Nal inside a land plant, regardless of their evolutionary separation by over 700 million years22. Therefore it may be attainable to mix and match the regulatory elements of light harvesting from various clades of photosynthetic organisms to proficiently tune photosynthetic efficiency and raise photosynthetic productivity.HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptMETHODSGenetic mapping and PCR analysis The fine mapping of the npq1 mutation was completed by scoring PCR-based markers on chosen tetrad mutant progeny derived from a cross among npq1 (137c strain background) and the polymorphic wild-type strain S1D2 (CC-2090). Markers have been developed according to info in Kathir et al.23 along with the marker list from David Stern offered at www.chlamy.org. To identify the mutation in the CVDE gene, genomic DNA PCR was performed using a series of primer pairs that collectively span the whole gene, plus the PCR items have been sequenced for comparison between the wild variety plus the npq1 mutant. The primers that resulted in various length items involving wild variety and npq1 have been RMD345 (5-CTTGGCGGAAGCAGAGTATGGC-3) and RMD346 (5CGGCCTCCCTTCATCCCTCCCAC-3).Nat Plants. Author manuscript; readily available in PMC 2017 March 12.Li et al.PagePhylogenetic evaluation CVDE homologs and CruP homologs have been identified by browsing via BlastP and tBlastN against the sequenced proteome and genome database, respectively, with an evalue cutoff of 1e-90.The prospective chloroplast transit peptides for CVDE homologs or CruP homologs had been predicted by aligning respective homologs from organisms with or without the need of chloroplasts utilizing the Clustal Omega program (version1.2.1; http://www.ebi.ac.uk/Tools/msa/clustalo/). The predicted mature proteins have been aligned working with Clustal Omega and BoxShade (version 3.21; www.ch.embnet.org/software/BOX_form.html). The phylogenetic tree was constructed at Phylogeny.2227206-09-7 Chemical name fr (http://phylogeny.1612287-20-3 Data Sheet lirmm.PMID:23916866 fr/phylo_cgi/advanced.cgi) with Gblocks for alignment curation, PhyML for building of Phylogenetic tree, and Tree Dyn for visualization of phylogenetic tree. Complementation of Chlamydomonas npq1 mutant For complementation of npq1, an 11.5-kb EcoRV/NotI fragment of BAC clone 33B9 containing the CVDE gene was subcloned in to the pBC1 vector24 to generate pCVDEg. For complementation of npq1 using a carboxyl-terminal FLAG-tagged version with the CVDE protein, the 1.4 kb SbfI/BglII fragment of pCVDEg containing the three terminus on the CVDE gene was subcloned into the pUC19-BglII vector to create pUC19-BglII-pCVDE. The 0.4 kb NcoI/BglII fragment of pUC19-BglII-pCVDE was then replaced by a synthesized version (Integrated DNA Technologies, Inc.), which contains a carboxyl-terminal FLAG-tag linked together with the CVDE protein by means of two glycines to produce plasmid pUC19-BglII-pCVDEFLAG. The 1.four kb SbfI/BglII fragment of pUC19-BglII-pCVDE-FLAG was then ligated into pCVDEg double-digested with same enzymes to generate pCVDEg-FLAG. Each pCVDEg and pCVDEg-FLAG have been separately transformed into the npq1 mutant making use of the glass bead process as described previously25. The positive transformants have been selected on paromomycin then screened for zeaxanthin accumulation following high light exposure by HPLC as previously described26. Complementation of Arabidopsis vde1 mutation by Chlamydomonas CVDE The predicted protein sequences of Chlamydomonas CVDE have been retrieved from both Phytozome at http://www.phytozome.net (protein ID: Cre04.g221550.t1.2) and the Joint Genome Institute a.