Ring/U-Box Protein AtUSR1 Functions in Promoting Leaf Senescence Through JA Signaling Pathway in Arabidopsis

Abstract

Leaf senescence is regulated by a large number of internal and environmental factors. Here, we report that AtUSR1 (U-box Senescence Related 1) which encodes a plant Ring/U-box protein, is involved in age-dependent and dark-induced leaf senescence in Arabidopsis. Expression of AtUSR1 gene in leaves was up-regulated in darkness and during aging. Plants of usr1, an AtUSR1 gene knock-down mutant, showed a significant delay in age-dependent and dark-induced leaf senescence and the delayed senescence phenotype was rescued when the AtUSR1 gene was transferred back to the mutant plants. Meanwhile, overexpression of AtUSR1 caused accelerated leaf senescence. Furthermore, the role of AtUSR1 in regulating leaf senescence is related to MYC2-mediuated jasmonic acid (JA) signaling pathway. MeJA treatments promoted the accumulation of AtUSR1 transcripts and this expression activation was dependent on the function of MYC2, a key transcription factor in JA signaling. Dual-luciferase assay results indicated that MYC2 promoted the expression of AtUSR1. Overexpression of AtUSR1 in myc2 mutant plants showed precocious senescence, while myc2 mutation alone caused a delay in leaf senescence, suggesting that AtUSR1 functions downstream to MYC2 in the JA signaling pathway in promoting leaf senescence.

Keywords: AtUSR1; JA; MYC2; leaf senescence; ring/U-box.

FIGURE 1

The expression pattern of AtUSR1. (A) The transcript level of AtUSR1 was enhanced in an age dependent manner and reached its peak at the ES stage. According to the description of Guo and Gan (2006), gene expression was detected at four different stages of leaf senescence in Arabidopsis. YL, young leaves; NS, fully expanded mature leaves without senescence symptoms; ES, early senescent leaves; and LS, late senescent leaves. The relative values of gene expression (AtUSR1 and SAG12) were calculated based on comparison with gene expression from YL, which was set as 1. (B) The expression pattern of AtUSR1 in indifferent parts of a yellowing leaf; B, Base; M, Middle; and T, Tip. (C) The expression pattern of AtUSR1 under dark conditions. The relative values of gene expression were calculated based on comparison with gene expression from B, which was set as 1. (D) Histochemical staining of GUS activity detection in leaves at different timepoints as indicated. (E) The expression of AtUSR1 was induced by MeJA and ABA treatments. * and ** indicate significant difference at 0.01 < P < 0.05 and P < 0.01 levels using student’s t-test. Data are shown as the mean ± SD from three independent experiments. Significance analysis was only performed on AtUSR1 expression.

FIGURE 2

The usr1 mutant plants show delayed leaf senescence. (A) The senescence phenotype of plants with different genotype as indicated after grown under continuously light for 40 days. usr1 mutant shows delayed leaf senescence and complementation plants proUSR1::USR1/usr1 rescued the delayed leaf senescence phenotype of usr1 plants. (B) Senescence phenotypes of detached 1st–12th leaves of different genotypes as indicated. (C,D) Chlorophyll content and Fv/Fm detection of leaves from different leaf positions in Col-0, usr1, and proUSR1::USR1/usr1 plants as indicated. (E) Expression of SAGs genes including SAG12, SAG13, and RBCS in plants of different genotypes as indicated. Single and double asterisk indicate significant difference at 0.01 < P < 0.05 and P < 0.01 levels using student’s t-test. Data are shown as the mean ± SD from three independent experiments.

FIGURE 3

AtUSR1 overexpression accelerates leaf senescence. (A) The precocious senescence phenotype of AtUSR1 overexpression lines after grown under continuously light condition for 40 days. (B) The phenotype of 1st–12th leaves detached from plants with different genotypes. (C,D) Chlorophyll content and Fv/Fm detection at different leaf positions in Col-0, two independent AtUSR1 overexpression plants as indicated. (E) Expression pattern of SAG12, SAG13, and RBCS in plants overexpressing AtUSR1 and in Col-0. Single and double asterisk indicate significant difference at 0.01 < P < 0.05 and P < 0.01 levels using student’s t-test. Data are shown as the mean ± SD from three independent experiments.

FIGURE 4

AtUSR1 is involved in dark-induced leaf senescence. (A) AtUSR1 Overexpression lines displayed precocious leaf senescence while the usr1 mutant showed delayed senescence under dark conditions. (B,C) Total chlorophyll content and Fv/Fm of different genotypes as indicated. The data in (B,C) different letters above columns indicate significant differences according to Duncan’s multiple range test (P < 0.05). Data are shown as the mean ± SD from three independent experiments.

FIGURE 5

AtUSR1 affects ROS accumulation. (A) The NBT staining results of leaves from different genotypes. (B) Accumulation of H₂O₂ in detached leaves of different genotypes as indicated. *Indicate significant difference at 0.01 < P < 0.05 levels using student’s t-test. Data are shown as the mean ± SD from three independent experiments.

FIGURE 6

AtUSR1 plays as positive role in JA mediated leaf senescence. (A) AtUSR1 overexpression enhanced JA-induced leaf senescence while usr1 mutation delayed this process. Detached Sixth leaves of different genotypes were treated with 50 μM MeJA for 5 days. (B,C) Total chlorophyll content and Fv/Fm in the leaf samples of different genotypes as indicated. The data in (B,C) different letters above columns indicate significant differences according to Duncan’s multiple range test (P < 0.05). Data are shown as the mean ± SD from three independent experiments.

FIGURE 7

AtUSR1 functions downstream to MYC2. (A) The expression pattern of AtUSR1 in Col-0 and the myc2 mutant under 100 μM MeJA treatments. (B) Schematic diagram of the reporter and effector constructs used in the dual luciferase assay. (C) MYC2 increased the activities of the AtUSR1 promoter. The promoter of AtUSR1 containing an E-box (CAGCGT) or a mutant version of the E-box (AAAAAA) fused with LUC was co-transformed with an effecter construct with or without 35S::MYC2 into Col-0 protoplasts. Ren LUC activity acted as internal control. The experiments presented here were done only with Col-0 plants (labeled as –MYC2). (D) MeJA induced the expression of AtUSR1 and this induction was MYC2-dependent. The reporter construct (proAtUSR1::LUC) was transformed into protoplasts of Col-0 or myc2, respectively. Ren Luc acted as an internal control. (E) The cross line myc2/35S::USR1 rescued the delayed senescence phenotype of myc2 under MeJA treatments. (F) Chlorophyll contents in different genotypes as indicated. (G) Membrane leakage rates in different genotypes as indicated. **Indicate significant difference at P < 0.01 levels using student’s t-test. Data are shown as the mean ± SD from three independent experiments. The data in (F,G) different letters above columns indicate significant differences according to Duncan’s multiple range test (P < 0.05).