dem-onstrated. For example, the induction of sunscreencompounds in higher plants has shown to be a result of

*To whom correspondence should be addressed.Email: [email protected] editor: U. Karsten.Received 26 September 2011; accepted 28 March 2012.doi: 10.1111/j.1440-1835.2012.00647.x

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the elevated expression of genes of the phenylpro-panoid biosynthetic pathway (Jordan 1996; Macker-ness & Jordan 1999; Agrawal et al. 2009; Takahashi &Ohnishi 2009; Lai et al. 2011; Tohge et al. 2011)

Regarding algae, only few studies deal with UV- andhigh-level radiation induced gene expression and mostof these focus on the unicellular green alga Chlamy-domonas spp. (Schroda et al. 1999; Im & Grossman2002; Liu et al. 2010; Zhang et al. 2011). A compre-hensive study on the expression of plastid and mito-chondrial genomes in C. reinhardtii by microarrayanalysis revealed extensive changes in gene expressionpatterns. Thereby the extent of UV induced changeswas notably higher than changes resulting from otherstresses (Lilly et al. 2002).

To study UV induced effects on gene expression inmarine macroalgae, Acrosiphonia arcta was chosen as amodel organism due to its widespread occurrence oncoastlines of temperate and polar regions of both hemi-spheres and the vertical distribution up to the eulittoralzone with temporarily high intensities of UVR. (Bischoff& Wiencke 1995)

Due to the scarcity of molecular studies on macroal-gae and the special characteristics of these organisms(high content of salt and other interfering compounds,massive cell walls, low numbers of referencesequences), an important part of the present study wasdedicated to the development and optimization of theappropriate techniques. This included the extraction ofsufficient amounts of high quality RNA, generation ofpreferably long cDNA clones, production of microarraysand application of subtraction and normalization tech-niques that allowed for highly efficient screening of theclone libraries.

The aim of this study was to provide a roadmap forsuccessful analysis of gene expression in marine mac-roalgae and to apply these techniques to elucidateeffects that ecological relevant doses of UV radiationmay exert on gene expression in the widespread greenalga A. arcta.

MATERIALS AND METHODS

Algal material

Acrosiphonia arcta (Dillw.) J. Ag. (Chlorophyta, Acrosi-phoniales) was raised from stock cultures from thealgal culture collection of the Alfred-Wegener-Institute(Bremerhaven; culture-no: 1121, isolate from DiskoIsland, Greenland). The algae were cultured in 2 Lbeakers in nutrient enriched seawater (after Provasoli1968) under continuous aeration with filtered air. Algalcultures were kept at 5°C under light-dark cycles of18:6 h and photon fluence rates of 8–15 mmol photonsm-2 s-1 for several weeks until final use. A completewater change was carried out every two weeks.

Radiation treatments

Algal thalli were exposed to artificial radiation producedby three daylight fluorescent tubes (True-Lite, 36/40 W,d-lite Lichttechnik, Berlin, Germany) and three UV fluo-rescent tubes (Q-Panel, UVA-340, Cleveland, OH, USA)in plastic trays at a constant temperature of 5 � 1°C.

The algae were exposed for different time periods(4–48 h) to a photon flux density of 43.8 mmol photonsm-2 s-1 of photosynthetically active radiation (PAR) andUV-irradiances of 8.38 W m-2 and 0.43 W m-2 of UV-Aand UVBR, respectively. This UV exposure is compa-rable to realistic outdoor conditions in the High Arcticof 13,1 W m-2 for UV-A and 0.4 W m-2 UV-B as mea-sured in August in Spitsbergen (Bischof et al. 1999;Hanelt et al. 2001). Radiation measurements werecarried out by a LiCor LI-1000 datalogger withLI-190-SB cosine sensor (LiCor Biosciences, Homburg,Germany) for PAR and a Solar-Light-Radiometer(PMA2100, Solar Light Co., Glenside, USA) forUVA + UVB. To obtain control samples, parts of theexposed plants were covered with a filter foil (Folex,Dreieich, Germany) ensuring a complete exclusion ofUVR.

Directly after the radiation treatment, algal sampleswere briefly rinsed in distilled water, dried by useof tissue paper and frozen in liquid nitrogen. Priorto RNA-extraction, algae were stored at -80°C in adeep-freezer.

RNA extraction and purification

The extraction of RNA from algal tissues rich in cell-wall components, polysaccharides and other interferingcompounds had to be optimized to obtain ampleamounts of high-quality mRNA suitable for cDNA syn-thesis, polymerase chain reaction (PCR) and libraryconstruction. For this purpose a modified protocoldescribed by Chang et al. (1993) was used. Up to 1 mgof total RNA was obtained from 100 mg (fresh weight)of algal tissue. Integrity of the RNA was verified by gelelectrophoresis. Briefly, algal material was ground to afine powder in liquid nitrogen and after addition of1 mL extraction buffer, samples were incubated for10 min on a shaker at 1800 rpm. After addition of0.8 mL of chloroform and manual agitation for 3 minthe aqueous phase was separated by centrifugation andtransferred to a new reaction tube. Selective precipita-tion of RNA was achieved by addition of 1/4 volume ofLiCl (10 M) and incubation at -20°C over night. Fol-lowing a centrifugation step, the supernatant wasremoved and the resulting pellet was dissolved in0.5 mL of RNase-free water. After a second extractionwith chloroform the aqueous phase was spiked with 2.5volumes of ethanol and 0.1 volume of sodium acetate(3 M, pH 4.8) to precipitate the RNA at -80°C for 1 h.


2 S. Kremb et al.

Following a centrifugation and a washing step the RNAwas dissolved in RNase-free water. For quality control,aliquots of 1 mg were separated on 1.5% agarose gels tovisualize ribosomal bands and exclude degradation ofthe RNA.

For further enrichment of mRNA from total RNApreparations, the Oligotex mRNA Mini Kit was usedaccording to the manufacturer’s instructions (Qiagen,Hilden, Germany).

