Synthesis of chlorophyll b: Localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit

BACKGROUND: Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. RESULTS: Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of (125)I(- )identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. CONCLUSION: CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis

Genome-Wide Evaluation of Histone Methylation Changes Associated with Leaf Senescence in Arabidopsis

Leaf senescence is the orderly dismantling of older tissue that allows recycling of nutrients to developing portions of the plant and is accompanied by major changes in gene expression. Histone modifications correlate to levels of gene expression, and this study utilizes ChIP-seq to classify activating H3K4me3 and silencing H3K27me3 marks on a genome-wide scale for soil-grown mature and naturally senescent Arabidopsis leaves. ChIPnorm was used to normalize data sets and identify genomic regions with significant differences in the two histone methylation patterns, and the differences were correlated to changes in gene expression. Genes that showed an increase in the H3K4me3 mark in older leaves were senescence up-regulated, while genes that showed a decrease in the H3K4me3 mark in the older leaves were senescence down-regulated. For the H3K27me3 modification, genes that lost the H3K27me3 mark in older tissue were senescence up-regulated. Only a small number of genes gained the H3K27me3 mark, and these were senescence down-regulated. Approximately 50% of senescence up-regulated genes lacked the H3K4me3 mark in both mature and senescent leaf tissue. Two of these genes, SAG12 and At1g73220, display strong senescence up-regulation without the activating H3K4me3 histone modification. This study provides an initial epigenetic framework for the developmental transition into senescence

Analysis of bivalently modified genes.

<p>Bivalent (H3K4me3 and H3K27me3) modifications were observed for a subset of genes at 23 d. These genes were classified into those that lost both modifications at 52 d (K4+K27- None, dark blue), those that retained the H3K27me3 mark at 52 d (K4+K27 – K27, light blue), those that retained the H3K4me3 mark at 23 d (K4+K27- K4, yellow) and those that retained the bivalent marks (K4+K27- K4+K27, brick red). Genes thus classified were then placed into the five groups shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone-0033151-g004" target="_blank">Figure 4</a>. Genes that retained the H3K27me3 mark were restricted to groups A and B while genes that retained the H3K4me3 mark were restricted to groups C–E.</p

Expression of Arabidopsis <i>KDM5B</i>–like genes in senescent leaf tissue.

<p><i>KDM5B</i> genes encode H3K4me3 demethylases, and eight KDM5B-like genes were identified in Arabidopsis. The same tissue used in the ChIP-seq analysis was analyzed for expression of the <i>KDM5B-like</i> genes using real-time qPCR. <i>ACT2</i> was the reference gene and relative expression at 52 d compared to 23 d is shown. Two <i>KDM5B-like</i> genes affect flowering time <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Jeong1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Noh1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Yang2" target="_blank">[43]</a>, and their published names as well as known target gene are shown. <i>At2g34880</i> mRNA was undetectable in both RNA samples, and is not shown.</p