Planta (2007) 225:615���624 DOI 10.1007/s00425-006-0383-0 123 ORIGINAL ARTICLE Magnetic intensity aVects cryptochrome-dependent responses in Arabidopsis thaliana Margaret Ahmad �� Paul Galland �� Thorsten Ritz �� Roswitha Wiltschko �� Wolfgang Wiltschko Received: 27 January 2006 / Accepted: 8 August 2006 / Published online: 6 September 2006 �� Springer-Verlag 2006 Abstract Cryptochromes are blue-light absorbing photoreceptors found in many organisms where they have been involved in numerous growth, developmen- tal, and circadian responses. In Arabidopsis thaliana, two cryptochromes, CRY1 and CRY2, mediate several blue-light-dependent responses including hypocotyl growth inhibition. Our study shows that an increase in the intensity of the ambient magnetic Weld from 33���44 to 500 T enhanced growth inhibition in A. thaliana under blue light, when cryptochromes are the mediat- ing photoreceptor, but not under red light when the mediating receptors are phytochromes, or in total darkness. Hypocotyl growth of Arabidopsis mutants lacking cryptochromes was unaVected by the increase in magnetic intensity. Additional cryptochrome-depen- dent responses, such as blue-light-dependent anthocyanin accumulation and blue-light-dependent degradation of CRY2 protein, were also enhanced at the higher mag- netic intensity. These Wndings show that higher plants are sensitive to the magnetic Weld in responses that are linked to cryptochrome-dependent signaling pathways. Because cryptochromes form radical pairs after photo- excitation, our results can best be explained by the rad- ical-pair model. Recent evidence indicates that the magnetic compass of birds involves a radical pair mechanism, and cryptochrome is a likely candidate for the avian magnetoreception molecule. Our Wndings thus suggest intriguing parallels in magnetoreception of animals and plants that appear to be based on common physical properties of photoexcited crypto- chromes. Keywords Anthocyanin �� Arabidopsis �� Cryptochromes �� Cryptochrome stability �� Hypocotyl growth �� Magnetic Weld �� Radical-pair mechanism Abbreviations cry Cryptochrome FAD Flavin adenindinucleotide phy Phytochrome Trp Tryptophan Introduction The geomagnetic Weld is an omnipresent source of information for magnetosensitive organisms (Wiltschko and Wiltschko 1995). Migratory birds are known to use a magnetic compass their responses in orientation M. Ahmad (&) Universit�� Paris VI, PCMP, Casier 156, 4 Place Jussieu, Paris 75005, France e-mail: ahmad@ccr.jussieu.fr M. Ahmad Penn State University, 25 Yearsley Mill Road, Media, PA 19063, USA R. Wiltschko �� W. Wiltschko (&) Fachbereich Biowissenschaften, J.W.Goethe-Universit��t, Siesmayerstr. 70, 60054 Frankfurt am, Germany e-mail: wiltschko@zoology.uni-frankfurt.de R. Wiltschko e-mail: wiltschko@zoology.uni-frankfurt.de P. Galland Fachbereich Biologie, Philipps-Universit��t Marburg, Karl-von-Frischstr. 8, 35032 Marburg, Germany T. Ritz Department of Physics and Astronomy, University of California, Irvine, CA 92697, USA

616 Planta (2007) 225:615���624 123 tests depend on the direction and intensity of the local magnetic Weld. Moreover, the magnetic compass responses of birds have been found to depend on the wavelength of the ambient light: under light from the blue to green part of the spectrum, they headed into the normal migratory direction, whereas they were dis- oriented under yellow and red light (Wiltschko and Wiltschko 2002). The sensitivity to magnetic Welds as weak as the geomagnetic Weld of 25���60 T is linked to the eVect of magnetic Welds on electron transfer reac- tions of photoreceptors through radical-pair processes (Schulten 1982 Ritz et al. 2000), with the blue-light receptor cryptochrome suggested as a promising candi- date for the receptor molecule (Ritz et al. 2000). The eVect of magnetic Welds in this model would modulate photoreceptor signaling and could manifest itself by forming direction- and intensity-dependent activation patterns (Ritz et al. 2000). In birds, reception of mag- netic compass information takes place in the eye (Semm and Demaine 1986 Wiltschko et al. 2002) the proposed function