Subtractive suppression hybridization

For the analysis of differential gene expression, thePCR-based subtractive suppression hybridization(SSH) has been shown to be a powerful tool for theenrichment of rare or weakly regulated mRNA speciesand was adjusted to match the special requirementsof macroalgae (Diatchenko et al. 1996; Sävenstrandet al. 2000; Heidenreich et al. 2001; Sahr et al.2005).

Subtractive suppression hybridization was carriedout by use of the PCR-Select cDNA Subtraction Kit(Clontech-Takara Bio, Saint-Germaine-en-Laye, France)following the guidelines of the manufacturer. Thismethod used two restriction enzyme-digested cDNA-populations ligated with different adaptors. One popu-lation contains specific transcripts (tester cDNA),whereas the other population encompasses the refer-ence transcripts (driver cDNA). Following the subtrac-tion of the tester cDNA population from the driver cDNApool, suppression PCR was carried out. This resulted ina selective amplification of transcripts more abundantin the tester cDNA population.

Microarray production and application

Prior to production of microarrays, PCR products werepurified by use of filter plates (Multiscreen PCR96filter plate, Millipore, Schwalbach, Germany). PurifiedcDNAs were spotted onto nylon membranes (Hybond-N+, GE Healthcare, München, Germany) by an auto-mated DNA-spotter (Microgrid II, Biorobotics, DigilabGenomic Solutions, Ann Arbor, MI, USA). After neutral-ization, washing and drying the PCR products werefixed by UV cross linking.

For preparation of probes, 20 mg of total RNA weremixed with 2 mL random primers and denaturized at70°C for 10 min. After cooling on ice, 6 mL of 5¥ firststrand buffer, 1 mL 100 mM dithiothreitol (DDT),1.5 mL dNTPs mixture (dATP, dTTP and dGTP, 20 mMeach) and 10 mL a33-dCTP (GE Healthcare, München,Germany) were added and samples were incubatedfor 10 min at 25°C. For reverse transcription, 1.5 mLSuperscript II reverse transcriptase (Invitrogen,Karlsruhe, Germany) were added and incubated for90 min at 42°C.

Screening of cDNA libraries

In a first approach, 672 clones obtained by a standardSSH-procedure with forward and reverse subtractionwere analyzed by microarray analysis. With every clonerepresented twice on the membranes and three repeatsof every hybridization, every single clone was ana-lyzed six times. Clones that showed differences insignal intensities (forward-subtracted versus reverse-subtracted) with a factor of >2 or <0.5 were consideredas positive.

Production of size-selected cDNA-libraries

To avoid the problem of short sequences resulting inpoor annotation success, cDNA-libraries enriched forlonger transcripts were established. To generate suit-able and sufficient starting material for the construc-tion of a cDNA-library, polyA+-RNA extracted fromUV-exposed (12 h) thalli of A. arcta was subjected toSMART-amplification of cDNA. The procedure wascarried out according to the manufacturer’s instructions(Clontech-Takara Bio). PCR products were separated on1% agarose gels, bands larger than 500 bp wereexcised and eluted from the agarose gel using theQiagen Gel Extraction Kit (Qiagen). The cDNA frag-ments were inserted into pGEM-T vectors (Promega,Mannheim, Germany) by TA-directed cloning. Bacterialclones were stored at -80°C as glycerol stocks for PCRamplification of the cDNA inserts. For microarray pro-duction, cDNA inserts of 1440 bacterial clones werePCR amplified from 1 mL of overnight culture usingM13 primers at standard PCR conditions. PCR prod-ucts were visualized on 1.4% agarose gels and weresubsequently spotted onto microarray membranes.

Screening of size-selected cDNA-libraries

The microarrays carrying the size-selected cDNA-libraries were hybridized to radiolabeled probes gener-ated by either SSH or un-subtracted cDNA originatingfrom total RNA extracted directly from UV-exposedalgal material. The approach using SSH-derivedprobes was used to screen the size-selected (butun-subtracted) cDNA-library with subtracted (andtherefore enriched for rare and weakly regulated tran-scripts) probe material. The other approach to screenthe size-selected library for differentially expressedgenes used cDNA derived from total RNA extractedfrom algae exposed to UVR or control plants treatedonly with PAR for different time periods (4, 12 and24 h). This approach was used to identify more abun-dant and strongly regulated genes by use of non-subtracted probes generated directly from RNA ofUV-treated algae. Furthermore this approach can be


3Gene expression analysis in Acrosiphonia arcta

used to reveal time-dependent expression of UV-responsive genes in a short-term experiment.

Sequencing and bioinformatic analyses

The cDNA inserts of selected bacterial clones wereamplified by standard PCR using M13 primers, purifiedvia filter plates (Multiscreen PCR96 filter plate, Milli-pore). Subsequently, 1 mL was added to 3 mL of asequencing mix (Big Dye Terminator Sequencing Kit,Applied Biosystems, Cleveland, OH, USA) containingT7 primer for initiation of the sequencing reaction.Following the sequencing reaction, products were pre-cipitated by addition of ethanol and dissolved in 25 mLof high-purity water. Sequencing was carried out in anABI 3100 sequencer (Applied Biosystems). The analy-sis of the obtained sequences was carried out by useof the blastx search tool provided by National Centerfor Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/). The identified genes weregrouped into different classes according to theirfunction, including energy metabolism, transport &homeostasis, transcription & translation, protein-related processes (folding, modification and degrada-tion), defence & stress and cellular metabolism.

Semiquantitative real-time-PCR

To confirm the reliability of the results of the microar-ray analysis, a set of selected genes was tested fortheir expression status using semiquantitative realtime reverse transcription (RT)-PCR. For this purpose,gene specific primers were designed using PrimerExpress 2.0 software (Applied Biosystems). As a tem-plate for real-time RT-PCR, total RNA extracted fromUV- and control-exposed algae was used. PCR reac-tions were carried out in a 7500 Real-Time-PCRsystem (Applied Biosystems) using SYBR green chem-istry (Absolute QPCR SYBR Green Low ROX Mix,ABgene, Hamburg, Germany). Briefly, 12.5 mL ofready-to-use SYBR green mixture were combined with1 mL of forward and reverse gene specific primers and10.5 mL of diluted cDNA. The temperature profileused for real time-PCR was applied according to themanufacturer’s guidelines for the SYBR Green LowROX Mix and included a combined annealing/extension step for 1 min at 60°C. Every combinationof cDNA preparation and gene specific primer pair wastested in triplicate and a no template control (NTC)was implemented in every run. As an internal controlthe housekeeping gene 18S rRNA was used and testedfor every single cDNA. Ct-values were automaticallycalculated and relative quantification was expressedaccording to a mathematical model established byPfaffl et al. 2002.

