Elucidating the biological function of Arabidopsis DASH-type cryptochrome
Final Report Abstract
From a structural, spectroscopic and in vitro functional point of view cry3 from Arabidopsis thaliana is among the best characterized members of the DASH-type cryptochrome subclade. However, its biological function remained elusive. This study was undertaken to characterize the in vivo function of cry3 using a genetic approach. A knock-out transposon insertion mutant of Arabidopsis was identified and compared to wild-type in respect to different biological responses including hypocotyl growth inhibition and protein composition of chloroplasts. Slightly longer hypocotyls were observed for the cry3 mutant in particular under low fluence rates of blue light. Comparison of the 2-D chloroplast protein patterns from wild-type and the cry3 mutant revealed 19 differentially expressed spots, which except one could be identified by MALDI/TOF or MS/MS. All of these proteins are encoded in the nucleus. The abundance of 12 of these spots was higher in the cry3 mutant than in wild-type suggesting that cry3 could act as a repressor. Previous data have shown that cry3 is located in organelles. Thus, the effects seen on expression of nuclear genes could be in line with retrograde signaling from the chloroplast to the nucleus. However, localization of cry3 was reinvestigated in this study and showed that cry3 is located in the nucleus, too. Thus, retrograde signaling by cry3 is not evidenced by our data. Unfortunately, the effects seen in the cry3 mutant on hypocotyl growth inhibition and chloroplast protein composition could not be complemented by CaMV 35S-promoter-driven expression of HA-tagged cry3. We assume that lack of strong phenotypes of the cry3 mutant may relate to functional redundancy possibly with other cryptochrome photoreceptors. Thus, future studies are required comparing the cry3 single with cry1/2/3 triple and cry1/3 and cry2/3 double mutants. Y2H library screens using cry3 as bait let to the identification of nine putative cry3 partners. Three of them (EMB3142, EMBRYO DEFECTIVE 3142, At5G51200; DPE2, DISPROPORTIONATING ENZYME 2, At2G40840; Glutamyl/glutaminyl-tRNA synthetase, gluRS, At5G26710) are most promising since they did not show any or very little self-activation, and, except gluRS, these proteins are located in the same compartments as cry3. Future studies such as BiFC are required to confirm the Y2H data. Another aspect addressed in this study is regulation of CRY3 expression. It was found that CRY3 expression is strongly and rapidly induced by blue, red, far-red and UV-B light. Analysis of light induction in different photoreceptor mutants demonstrated that phyA, cry1 and/or cry2 and UVR8 but not phyB control CRY3 expression. Thus, the pattern of CRY3 light-regulation is quite complex and supports the notion that cry3 function may strongly depend on other photoreceptors. Finally, the transgenic lines expressing the cry3-GFP fusion were used to affinity-purify cry3 from plant extracts and to determine its chromophore composition. The protein contained the same cofactors as the 6xHis-tagged protein expressed in E. coli, i.e. flavin adenine dinucleotide and N5, N10- methenyltetrahydrofolate. This demonstrated that the heterologously expressed cry3 protein is a valid surrogate of the corresponding protein expressed in plants. This is, to our knowledge, the first conclusive analysis of cofactors bound to an Arabidopsis protein of the cryptochrome/photolyase family and purified from plant tissue.
Publications
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(2013) Lifetimes of Arabidopsis cryptochrome signaling states in vivo. Plant J. 74, 583-592
Herbel V., Orth C., Wenzel R., Ahmad M., Bittl R., Batschauer A.
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(2014). Cellular metabolites enhance light sensitivity of Arabidopsis cryptochrome through alternate electron transfer pathways. Plant Cell 26, 4519-4531
Engelhard C., Wang X., Robles D., Moldt J., Essen L.-O., Batschauer A., Bittl R., and Ahmad M.
