Fig 1.
REF6 directly binds to both the promoters and coding regions of NYE1 and NYE2.
(A) Physical interactions of REF6 with NYE1 promoter in Y1H assays and blue-white spotting tests. (B) ChIP assays of in vivo association of REF6-HA with NYE1/2 genes in the 10-day-old seedlings of ref6-1+PREF6::REF6-HA and Col-0. Fold enrichments were calculated as the ratios of the signals in ref6-1+PREF6::REF6-HA to the signals in Col-0. Primers are listed in S4 Table. GPAT4 and NAC004 genes were used as positive controls, with TUB as a negative control. Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by paired Student’s t test. (C, D) EMSAs of in vitro binding of REF6 to the CTCTGYTY motifs within the ChIP-PCR fragments P4 and P10 of NYE1 (C) and NYE2 (D) promoters (indicated with red arrows). Probe sequences used in EMSA are shown in (C) and (D). GST-tagged REF6 was incubated with the biotin-labeled wild-type DNA probe. Competition experiments were performed by adding the excessive amounts (40× and 200×, respectively) of unlabeled DNA probe. A mutated probe was used to test binding specificity. Shifted bands, indicating the formation of DNA-protein complexes, are indicated by arrows. “-” represents absence, “+” represents presence. Sequences of both the wild-type and mutated probes are shown on the bottom of the images.
Fig 2.
REF6 promotes both Chl degradation and general leaf senescence process.
(A, B) Changes in the transcript levels of NYE1/2 in ref6-1 and ref6-1+PREF6::REF6-HA relative to those in Col-0 with aging (A) and during dark treatment (B) (DAD, days after dark treatment). Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by paired Student’s t test. (C, D) Plants of indicated genotypes (ref6-1, ref6-1+PREF6::REF6-HA, nye1 nye2 as well as Col-0) grown under long day-growth conditions for 40 days (C). Chl contents (D) in the leaves of the plants shown in (C). (E, F) Phenotypes of indicated genotypes on 4 DAD. Detached leaves were obtained from 25-day-old plants under long day-growth conditions. Unless stated otherwise, the 3rd and 4th detached rosette leaves were used for physiological and molecular analyses. (G) Leaf phenotypes and Chl contents of ref6-1 after infiltration with Agrobacterium containing 35S::NYE1 or empty vector pCHF3. (H) Leaf phenotypes and Chl contents of nye1-1 or nye1 nye2 after infiltration with Agrobacterium containing 35S::REF6 or empty vector pCHF3.
Fig 3.
Overexpression of REF6 and NYE1 in each other’s mutants confirms their roles in promoting the general leaf senescence process.
(A) Leaf phenotypes of ref6-1+PiDEX::NYE1 transgenic plants treated with or without DEX on 3 DAD. -1, -3, and -4 represent different transgenic lines. (B, C) Chl contents (B) and Fv/Fm ratios (C) in the leaves shown in (A). (D) Leaf phenotypes of nye1-1+PiDEX::REF6 transgenic plants treated with or without DEX on 3 DAD. -1, -2, and -5 represent different transgenic lines. (E, F) Chl contents (E) and Fv/Fm ratios (F) in the leaves shown in (D). (G-I) H2O2 levels detected by DAB staining (G), Fv/Fm ratios (H), and Ion leakage (I), in ref6-1, Col-0, and ref6-1+PREF6::REF6-HA plants on 4 DAD. Data are mean ± SD (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test.
Fig 4.
Transcriptome analysis of Col-0 and ref6-1 mutant.
(A) Principle component analysis of log2-transformed transcriptome data of 12 samples. (B) Volcano plot of the differentially expressed genes (DEGs) for Col-0 and ref6-1 investigated in this work (10-day-old seedlings, left; 40-day-old rosette leaves, right). The y-axis corresponds to the mean expression value of -log10 (q-value), and the x- axis displays the log2-fold change value. (C-E) Heat map showing the expression of chlorophyll biosynthesis, subunit genes of photosystems I and II (C), senescence associated genes (D), and ethylene related genes (E) in 10-day-seedlings and 40-day-old leaves of Col-0 and ref6-1.
