Improvement of light emission by SuperhRLUC in Arabidopsis and recombination vectors. (A) In vivo luminescence measurement of transgenic Arabidopsis expressing regular RLUC (2 lines), hRLUC, or SuperhRLUC. Asterisk represents the significant increase of luciferase activity of SuperhRLUC over hRLUC (P < 0.01, two tailed t-test; n = 4 repeats with 25 seedlings each). The immunoblot below probed with RLUC antibody confirms that hRLUC and SuperhRLUC (36 kDa) accumulate to similar, high, levels; the original RLUC can only be detected on the immunoblot after prolonged development. The arrowhead indicates a non-specific immunoreaction. (B) Photon-counting images of representative seedling roots. Photon emission in the primary roots of SuperhRLUC transgenic Arabidopsis was stronger compared to regular hRLUC (lower panels). The upper panels show a 3-dimensional version of the images below in which photon intensity is encoded in the third axis. Seedlings were incubated in 2 μM coelenterazine and imaged for 5 min. (C) pBS-SuperhRLUC-attR and pBS-attR-SuperhRLUC are recombination vectors for expression of SuperhRLUC fusion proteins, which contain the lambda att recombination sites utilized by the Gateway™ (Invitrogen) system . Sequence elements flanking an insert (target cDNA) are shown before (pENTR) and after (Destination) attL × attR recombination. The 35S indicates a strong promoter in plants, which can be replaced by restriction digestion with KpnI and SwaI or AvrII. The ccdB gene provides for counter-selection of non-recombinants.