of cryptochromes in magnetorecep- tion is supported by their occurrence in the retina (M��ller et al. 2004 Mouritsen et al. 2004). Cryptochrome photoreceptors were Wrst identiWed in higher plants where they are ubiquitous and medi- ate a number of blue-light-dependent developmental and growth responses (Ahmad and Cashmore 1993 Briggs and Olney 2001 Ahmad 2003) they have since been identiWed in animals and prokaryotes (Lin and Shalitin 2003). In Arabidopsis, cryptochromes are encoded by two similar genes, cry1 and cry2, and show partial functional overlap in mediating responses such as blue-light-dependent inhibition of hypocotyl elon- gation, anthocyanin accumulation, vegetative growth, Xoral initiation, maintenance of circadian rhythms and expression of blue-light regulated genes. In addi- tion, CRY2 protein levels in seedlings decrease rap- idly upon illumination by blue light, presumably as a result of protein degradation of the light-activated form of the receptor (Ahmad et al. 1998 Lin et al. 1998). Like photolyases, plant cryptochromes have been shown to undergo a light-dependent electron transfer reaction, known as photoactivation, that leads to photoreduction of the Xavin cofactor, FAD (Giovani et al. 2003). This photoreduction presum- ably plays a role in signaling, as mutants that inacti- vate this reaction in the puriWed protein in vitro also show impaired photoreceptor function in vivo (Zeug- ner et al. 2005). Given that light-dependent electron transfer may play a role in cryptochrome signaling, it appeared to be an intriguing possibility that crypto- chrome-controlled responses in plants may also be aVected by weak magnetic Welds. In the present study, we wanted to test (1) whether a change in ambient magnetic conditions aVects physio- logical responses in plants, and (2) whether any observed magnetic eVects are speciWc to cryptochrome- mediated pathways. For this purpose we choose hypo- cotyl growth, anthocyanin accumulation and CRY2 protein stability as reference responses, exposing seed- lings to two distinct magnetic conditions: the local mag- netic conditions of 33, 40 or 44 T and a more than ten times increased Weld of 500 T. Materials and methods Plant materials and growth conditions As wild-type strain of Arabidopsis thaliana (L.) Heynh, we used the ecotype Landsberg erecta (Ler) (Redei 1962), originally obtained from Lehle Seeds, Tucson, AZ, USA. The double mutant hy4-3 fha1 used in the hypocotyl growth tests is defective in both cry1 and cry2 (Ahmad et al. 2002). Arabidopsis seeds were ster- ilized by rinsing them in 70% (v/v) ethanol and in 5% (v/v) sodium hypochloride solution. Seeds were sown on half-strength Murashige and Skoog salts medium (Sigma) on Petri plates containing 2% (w/v) sucrose and 0.9% (w/v) agar. Plates were maintained for 2 days at 5��C, and then placed at room temperature for 2 days, during which time the seedlings for studying hypocotyl growth and anthocyanin accumulation were irradiated under white Xuorescent light for typically 24 h (100 mol m��2 s��1) to induce germination, and later transferred to the relevant light condition at the respective magnetic Weld intensity or, in the case of the seedlings used for the CRY2 stability experiments, returned to darkness at the local magnetic Weld for an additional 48 h. For all experiments, the seedlings received identi- cal growth and germination treatment and were sub- sequently placed simultaneously in the two diVerent magnetic Welds. The initial series of tests on hypocotyl growth (tests F1���F6) was carried out in Frankfurt am, Germany, where the technique to generate the 500 T magnetic Weld was available. This required shipping plates of seedlings from the Paris laboratory to Frankfurt and often storing them at 5��C before the tests, resulting in some variation in the state of germi- nation of seedlings (between radicle emergence and early cotyledon emergence) from one trial to the next. Therefore, to avoid this source of variation between trials, the equipment for the magnetic Weld and the light sources (see below) were moved to Paris, to achieve greater uniformity in germination