Fig 5.
REF6 directly up-regulates the transcription of EIN2, ORE1, and NAP.
(A) Relative transcript levels of EIN2, ORE1, and NAP genes in the leaves detached from 25-day-old or 40-day-old Col-0, ref6-1, and ref6-1+PREF6::REF6-HA plants grown under long day-growth conditions. (B) ChIP assays of in vivo binding of REF6-HA to EIN2, ORE1, and NAP promoters and coding regions in the 10-day-old seedlings of ref6-1+PREF6::REF6-HA and Col-0 plants. Fold enrichments were calculated as described previously. Primers are listed in S4 Table. NAC004 gene was used as a positive control, whereas TUB as a negative control. In (A) and (B), data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by paired Student’s t test. (C) EMSAs of in vitro binding of REF6 to the CTCTGYTY motifs within the ChIP-PCR fragments P17, P22, and P24 of EIN2, ORE1, and NAP coding regions (indicated with red arrows). Probe sequences used in EMSA are: 5’-AGGAACCATTCTCTGGATAAACCCTAGC-3’ for EIN2, 5’-TACTCGGATCCTCTGTTTTTACAAGACA-3’ for ORE1, and 5’-TTTCTCCAAACTCTGTTTTCTCTGTAAA-3’ for NAP.
Fig 6.
Loss-of-function of REF6 increases H3K27me3 levels at NYE1/2, EIN2, ORE1, and NAP genes.
(A) Schematic diagrams of NYE1/2, EIN2, ORE1, and NAP genes and positions of the primers used for determining their H3K27me3 levels. Primer sequences are listed in S4 Table. (B) H3K27me3 levels of NYE1/2, EIN2, ORE1, and NAP genes, expressed as the percentage of input, in the 10-day-old seedlings of Col-0, ref6-1, and ref6-1+PREF6::REF6-HA grown under long day-growth conditions. (C) H3K27me3 levels of NYE1/2, EIN2, ORE1, and NAP genes in the leaves detached from the 40-day-old plants of Col-0, ref6-1, and ref6-1+PREF6::REF6-HA grown under long day-growth conditions. In (B)—(C), data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by paired Student’s t test.
Fig 7.
REF6 regulates leaf senescence independently of plant development.
(A) Phenotypes of the leaves detached from 40-day-old ref6-1 and Col-0 plants grown under short day-growth conditions on 4 DAD. (B, C) Chl contents (B) and Fv/Fm ratios (C) in the leaves shown in (A). (D) Phenotypes of the leaves detached from 14-day-old ref6-1 and Col-0 plants grown under long day-growth conditions on 4 DAD. (E, F) Chl contents (E) and Fv/Fm ratios (F) in the leaves shown in (D). (G) Leaf phenotypes of ref6-1+PiDEX::REF6 transgenic plants treated with or without DEX on 3 DAD. -2, -3, and -5 represent different transgenic lines. (H, I) Chl contents (H) and Fv/Fm ratios (I) in the leaves shown in (G). In (B), (C), (E), (F), (H), and (I), data are mean ± SD (n = 10). ***P < 0.001 by unpaired Student’s t test.
Fig 8.
Major target SAGs of REF6 in the regulation of leaf senescence.
ORE1 and NAP, acting downstream of EIN2 and EIN3, are the two major NAC transcription factors responsible for promoting Chl degradation and leaf senescence by up-regulating the expression of NYE1/2 as well as other SAGs. EIN3 is also involved in a feed-forward regulation by directly suppressing the expression of miR164, which targets ORE1 at the post-transcriptional level [46, 49, 51–54]. In this study, it is revealed that, during leaf senescence, REF6 directly facilitates the activation of the whole pathway by reducing the H3K27me3 level at EIN2, ORE1, and NAP [51] as well as AtNAC3, NTL9, LOX1, PAD4 and PPDK [68–72].