Fig 1.
The positions of mutations and expression of DRD1 gene.
(A) Domain structures of DRD1 and DDM1 and positions of drd1-6, drd1-p and ddm1-2. Amino acid sequence change from tryptophan (W) to stop codon in helicase superfamily C-terminal (HELICc) domain in drd1-6. The triangle indicates the position of T-DNA insertion in drd1-p mutant. In case of ddm1-2, substitution of G to A in the splice donor site of intron 11 brings about lack of helicase superfamily C-terminal (HELICc) domain. (B) RT-PCR analysis of DRD1 and control ACTIN2 genes in WT and drd1-p mutant leaves. The drd1-p mutant displayed a decrease in DRD1 expression levels compared to WT.
Fig 2.
Delayed leaf senescence symptoms in the drd1-6 mutant.
(A) Phenotypes of 28-day-old and 55-day-old wild-type (WT) and drd1-6 mutant whole plants. (B) Individually darkened leaf (IDL) senescence of WT (left) and drd1-6 (right) plants. Rosette leaves of 28-day-old WT and drd1-6 mutant (IDL 0 d) were induced to undergo senescence for 5 d under dark conditions (IDL 5 d). The red and blue arrows indicate 5 d IDL of WT and drd1-6 plants, respectively. (C) Phenotypes of detached WT and drd1-6 leaves after 5-d dark incubation. (D) Photochemical efficiency of photosystem II (Fv/Fm) and (E) maximal electron transport rate (ETRmax) in WT and the drd1-6 leaves were examined at the indicated days during dark-induced senescence (DIS). Data represent average values ± SE (n = 27) of three independent experiments. * indicates P < 0.01 by student’s t-test.
Fig 3.
Delayed leaf senescence symptoms in the drd1-6 mutant at later developmental stages.
Rosette leaves of 28-day-old WT and the 34, 36, and 38-day-old drd1-6 mutants were detached and darkened for 0, 3, 5 days. Photochemical efficiency of photosystem II (Fv/Fm) in WT and mutant leaves was examined at the indicated days. Data represent average values ± SE (n = 20) of independent experiments. Bars with the same letter are not significantly different at P < 0.05 by Tukey’s honestly significant difference (HSD) test.
Fig 4.
Effects on chlorophyll and protein degradation in the drd1-6 plants during DIS.
(A) Chlorophyll content was measured using rosette leaves after 0d, 3 d, 5 d, and 7 d of DIS, as indicated. (B) Protein contents from the WT and the drd1-6 mutant leaves before (0 d) and after 3 d, 5 d, and 7 d of DIS. (C) Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE) of thylakoid protein complexes. Dodecylmaltoside-solubilized thylakoid membrane proteins corresponding to equal amounts of fresh weight were subjected to BN-PAGE. (D) Immunoblot analysis of thylakoid protein complexes. Thylakoid membranes were solubilized and subjected 15 μg of protein per well to SDS-PAGE. The band indicated by the arrow head was identified by LC MS/MS (see S2 Table). (E) D1 protein was identified by immunoblot analysis using anti-D1 antiserum.
Fig 5.
Expression of senescence-associated genes in the drd1-6 mutant during DIS.
(A) RT-PCR and (B) Quantitative real-time PCR (qRT-PCR) analysis of gene expression in WT and the drd1-6 mutant leaves at the indicated days. SAG12, senescence-associated gene 12; ANS, antocyanidin synthase gene; CBR, chlorophyll b reductase gene; and PAO, pheophorbide α oxygenase gene. The values are normalized to ACTIN2 expression. Data indicate the mean ± SD (n = 9) of three independent experiments.
Fig 6.
Transcript level changes in rosette leaves during DIS.
(A) Microarray analysis represents that the numbers of genes show two- (black), three- (gray), or fourfold (white) up- (upper) or down-(lower) regulation in expression of 0 d, 3 d, and 5 d DIS drd1-6 mutant compared to WT. (B) The number of genes with two-, three-, or fourfold increase or decrease in gene expression during 3 d and 5 d DIS compared to control (0 d) in the WT and the drd1-6 mutant are represented by black, gray, and white bars, respectively. Data represent the means of two independent Affymetrix Gene Chip analyses. FD, fold difference.
Table 1.
Gene ontology analysis of differentially expressed genes (DEGs) between the WT and the drd1-6 after 5-d DIS.
Table 2.
Gene lists closely associated with the phenotypic differences between the WT and the drd1-6 by DIS.
Fig 7.
Expression of transcriptional gene silencing markers and histone methyltransferase/acetyltransferase genes in the drd1-6 mutant.
(A) Expression analysis using transcriptional gene silencing markers 180-bp centromeric repeats (CEN) and (B) transcriptionally silent information (TSI) of WT and the drd1-6 mutant during DIS. Relative RNA levels were measured by qRT-PCR and the values are normalized to ACTIN2 expression. Data indicate the mean ± SD (n = 9) from three independent experiments. Bars with the same letter are not significantly different at P < 0.05 by Tukey’s honestly significant difference (HSD) test. (C) RT-PCR analysis in the WT and the drd1-6 mutant leaves at the indicated days. SDG8, set domain group 8; SDG27, set domain protein 27; and HAC1: histone acetyltransferase of the CBP family 1. ACTIN2 was used as control. Representative data from three or more independent experiments with similar results are shown.
Fig 8.
Delayed leaf senescence symptoms in the ddm1-2 as well as in the drd1-6 mutant.
(A) Phenotypes of detached WT, drd1-6, drd1-p and ddm1-2 leaves after 0, 3, and 5-d dark incubation. (B) Photochemical efficiency of photosystem II (Fv/Fm) in WT and mutant leaves in (A). Data represent average values ± SE (n = 27) of three independent experiments. Bars with the same letter are not significantly different at P < 0.05 by Tukey’s honestly significant difference (HSD) test.