RESULTS AND DISCUSSION

Method optimization

In this study, molecular techniques for the analysis ofdifferential gene expression were adapted to the spe-cific requirements of marine macroalgae and success-fully used to identify UV-regulated genes in the marinegreen alga A. arcta.

To analyze differential gene expression we used acombination of PCR-based SSH and microarray analy-sis. Subtractive hybridization was shown to be anappropriate tool for the enrichment of differentiallyexpressed transcripts using normalization and selec-tive amplification of rare transcripts (Diatchenko et al.1996; Sahr et al. 2005; Park et al. 2007). Rare andweakly expressed transcripts turned out to be underes-timated in standard non-subtracted cDNA libraries(Heidenreich et al. 2001; Nguyen et al. 2005; Sahret al. 2005). However, standard SSH suffers fromtwo limitations: first, the enrichment of rarely expressedtranscripts shifts the natural expression pattern makinginterpretation of the results more complicated. Second,SSH generates short cDNA fragments (80–300 bp) forsequencing. This impedes sequence identification byannotation, particularly if species with a low sequencedata background are under investigation (Levesqueet al. 2003). In fact, standard SSH in combination withmicroarray analysis revealed only few differentiallyexpressed genes in our hands. After sequencing of 104clones, only nine of the corresponding cDNAs could beidentified by blastx analysis. The majority of thesesequences turned out to be short (most sequencesranging between 80–200 bp) as a result of restric-tion digestion of cDNAs by RsaI in the standardSSH-procedure.

We addressed these problems by creating anadvanced protocol that combines the advantage of longsequences with the benefits of subtractive hybridiza-tion. This protocol included microarrays produced fromlarge-fragment enriched cDNA and screening by a two-pronged approach using both, un-subtracted (andtherefore unbiased) cDNA as well as SSH-derived (nor-malized and enriched) hybridization probes.

The screening of the size-selected cDNA-librarieswith forward- and reverse-subtracted cDNAs from SSHresulted in 230 clones, whereas the application ofun-subtracted probes yielded 202 clones. In sum-mary, this approach resulted in 432 clones with 114(26%) identified sequences representing a significantimprovement compared to the standard SSH protocolwhere only 9% of sequences could be identified. More-over, a substantial number of transcripts (particularlywith regard to genes involved in regulatory processesas well as transport and homeostasis) could only beidentified by using SSH-derived hybridization probes


4 S. Kremb et al.

indicating the advantages of normalization and subtrac-tion. A similar approach was successfully used for theidentification of differentially expressed transcripts inbreast cancer cells (Yang et al. 1999).

Functional relation of transcripts toelevated UV radiation

The UV-responsive genes identified by the differentapproaches were grouped into different classes accord-ing to their function, including energy metabolism,transport & homeostasis, transcription & translation,protein-related processes (folding, modification anddegradation), defence & stress and cellular metabo-lism. Table 1 summarizes blastx search results (e-valueand best match), expression status in the analyzed algalmaterial, the approach used to identify the particulartranscript and the number of clones corresponding tothe same gene.

Several genes encoding components of the photo-systems (cab = chlorophyll a/b binding proteins andproteins constituting the light harvesting complexes)turned out to be regulated by UVR. Generally thesegenes seem to be repressed shortly after onset of UVexposure and show a strong induction in the later phaseof the experiment. This may indicate an adjustment ofthe photosystems to the altered light conditions anddisplacement of potentially damaged components. Inaddition, a protective role of cabs has been speculated(Lai et al. 2011). In contrast, CP26, a photosyntheticantenna protein and member of the cab family appearsto be weakly regulated as was also demonstrated forChlamydomonas reinhardtii (Minagawa et al. 2001).The elevated expression of glyceralehyde-3-phosphatedehydrogenase points to an induction of the Calvincycle to abolish excess energy generated by UVR in thephotosystems. A similar response was observed in Ara-bidopsis thaliana exposed to oxidative stress (Sweetloveet al. 2002).

Genes involved in transport processes and homeosta-sis show a different expression pattern. Nearly all ofthese transcripts were exclusively identified by SSH-derived hybridization probes indicating a tight regulationof these genes. The induction of a high affinity nitratetransporter points to an increased nitrogen demand(Miller et al. 2007) whereas the increased expression ofa chloroplast w6-desaturase in A. arcta might contributeto the adaptation of membrane systems and the dis-placement of membrane lipids damaged by lipid peroxi-dation, an important mechanism in stress tolerance(Kirsch et al. 1997; Khodakovskaya et al. 2006; Zhanget al. 2011).

Regarding the group of genes involved in transcrip-tion and translation, differential expression of allmembers of this group could only be identified by useof SSH derived probes. This argues in favor for a weak

expression of the identified transcription factor andseveral translation initiation factors. A DEAD-box RNAhelicase might also participate in translation regulationand plays a crucial role in development and stressreactions as has been demonstrated in A. thaliana(Gong et al. 2005). The pronounced induction of thisgene indicates a role in stress tolerance and is inagreement with the light-dependent induction of thisgene in Synechocystis (Kujat & Owttrim 2000).

In the group of genes related to communication andsignaling, two of the identified transcripts, encoding aserine/threonine protein kinase and another one withhomology to G protein b subunit belong to signalingpathways, whereas Gbp1p binds to the G-strand oftelomeres and interacts specifically with RNA. Thestrong induction could be explained as protection oftelomeres being sensitive to oxidative stress (Johnstonet al. 1999; von Zglinicki et al. 2000). However, Gbp1was also found to be involved in transcriptional regula-tion in Chlamydomonas (Seitz et al. 2010).

