The Reduced Plastid-Encoded Polymerase-Dependent Plastid Gene Expression Leads to the Delayed Greening of the <i>Arabidopsis fln2</i> Mutant

<div><p>In <i>Arabidopsis</i> leaf coloration mutants, the delayed greening phenomenon is common. Nonetheless, the mechanism remains largely elusive. Here, a delayed greening mutant <i>fln2–4</i> of FLN2 (Fructokinase-Like Protein2) was studied. FLN2 is one component of Transcriptionally Active Chromosome (TAC) complex which is thought to contain the complete plastid-encoded polymerase (PEP). <i>fln2–4</i> displayed albino phenotype on medium without sucrose. The PEP-dependent plastid gene expression and chloroplast development were inhibited in <i>fln2–4</i>. Besides interacting with thioredoxin z (TRX z), we identified that FLN2 interacted with another two members of TAC complex in yeast including its homologous protein FLN1 (Fructokinase-Like Protein1) and pTAC5. This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. <i>fln2–4</i> exhibited delayed greening on sucrose-containing medium. Comparison of the PEP-dependent gene expression among two complete albino mutants (<i>trx z</i> and <i>ptac14</i>), two yellow mutants (<i>ecb2–2</i> and <i>ys1</i>) and the <i>fln2–4</i> showed that <i>fln2–4</i> remains partial PEP activity. FLN2 and FLN1 are the target proteins of TRX z involved in affecting the PEP activity. Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1–FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in <i>fln2–4</i>. We proposed the partial PEP activity in the <i>fln2</i> mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.</p></div

Changes in the transcript levels of PEP-dependent genes during the greening process of <i>fln2–4</i> mutant.

<p>The expression levels of <i>rbcL</i>, <i>psbA</i> and <i>psbB</i> genes in 7-day-old and 14-day-old <i>fln2–4</i> mutants were determined by Northern blot as compared with WT, respectively. The experimental WT and <i>fln2–4</i> seedlings were grown on sucrose-containing MS medium.</p

Chlorophyll accumulation in the WT and <i>fln2–4</i> mutants.

*<p>Averages ± standard deviations of chlorophyll (Chl) concentrations for 3 independent measurements. FW: fresh weight.</p

Expression analysis of the plastid encoded genes in <i>fln2–4</i> seedlings.

<p>Northern blot was performed to detect the plastid gene transcript levels in the 7-day-old <i>fln2–4</i> seedlings and WT grown on MS medium without sucrose. Three classes of genes were examined, <i>psbA, psbB</i>, and <i>rbcL</i> were selected as PEP-dependent genes, <i>clpP</i> and <i>rrn16</i> were selected as PEP- and NEP-dependent genes, <i>accD</i> and <i>rpoA</i> were selected as NEP-dependent genes.</p

Comparison of PEP-dependent plastid gene expression between <i>fln2–4</i> and four leaf coloration mutants.

<p>(A) The phenotypes of WT and four leaf coloration mutants grown with or without sucrose. (B) qRT-PCR analysis the transcript levels of four PEP-dependent plastid genes in <i>fln2–4</i> seedlings and other four leaf coloration mutants grown on MS medium without sucrose for 7 days. These PEP-dependent genes refer to <i>psbA</i>, <i>psbB</i>, <i>psaB</i>, <i>petD</i>. Expression levels are presented as the percentage relative to WT. Data are means ± SD (n = 3). (C) The accumulation of <i>psbA</i> and <i>psbB</i> transcripts detected by Northern blot.</p