Pale-Green Phenotype of <i>atl31</i><i>atl6</i> Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in <i>Arabidopsis thaliana</i>

<div><p>Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both <i>atl31</i> and <i>atl6</i> developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the <i>atl31/atl6</i> mutant showed normal Fv/Fm. In the pale-green leaves of the <i>atl31/atl6</i>, the expression of <i>HEMA1</i>, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor <i>GLK1</i>, which directly promotes <i>HEMA1</i> transcription, was also significantly decreased in <i>atl31/atl6</i> mutant. Finally, application of 5-aminolevulinic acid to the <i>atl31/atl6</i> mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of <i>atl31/atl6</i> mutant.</p></div

Expression of photosynthesis-related genes.

<p>Total RNA was extracted from 10-day-old WT and <i>atl31–1/atl6–1</i> plants grown under LL and ML conditions, and transcript levels of photosynthesis-related genes, <i>CAB2</i>, <i>HEMA1</i>, <i>rbcL</i>, <i>psaA</i>, <i>rpoA</i> and <i>accD</i> (A), <i>GLK1</i> and <i>GLK2</i> (B) were measured by quantitative real-time RT-PCR. <i>EF1α</i> was used as an internal control. The ratio of each transcript in <i>atl31–1/atl6–1</i> plants grown under ML vs. LL conditions were normalized relative to the ratio in WT plants. Error bars represent SD (n = 3). Asterisks indicate statistically significant differences between the mutants and WT plants by Student’s <i>t</i>-test (*, <i>P</i> < 0.001).</p

BN-PAGE and immunoblot analyses.

<p>(A) Separation and identification of the pigment-protein complexes in first and second true leaves of 2-week old WT and <i>atl31–1/atl6–1</i> plants grown under ML and LL conditions. The BN-PAGE gels were destained with 20% MeOH and 7% acetic acid to clarify the bands of the pigment-protein complexes. The PSII dimer (PSII-D), PSI-LHCI supercomplex, RubisCO complex, CP29-CP24-LHCII trimer (LHCII assembly), LHCII trimer (LHCII-T) and LHCII monomer (LHCII-M) were visualized in the BN-gel. Proteins were identified as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117662#pone.0117662.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117662#pone.0117662.ref032" target="_blank">32</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117662#pone.0117662.ref033" target="_blank">33</a>]. The positions of the molecular markers are indicated on the right. (B) Immunoblot analyses and CBB staining of the plastid proteins in first and second true leaves of 2-week-old WT and <i>atl31–1/atl6–1</i> plants grown under ML conditions. Total leaf extracts were loaded onto 14% SDS—PAGE gels containing 4M urea. Immunoblot analyses were performed using antibodies to PsbC, PsbD, Lhcb1 and PsaA/B, whereas RbcL was visualized by CBB staining. A dilution series of the WT samples is indicated in percentage.</p

Fv/Fm measurement.

<p>Effect of light conditions on the Fv/Fm of first or second true leaves of 2-week-old WT and <i>atl31–1/atl6–1</i> plants grown under ML or LL conditions. Error bars represent SD (n = 5). Statistical significance was determined by ANOVA, followed by post-hoc Tukey’s tests. Means that differed significantly (<i>P</i><0.05) are indicated by different letters.</p

Plastid ultrastructure.

<p>Transmission electron microscopic images of first or second true leaves of 2-week old plants <i>atl31–1/atl6–1</i> (A-D) and WT (E-H) plants grown under ML (A-C and E-G) and LL (D and H) conditions. A red line on the left below representative leaves indicated the section of each sample. Scale bar: 1 μm.</p

Manifestation of the pale-green phenotype in the <i>atl31–1/atl6–1</i> double mutant.

<p>Photographs of representative <i>atl31–1/atl6–1</i> and WT plants over time. Scale bar: 5 mm.</p

Dependence of the pale green phenotype of <i>atl31–1/atl6–1</i> double mutants on light intensity.

<p>WT and <i>atl31–1/atl6–1</i> plants were grown at the indicated light intensity for 2 weeks. (A) Photographs of representative first or second leaves of each plant. Scale bar: 5 mm. (B) and (C) Chl amount and Chl a/Chl b ratio (Chl a/b) in first or second leaves of each plant respectively. Error bars represent SD (n = 5). Statistical significance was determined by ANOVA, followed by post-hoc Tukey’s tests. Means that differed significantly (<i>P</i><0.05) are indicated by different letters.</p

The <i>rpt2a</i> mutant shows transcriptional gene silencing.

<p>(A) 35S::HPT in the Col-0 (WT) and <i>rpt2a-2</i> mutant on MS medium. Col-0 plants without any transgene indicate “Col-0”. (B) 35S::HPT in the WT and <i>rpt2a-2</i> mutant on MS medium containing 50 µM hygromycin. (C) Relative luminescence intensity of 35S::LUC2 in WT, <i>rpt2a-2</i> and <i>rpt2b-1</i> mutants. 35S::LUC2 in WT is set as 100%. *t-test P<0.05, error bar = S.D., n = 20. (D) Quantification of LUC2 gene expression in 35S::LUC2 in WT, <i>rpt2a-2</i> and <i>rpt2b-1</i> mutants. 35S::LUC2 in WT is set as 1. Values are the averages of the three experiments, and the level of <i>18S rRNA</i> was used as an internal control.</p

DNA methyltransferase mutants release gene silencing in the <i>rpt2a</i> mutant.

<p>(A) Relative luminescence intensity of WT, <i>rpt2a-2</i>, <i>met1-1</i> and <i>rpt2a-2met1-1</i> double mutants. 35S::LUC2 in WT is set as 100%. *t-test P<0.05, error bar = S.D., n = 10. (B) Relative luminescence intensity of WT, <i>rpt2a-2</i>, <i>drm1 drm2 cmt3</i> triple mutant (<i>ddc</i>) and <i>rpt2a-2 drm1 drm2 cmt3</i> quadruple mutant (<i>rpt2a/ddc</i>). 35S::LUC2 in WT is set as 100%. *t-test P<0.05, error bar = S.D., n = 20.</p

DNA methylation level of transposons is increased in the <i>rpt2a</i> mutant.

<p>(A) McrBC PCR of transposons at Col-0 and <i>rpt2a-2</i> (no transgene: <i>rpt2a</i>-NT). McrBC-digested genomic DNA is amplified by PCR with primers for the indicated transposons. Input DNA was normalized for each genotype with <i>Actin2</i>. (B) Mean levels of DNA methylation in different cytosine context at the <i>AtGP1</i> in Col-0 and <i>rpt2a-2</i> (no transgene). (C) Mean levels of DNA methylation in different cytosine context at the <i>MEA-ISR</i> in Col-0 and <i>rpt2a-2</i> (no transgene).</p