Several genes with relation to protein folding, modi-fication and degradation were identified, pointing at theimportant role of degrading damaged proteins in stressresponse. In addition to heat shock proteins and cyclo-philin that are acting as molecular chaperones, thetypically stress-regulated ATP-dependent Clp protease(Porankiewicz et al. 1998) and several members of theubiquitin-proteasome pathway were found to be regu-lated by UV treatment. The ATP-dependent Clp pro-tease plays a crucial role in plastid homeostasis andmaintenance of a functional thylakoid electron trans-port chain (Olinares et al. 2011). The ubiquitin-proteasome system is involved in the degradation ofdamaged proteins (Kurepa et al. 2009) and is poten-tially used by A. arcta as a first-line strategy directlyafter onset of the radiation treatment.

Moreover, a number of genes with a role in defence,stress response and detoxification were foundto be differentially regulated. The induction ofS-adenosylmethionine-dependent methyltransferasemight be related to detoxification and protectionagainst herbivores by formation of organic thiolates andthiocyanates (Attieh et al. 2002). In addition to thetypically stress-related glutathione S-transferase (Hayes& Strange 1995; Huang et al. 2002; Foyer & Noctor2011) and glutaredoxin (Dietz 2011), a potential regu-lator of apoptosis (Kawai-Yamada et al. 2001), baxinhibitor-1 like protein, was found to be differentiallyregulated. Anticipation of UV-induced apoptosis mightplay an important role in the reaction towards UV radia-tion in A. arcta. The induction of a putative flavonol3-O-gucosyltransferase is potentially related to stressresponse in that this enzyme is involved in flavonoidbiosynthesis and therefore in the formation of UV pro-tective substances. However, no evidence for the occur-rence of flavonoid-like compounds could be found by



Table 1. Concise list of all identified genes found to be regulated in response to an increased UV exposure. The table includes National

Center for Biotechnology Information (NCBI) accession numbers for the best matches at blastx search with related e-values, the expression

status at a certain point of time within the UV treatment (+ = induced; - = repressed; 0 = no regulation), the approach by which the

corresponding gene was identified (RNA, RNA-derived hybridization probes in combination with a size-selected cDNA library; SSH,

standard SSH procedure; SSH-p, use of SSH-derived hybridization probes in combination with a size-selected cDNA library) and the

number of clones corresponding to the certain gene

Gene Best match blastx E value Expression status Approach #

Energy metabolism 4 h 12 h 24 hChlorophyll a/b binding protein AAB70556 4e-87 - 0 +++ RNA 6Chlorophyll a/b binding protein AAC79711 1e-90 - 0 +++ RNA 5Chlorophyll a/b binding protein CP26 BAB20613 9e-10 - SSH 3Chloroplast light harvesting complex I protein ABA01130 1e-29 - 0 +++ RNA 1Major light-harvesting complex II protein m1 AAM18057 7e-57 - 0 +++ RNA 1Photosystem I subunit XI AAO85557 2e-07 0 0 + RNA 1Glyceralehyde-3-phosphate dehydrogenase gi1181548 3e-17 0 + + RNA 1Putative NADH-ubiquinone oxidoreductase 30.4 kDa subunit AAW44102 2e-28 - SSH-p 1

Transport and homeostasisHigh affinity nitrate transporter AAU87579 8e-13

0+ 0 RNA 1

Amino acid transport protein (putative) AAM65327 4e-21 + SSH-p 1Oxoglutarate/malate translocator-like protein AAM63113 2e-32 - SSH-p 1Vacuolar membrane ATPase subunit G AAF24609 1e-12 + SSH-p 1Chloroplast w6 desaturase BAA23881 5e-18 +++ SSH-p 1

Transcription and translationTranscription factor homolog BTF3-like protein CAE45592 2e-26 - SSH-p 1DEAD-box RNA helicase AAN74636 1e-35 +++ SSH-p 1Eukaryotic initiation factor 5A XP_750156 2e-35 0 0 - SSH-p 1Eukaryotic initiation factor 5A (2) CAA45104 8e-44 - SSH-p 1Eukaryotic initiation factor 5A (3) AAM64601 9e-46 - SSH-p 1Eukaryotic translation init. factor 2g subunit ABA99260 7e-23 + SSH 1

Communication and signallingSer/Thr protein kinase isolog BAD94000 7e-68 0 0 - RNA 2Homology to G protein b subunit (putative) CAA37638 2e-78 ++ SSH-p 1G-strand binding protein Gbp1p gi1076208 2e-37 +++ SSH-p 2

Protein folding, modification and degradationHSP17.7-a protein CAB93512 1e-12 + - n.d. RNA 1Small heat shock protein AAR99375 2e-04 - - - RNA 1Cyclophilin AAK21908 3e-29 - SSH-p 1ATP-dependent Clp protease proteolytic su. 3 AAP55198 1e-04 +++ SSH-p 1OTU-like cysteine protease-like BAD45450 5e-18 0 0 - RNA 1UPL6 ubiquitin-protein ligase NP_188346 4e-09 ++ SSH 120S proteasome subunit C8 AAM66932 7e-56 0 0 - RNA 120S proteasome subunit a3 AAK53380 4e-52 + 0 - RNA 126S proteasome regulatory subunit AAM13367 1e-34 0 0 - RNA 1

Defence, stress response and detoxificationGlutathione S-transferase ZP_00691142 4e-15 0 - n.d. RNA 1Glutaredoxin AAL04507 2e-19 0 0 - RNA 1Bax inhibitor-1 like protein AAM65074 4e-24 ++ SSH-p 1Early light inducible protein ELIP AAK52823 0,1 - - n.d. RNA 1Flavonol 3-O-glucosyltransferase (putative) AAO63909 8e-32 0 0 ++ RNA 1

