One step and tandem-purification of HCF208.

<p>100 (A) or 200 µg (B, C) chlorophyll aliquots of solubilized membrane proteins were applied for purification. Aliquots of 20 µg chlorophyll from extracts and total amounts of eluates were separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunodecorated with antibodies against the HA tag (Anti-HA-Peroxidase) and ATP-Synthase as a control. (A) One step purification of proteins from wild type and HCF208pAUL1 via the HA epitope and competitive elution. (B) Tandem purification of proteins from wild type and HCF208pAUL2 via Strep-tag<i>III</i> and 3xHA (C) Tandem purification of proteins from wild type and HCF208pAUL3 via ProtA tag +3C protease cleavage and Strep-tag<i>III</i>.</p

Schematic illustration of the Gateway compatible pAUL destination vector series, showing expression cassettes.

<p>(A) C-terminal fusion vectors pAUL1-16. Expression is driven by either 2x p<i>35 </i><i>S CaMV</i> or endogenous promoter sequences from <i>A. thaliana</i> (p<i>HCF107</i>, p<i>HCF136</i>, p<i>HCF173</i>): pAUL1-3 and pAUL13 carry <i>p35S CaMV</i>; pAUL4-6 and pAUL14 carry p<i>HCF107</i>; pAUL7-9 and pAUL15 carry p<i>HCF136</i>; pAUL10-12 and pAUL16 carry p<i>HCF173</i>. Protein tags are: 3xHA single tag (pAUL1, 4, 7, 10); Strep-tag<i>III</i> single tag (pAUL13-16); 3xHA/Strep-tag<i>III</i> double tag (pAUL2, 5, 8, 11); and 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL3, 6, 9, 12). (B) N-terminal fusion vectors pAUL17-20. Vectors carry coding sequences for 3xHA single tag (pAUL17); 3xHA/Strep-tag<i>III</i> double tag (pAUL18); 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL19); and Strep-tag<i>III</i> single tag (pAUL20).</p

Complementation analysis of <i>hcf208</i>and <i>hcf107.2</i> with a representative pAUL vector set.

<p>(A) Schematic illustration of promoter/cDNA/tag combinations generated for transformation of <i>hcf107.2</i> and <i>hcf208</i>. (B) Fluorometric analysis of HCF208- and HCF107 complemented plants, wild type and <i>hcf208/hcf107.2</i> mutant plants. Pseudo-color images of maximum quantum efficiency of photosystem II (Fv/Fm) are displayed for HCF107 and of photochemical quenching efficiency (qP) are displayed for HCF208. 3 independent transformants were tested for each construct. Values for each line investigated are illustrated in diagrams. (C) Western blot analysis of complemented lines, wild type and mutant plants. 50 µg of crude protein extract were loaded. Membranes were decorated with Anti-HA-Peroxidase antibody for HCF208 and HCF107; HCF107 was also visualized by an HCF107-specific antibody.</p

Oligonuleotides used for cloning of target genes.

<p>Oligonuleotides used for cloning of target genes.</p

Characterization of promoters 2x p<i>35S CaMV</i>, p<i>HCF107</i>, p<i>HCF136</i>, and p<i>HCF173</i> fused to the <i>GUS</i> reporter gene.

<p>(A) GUS staining of 5-day-old transgenic <i>A. thaliana</i> seedlings. (B) Histochemical localization of GUS activity in leaf sections of 3-week-old transgenic <i>A. thaliana</i> plants. UE, upper epidermis; PM, palisade mesophyll; SM, spongy mesophyll; LE, lower epidermis. (C) GUS activities in transgenic <i>A. thaliana</i> lines. In each case, 10 independent transgenic lines were tested 15 or 30 days after germination. Median values are shown as black bars and indicated at the top of each column. MU, 4-methylumbelliferone.</p

Oligonucleotides used for pAUL vector construction.

<p>Oligonucleotides used for pAUL vector construction.</p