Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: Alignment of the DRONPA nucleotide sequence of the commercial vector DRONPA-GREEN pDG-S1 (MLB Group Company) and the codon-optimized DRONPA-s. Changes in DRONPA-s are highlighted in black. The deduced amino acid sequence is shown below the nucleotide sequences.

Figure S2: Photoswitching performance of DRONPA-s. A) CAI for the original DRONPA and the codon-optimized DRONPA-s, determined by GeneArt using their in-house proprietary software GENEOPTIMIZER®. The histograms show the percentage of sequence codons, which fall into a quality class (defined by the frequency of usage). The quality of the most often used codon for a given amino acid is set to 100, the remaining codons are scaled accordingly (42). B) Details on sequence motifs removed during the optimization process.

Figure S3: Photoswitching performance of DRONPA-s. A) Absorption spectrum (blue, broken line) and emission spectrum (green, solid line) of the synthetic DRONPA-s in solution. B) Decay in fluorescence intensity after irradiation with 488 nm (20 mW/cm2). Each data point corresponds to the integral of the emission spectrum between 500 and 580 nm.

Figure S4: Functional complementation of the atgrp7-1 T-DNA insertion line with the AtGRP7-DRONPA-s fusion protein. A) An immunoblot of total protein extracts from atgrp7-1, Col-0 wt and two independent transformants of AtGRP7-DRONPA-s in atgrp7-1 was probed with an antibody against DRONPA to detect the fusion protein (the asterisk indicates a degradation product) (top) and reprobed with a light-harvesting chlorophyll-binding protein (LHCP) antibody to confirm equal loading. In parallel, a blot was probed with an antibody against AtGRP7 (middle). The part corresponding to endogenous AtGRP7 protein is shown. The blot was reprobed with the LHCP antibody to confirm equal loading. On the blot probed with an antibody against AtGRP8 (bottom), unspecific cross-reaction with the large subunit of ribulose-1,5-bisphosphate carboxylase (RbcL) confirms equal loading. B) The flowering time of Col-0 wt, atgrp7-1 and AtGRP7-DRONPA-s in atgrp7-1 grown in short days was compared by determining total leaf number. Data are based on two independent experiments with n ≥ 20 each. The leaf number in atgrp7-1 and the complemented line is significantly different (p value = 0.000385).

Figure S5: Switching behavior of immobilized DRONPA-s. A) A 20 × 20-µm region of recombinant DRONPA-s embedded in a Mowiol matrix is switched off by exposure to 488 nm. Subsequently, a small spot in the region of the laser focus is switched back to the fluorescent state by irradiation with 404-nm light. Fluorescence intensity is indicated by the quantitative color code: yellow corresponds to the highest fluorescence intensity. B) Calculation of the spot size with the full width at half maximum (FWHM) convention. Shown are the fluorescence intensities along the indicated line in (A).

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