Cellular metabolismS-adenosylmethionine-dep. methyltransferase NP_181920 1e-55 - 0 + RNA 1Methyltransferase (putative) gi6491814 2e-24 0 + 0 RNA 1ASN1 NP_190318 7e-52 - SSH-p 2DIN2 NP_191573 2e-26 0 + 0 RNA 1Phosphoglycerate dehydrogenase (putative) AAG51802 3e-26 0 0 - RNA 1N-acetyl-g-glutamyl-phosphate reductase NP_849993 6e-69 - SSH-p 1g-glutamylcysteine synthetase gi50936911 3e-25 +++ SSH-p 1Stearoyl-acyl carrier protein desaturase BAA07681 2e-44 0 + 0 RNA 1Malate dehydrogenase, glyoxysomal precursor ABA99938 8e-52 + SSH 1Transketolase 1 (putative) XP_550612 6e-04 - SSH 1


6 S. Kremb et al.

high performance liquid chromatography (HPLC) andUV spectroscopy in UV treated A. arcta (data notshown). Flavonoids are products of phenolic metabo-lism and are well reported to act as UV filter aswell as antioxidants in terrestrial plants (Jaakola &Hohtola 2010; Fini et al. 2011). In algae, flavonoidswere not detected to date. According to our results,Roleda et al. (2010) did also not succeed in detectingflavonoid-like substances in the green macroalgaUrospora penicilliformis. It is likely that the completemetabolic pathway leading to the formation offlavonoids has not completely evolved in these organ-isms. However, the UV-induction of a putative flavonol3-O-gucosyltransferase could indicate that members ofthe flavonoid metabolic pathway are already present ingreen algae but do not lead to the formation of com-plete flavonoid structures. At an evolutionary point ofview, green algae have not completed the pathwayleading to the production of these highly effective UVscreens, most probably due to the comparably lower UVintensities in the aquatic environment.

The last functional group encompasses genesinvolved in a variety of cellular metabolic pathways. Inaddition to a methyltransferase, transketolase und aphosphoglycerate dehydrogenase, a malate dehydroge-nase was found to be induced. It serves as an importanteffector in the malate valve, catalyzing the antiport ofmalate/oxalacetate over the chloroplast membrane. Inthis process, excess reduction equivalents originatingfrom a partial decoupling of the thylakoid membrane inthe light are dissipated (Wilhelm & Selmar 2011). Thismechanism plays an important role in light stress con-ditions to dissipate excess energy and could also beused by UVR exposed A. arcta. Furthermore, malatemetabolism in general is considered to be important inUV stress and wounding reactions in plants (Casatiet al. 1999). In addition, several genes involved inglutathione and amino acid metabolism have beenfound to be regulated by UV treatment in A. arcta. Thetripeptide glutathione is an important reductive agentfor a variety of detoxifying and cell protecting processeswith particular regard to oxidative stress (Foyer &Noctor 2011). The g-glutamylcysteine synthetase par-ticipates in the biosynthesis of glutathione (Foyer &Noctor 2011) and was found to be induced in A. arcta.The repression of a N-acetyl-g-glutamyl-phosphatereductase can be seen in the same context as thisenzyme plays a role in the synthesis of ornithinefrom glutamate (Miura-Ohnuma et al. 2005), whereasglutamate is also needed for the synthesis of glu-tathione. Thus, the inhibition of ornithine biosynthesiscould favor the formation of glutathione. The slightrepression of an asparagine synthetase together withthe induction of an amino acid transport protein anda nitrate transporter point to an increased amino acidmetabolism. A light-dependent repression of aspar-

agine synthetase genes could also be observed in otherplants (Herrera-Rodriguez et al. 2004).

Verification of the microarray (hybridization)-basedresults by real-time RT-PCR with a set of four genesrevealed accordance of gene expression data (Fig. 1).In either case the tendency of regulation (up or down)could be verified by real-time RT-PCR but the extent ofup- or downregulation differs between the two methodsand was always lower as measured by SSH.

In summary, the results of this work show a distinctgene expression pattern in UV treated A. arcta andprovide evidence for specific responses to cope withthis high-energy radiation. This includes modulation ofthe photosynthetic apparatus, induction of glutathioneand amino acid metabolism, detoxification of photo-products and use of the malate valve to dissipate excessenergy from the chloroplast. Some more specific reac-tions could include the inhibition of UV-induced apop-tosis, induction of protective mechanisms againstherbivores and protection of telomeres against oxidativestress. These reactions on the molecular level might beconsidered as acclimation of the alga towards UVR andare in good concordance with previous findings onthe physiological level. Due to the vertical position ofA. arcta in the intertidal and upper sublittoral it peri-odically gets exposed to high levels of UVR. However,this alga shows a relatively high photosynthetic sensi-tivity towards elevated UV levels, which is related to itsmorphological characteristic (Aguilera et al. 1999).A. arcta grows in tufts where the inner parts of the algaare shaded by the outer branches protecting them fromdesiccation and excess sunlight at low tide. Due toself-shading, the inner parts of the alga are adapted tolow light conditions. Often, the outer parts appear

Fig. 1. Absolute gene regulation analyzed by real-time reverse

transcription-polymerase chain reaction (RT-PCR) of four selected

genes of Acrosiphonia arcta at three different points of time

during the UV treatment (4, 12 and 24 h). Expression data cal-

culated from ct-values according to the mathematical model

described by Pfaffl et al. 2002. The value 1 on the x axis (broken

line) means no gene regulation, whereas values lower and higher

than 1 indicate downregulation or upregulation, respectively.



brown or white, whereas the inner parts mostly show adark-green color (Aguilera et al. 1999). The fast inhi-bition and subsequent induction of most of the photo-synthetic genes is related to these physiologicalcharacteristics of A. arcta at the molecular level.

The vertical distribution of A. arcta also correlateswith its high antioxidative capacity indicated by highactivities of superoxide dismutase, ascorbate peroxi-dase and glutathione reductase (Aguilera et al. 2002).On the transcript level these biochemical data correlatewith the induction of several genes presumably involvedin glutathione synthesis pointing to an induction of theantioxidative system by UVR.

The use of cultured algal material instead of fieldmaterial is artificial and surely affects the ecologicalsignificance of the presented data. However, the use ofcultured material offered considerable advantages inthis case. Field material of A. arcta generally appearsto be heavily interspersed with epiphytic organisms,which are difficult to remove due to its strong ramifi-cation. This represents a source of contamination withundesirable genetic material interfering with cDNAlibrary construction and probe generation. Moreover,cultured material is readily available, easy to use in UVexposure experiments and generally ideally suited formethod optimization. The use of cultured materialwas intended to construct contamination-free cDNAlibraries as a basis for future experiments with fieldmaterial.

The radiation conditions used in this study wereselected to be consistent with previous studies onA. arcta (Aguilera et al. 1999) and are comparable torealistic outdoor conditions in the high Arctic as mea-sured in August in Spitsbergen (Bischof et al. 1999;Hanelt et al. 2001). However, the PAR : UV ratio isartificial and is likely to superimpose UV effects com-pared to PAR effects. Moreover, we did not discriminatebetween UV-A and UV-B-related effects on gene expres-sion. These factors clearly constrain the significance ofthe data with regard to the actual in situ conditions.However, as stated above, our intention was to optimizegene expression analysis for macroalgal species and toform a strong foundation for subsequent studies ratherthan to conduct a detailed mulitfactorial analysis ofUV-related gene expression in A. arcta.

In conclusion the presented data correlate withrecent findings on UV-related physiological effects onphotosynthesis and antioxidative strategies in A. arcta.Moreover, unexplored strategies of green macroalgaetowards UVR can potentially be assumed from the pre-sented data. However, in consideration of the artificialPAR : UV ratio and the use of cultured material in thisstudy, further studies are necessary for confirmationand more detailed analysis of the proposed mecha-nisms. The combination of the SSH-driven enrichmentof rare transcripts with size-selected cDNA libraries

prepared from cellular RNA used for the production ofmicroarrays proved to be successful in the identificationof differentially expressed genes and significantlyincreased the number of hits compared to the standardSSH procedure. We conclude that this method isgenerally applicable to improve the identification ofdifferentially expressed transcripts in genetically lesscharacterized organisms.

ACKNOWLEDGMENTS

We thank Evelyn Bieber, Susanne Stich, Claudia Danieland Andreas Wagner for expert technical assistance,Tobias Sahr, Inka Bartsch and Florian Battke for valu-able advices and help.

REFERENCES

Agrawal, S. B., Singh, S. and Agrawal, M. 2009. Ultraviolet-Binduced changes in gene expression and antioxidants inplants. Adv. Bot. Res. 52: 47–86.

Aguilera, C., Dummermuth, A., Karsten, U., Schriek, R. andWiencke, C. 2002. Enzymatic defences against photooxi-dative stress induced by ultraviolet radiation in Arcticmarine macroalgae. Polar Biol. 25: 432–41.

Aguilera, J., Karsten, U., Lippert, H. et al. 1999. Effects ofsolar radiation on growth, photosynthesis and respiration ofmarine macroalgae from the Arctic. Mar. Ecol. Prog. Ser.191: 109–19.

Attieh, J., Djiana, R., Koonjul, P., Etienne, C., Sparace, S. A.and Saini, H. S. 2002. Cloning and functional expressionof two plant thiol methyltransferases: a new class ofenzymes involved in the biosynthesis of sulfur volatiles.Plant Mol. Biol. 50: 511–21.

Bischof, K., Hanelt, D. and Wiencke, C. 1998. UV-radiationcan affect depth-zonation of Antarctic macroalgae. Mar.Biol. 131: 597–605.

Bischof, K., Hanelt, D. and Wiencke, C. 1999. Acclimation ofmaximal quantum yield of photosynthesis in the brownalga Alaria esculenta under high light and UV radiation.Plant Biol. (Stuttg) 1: 435–44.

Bischof, K., Krabs, G., Wiencke, C. and Hanelt, D. 2002.Solar ultraviolet radiation affects the activity of ribulose-1,5-bisphosphate carboxylase-oxygenase and the compo-sition of photosynthetic and xanthophyll cycle pigments inthe intertidal green alga Ulva lactuca L. Planta 215:502–9.

Bischof, K., Gomez, I., Molis, M. et al. 2006. Ultravioletradiation shapes seaweed communities. Rev. Environ. Sci.Biotechnol. 5: 141–66.

Bischoff, B. and Wiencke, C. 1995. Temperature ecotypesand biogeography of Acrosiphoniales (Chlorophyta) withArctic-Antarctic disjunct and Arctic/cold-temperate distri-butions. Eur. J. Phycol. 30: 19–27.

Casati, P., Drincovich, M. F., Edwards, G. E. and Andreo, C. S.1999. Regulation of the expression of NADP-malic enzyme


8 S. Kremb et al.

by UV-B, red and far-red light in maize seedlings. Braz. J.Med. Biol. Res. 32: 1187–93.

Chang, S., Puryear, J. and Cairney, J. 1993. A simple andefficient method for isolating RNA from pine trees. PlantMol. Biol. Rep. 11: 113–16.

Collen, J. and Davison, I. R. 1999. Reactive oxygen produc-tion and damage in intertidal Fucus spp. (Phaeophyceae).J. Phycol. 35: 54–61.

Diatchenko, L., Lau, Y. F., Campbell, A. P. et al. 1996. Sup-pression subtractive hybridization: a method for generatingdifferentially regulated or tissue-specific cDNA probes andlibraries. Proc. Natl Acad. Sci. U.S.A. 93: 6025–30.

Dietz, K. J. 2011. Peroxiredoxins in plants and cyanobacteria.Antioxid. Redox Signal. 15: 1129–59.

Fini, A., Brunetti, C., Di Ferdinando, M., Ferrini, F., Tattini, M.2011. Stress-induced flavonoid biosynthesis and the anti-oxidant machinery of plants. Plant Signal Behav. 6: 709–11.

Foyer, C. H. and Noctor, G. 2011. Ascorbate and glutathione:the heart of the redox hub. Plant Physiol. 155: 2–18.

Gong, Z., Dong, C. H., Lee, H. et al. 2005. A DEAD box RNAhelicase is essential for mRNA export and important fordevelopment and stress responses in Arabidopsis. PlantCell 17: 256–67.

Hanelt, D., Tüg, H., Bischof, K. et al. 2001. Light regime inan Arctic fjord: a study related to stratospheric ozonedepletion as a basis for determination of UV effects onalgal growth. Mar. Biol. 138: 649–58.

Hayes, J. D. and Strange, R. C. 1995. Potential contributionof the glutathione S-transferase supergene family to resis-tance to oxidative stress. Free Radic. Res. 22: 193–207.

Heidenreich, B., Mayer, K., Sandermann, J. R. H. and Ernst,D. 2001. Mercury-induced genes in Arabidopsis thaliana:identification of induced genes upon long-term mercuricion exposure. Plant Cell Environ. 24: 1227–34.

Herrera-Rodriguez, M. B., Maldonado, J. M. and Perez-Vicente, R. 2004. Light and metabolic regulation of HAS1,HAS1.1 and HAS2, three asparagine synthetase genes inHelianthus annuus. Plant Physiol. Biochem. 42: 511–8.

Huang, L., Mccluskey, M. P., Ni, H. and Larossa, R. A. 2002.Global gene expression profiles of the cyanobacterium Syn-echocystis sp. strain PCC 6803 in response to irradiationwith UV-B and white light. J. Bacteriol. 184: 6845–58.

Im, C. S. and Grossman, A. R. 2002. Identification andregulation of high light-induced genes in Chlamydomonasreinhardtii. Plant J. 30: 301–13.

Jaakola, L. and Hohtola, A. 2010. Effect of latitude on fla-vonoid biosynthesis in plants. Plant Cell Environ. 33:1239–47.

Jenkins, G. I. 2009. Signal transduction in responses to UV-Bradiation. Annu. Rev. Plant Biol. 60: 407–31.

Johnston, S. D., Lew, J. E. and Berman, J. 1999. Gbp1p, aprotein with RNA recognition motifs, binds single-strandedtelomeric DNA and changes its binding specificity upondimerization. Mol. Cell. Biol. 19: 923–33.

Jordan, B. R. 1996. The effects of Ultraviolet-B radiationon plants: a molecular perspective. Adv. Bot. Res. 22:97–162.

Karsten, U., Wulff, A., Roleda, M. Y. et al. 2011. Physiologi-cal responses of polar benthic algae to ultraviolet radiation.

Kawai-Yamada, M., Jin, L., Yoshinaga, K., Hirata, A. andUchimiya, H. 2001. Mammalian Bax-induced plant celldeath can be down-regulated by overexpression of Arabi-dopsis Bax Inhibitor-1 (AtBI-1). Proc. Natl Acad. Sci.U.S.A. 98: 12295–300.

Khodakovskaya, M., Mcavoy, R., Peters, J., Wu, H. and Li, Y.2006. Enhanced cold tolerance in transgenic tobaccoexpressing a chloroplast omega-3 fatty acid desaturasegene under the control of a cold-inducible promoter.Planta 223: 1090–100.

Kirsch, C., Takamiya-Wik, M., Reinold, S., Hahlbrock, K. andSomssich, I. E. 1997. Rapid, transient, and highly local-ized induction of plastidial omega-3 fatty acid desaturasemRNA at fungal infection sites in Petroselinum crispum.Proc. Natl Acad. Sci. U.S.A. 94: 2079–84.

Kujat, S. L. and Owttrim, G. W. 2000. Redox-regulated RNAhelicase expression. Plant Physiol. 124: 703–14.

Kurepa, J., Wang, S., Li, Y. and Smalle, J. 2009. Proteasomeregulation, plant growth and stress tolerance. Plant Signal.Behav. 4: 924–7.

Lai, Y., Xu, B., He, L. et al. 2011. Differential gene expressionin pepper (Capsicum annuum) exposed to UV-B. Indian J.Exp. Biol. 49: 429–37.

Langer, J. 1999. Messungen des arktischen strato-sphaerischen Ozons: Vergleich der Ozonmessungen in NyAlesund, Spitzbergen, 1997 und 1998. Ber. Polarforsch.322: 1–222.

Levesque, V., Fayad, T., Ndiaye, K., Nahe Diouf, M. andLussier, J. G. 2003. Size-selection of cDNA libraries forthe cloning of cDNAs after suppression subtractive hybrid-ization. Biotechniques 35: 72–8.

Lilly, J. W., Maul, J. E. and Stern, D. B. 2002. The Chlamy-domonas reinhardtii organellar genomes respond transcrip-tionally and post-transcriptionally to abiotic stimuli. PlantCell 14: 2681–706.

Liu, S., Zhang, P., Cong, B. et al. 2010. Molecular cloningand expression analysis of a cytosolic Hsp70 gene fromAntarctic ice algae Chlamydomonas sp. ICE-L. Extremo-philes 14: 329–37.

McKenzie, R., Smale, D. and Kotkamp, M. 2004. Relation-ship between UVB and erythemally weighted radiation.Photochem. Photobiol. Sci. 3: 252–6.

Mackerness, A.-H. and Jordan, B. R. 1999. Changes ingene expression in response to ultraviolet B-inducedstress. In Pessarakli, M. (Ed.) Handbook of Plant andCrop Stress. Marcel Dekker Inc, New York, Basel, pp. 749–68.

Madronich, S., McKenzie, R. L., Bjorn, L. O. and Caldwell,M. M. 1998. Changes in biologically active ultravioletradiation reaching the Earth’s surface. J. Photochem. Pho-tobiol. B 46: 5–19.



Manney, G. L., Santee, M., Rex, M. et al. 2011. Unprec-edented Arctic ozone loss in 2011. Nature 478: 469–75.

Miller, A. J., Fan, X., Orsel, M., Smith, S. J. and Wells, D. M.2007. Nitrate transport and signalling. J. Exp. Bot. 58:2297–306.

Minagawa, J., Han, K. C., Dohmae, N., Takio, K. and Inoue, Y.2001. Molecular characterization and gene expression oflhcb5 gene encoding CP26 in the light-harvesting complex IIof Chlamydomonas reinhardtii. Plant Mol. Biol. 46: 277–87.

Miura-Ohnuma, J., Nonaka, T., Katoh, S. et al. 2005.Improved expression, purification and crystallization of aputative N-acetyl-gamma-glutamyl-phosphate reductasefrom rice (Oryza sativa). Acta Crystallograph. Sect. FStruct. Biol. Cryst. Commun. 61: 1058–61.

Nguyen, B., Bowers, R. M., Wahlund, T. M. and Read, B. A.2005. Suppressive subtractive hybridization of and differ-ences in gene expression content of calcifying and noncal-cifying cultures of Emiliania huxleyi strain 1516. Appl.Environ. Microbiol. 71: 2564–75.

Olinares, P. D., Kim, J. and Van Wijk, K. J. 2011. The Clp pro-tease system; a central component of the chloroplast pro-tease network. Biochim. Biophys. Acta 1807: 999–1011.

Park, J. S., Choung, M. G., Kim, J. B. et al. 2007. Genesup-regulated during red coloration in UV-B irradiatedlettuce leaves. Plant Cell Rep. 26: 507–16.

Perez-Rodriguez, E., Aguilera, C., Gomez, I. and Figueroa, F.L. 2001. Excretion of coumarins by the Mediterraneangreen alga Dasycladus vermicularis in response to environ-mental stress. Mar. Biol. 139: 633–9.

Perez-Rodriguez, E., Aguilera, J. and Figueroa, F. L. 2003.Tissular localization of coumarins in the green alga Dasy-cladus vermicularis (Scopoli) Krasser: a photoprotectiverole? J. Exp. Bot. 54: 1093–100.

Pfaffl, M. W., Horgan, G. W. and Dempfle, L. 2002. Relativeexpression software tool (REST) for group-wise comparisonand statistical analysis of relative expression results inreal-time PCR. Nucleic Acids Res. 30: e36.

Porankiewicz, J., Schelin, J. and Clarke, A. K. 1998. TheATP-dependent Clp protease is essential for acclimation toUV-B and low temperature in the cyanobacterium Synecho-coccus. Mol. Microbiol. 29: 275–83.

Provasoli, L. 1968. Media and prospects for the cultivation ofmarine algae. In Watanabe, A. and Hattori, A. (Eds) Cul-tures and Collections of Algae. Proceedings of the Japa-nese Conference Hakone, Japanese Society of PlantPhysiology, Tokyo, pp. 63–75.

Rex, M., Harris, R. P., Von Der Gathen, P. et al. 1997. Pro-longed stratospheric ozone loss in the 1995–96 Arcticwinter. Nature 389: 835–38.

Roleda, M. Y., Campana, G., Wiencke, C., Hanelt, D., Quartino,M. and Wulff, A. 2009. Sensitivity of Antarctic Urosporapenicilliformis (Ulotrichales, Chlorophyta) to ultravioletradiation is life stage dependent. J. Phycol. 45: 600–9.

Roleda, M. Y., Luetz-Meindl, U., Wiencke, C. and Luetz, C.2010. Physiological, biochemical and ultrastructuralresponses of the green macroalga Urospora penicilliformisfrom Arctic Spitsbergen to UV radiation. Protoplasma 243:105–16.

Sahr, T., Voigt, G., Paretzke, H. G., Schramel, P. and Ernst, D.2005. Caesium-affected gene expression in Arabidopsisthaliana. New Phytol. 165: 747–54.

Sävenstrand, H., Brosche, M., Angenhagen, M. and Strid, A.2000. Molecular markers for ozone stress isolated by Sup-pression Subtractive Hybridization: specificity of geneexpression and identification of a novel stress-regulatedgene. Plant Cell Environ. 23: 689–700.

Schroda, M., Vallon, O., Wollman, F. A. and Beck, C. F. 1999.A chloroplast-targeted heat shock protein 70 (HSP70)contributes to the photoprotection and repair of photosys-tem II during and after photoinhibition. Plant Cell 11:1165–78.

Seitz, S. B., Voytsekh, O., Mohan, K. M. and Mittag, M. 2010.The role of an E-box element: multiple frunctionsand interacting partners. Plant Signal. Behav. 5: 1077–80.

Sweetlove, L. J., Heazlewood, J. L., Herald, V. et al. 2002.The impact of oxidative stress on Arabidopsis mitochon-dria. Plant J. 32: 891–904.

Takahashi, A. and Ohnishi, T. 2009. Molecular mechanismsinvolved in adaptive responses to radiation, UV light, andheat. J. Radiat. Res. 50: 385–93.

Tohge, T., Kusano, M., Fukushima, A., Saito, K. and Fernie,A. R. 2011. Transcriptional and metabolic programs fol-lowing exposure of plants to UV-B irradiation. Plant Signal.Behav. 6: 1987–92.

Von Zglinicki, T., Pilger, R. and Sitte, N. 2000. Accumulationof single-strand breaks is the major cause of telomereshortening in human fibroblasts. Free Radic. Biol. Med.28: 64–74.

Wiencke, C., Roleda, M. Y., Gruber, A., Clayton, M. N. andBischof, K. 2006. Susceptibility of zoospores to UV radia-tion determines upper depth distribution limit of Arctickelps: evidence through field experiments. J. Ecol. 94:455–62.

Wilhelm, C. and Selmar, D. 2011. Energy dissipation is anessential mechanism to sustain the viability of plants: thephysiological limits of improved photosynthesis. J. PlantPhysiol. 168: 79–87.

Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O. andWeigel, R. J. 1999. Combining SSH and cDNA microarraysfor rapid identification of differentially expressed genes.Nucleic Acids Res. 27: 1517–23.

Zhang, P., Liu, S., Cong, B. et al. 2011. A novel omega-3 fattyacid desaturase involved in acclimation processes of polarcondition from Antarctic ice algae Chlamydomonas sp.ICE-L. Mar. Biotechnol. (NY) 13: 393–401.


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