The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis

Abstract

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development and involves the extensive reprogramming of gene expression. Despite the importance of senescence in plants, the underlying regulatory mechanisms are not well understood. This study reports the isolation and functional analysis of RAV1, which encodes a RAV family transcription factor. Expression of RAV1 and its homologues is closely associated with leaf maturation and senescence. RAV1 mRNA increased at a later stage of leaf maturation and reached a maximal level early in senescence, but decreased again during late senescence. This profile indicates that RAV1 could play an important regulatory role in the early events of leaf senescence. Furthermore, constitutive and inducible overexpression of RAV1 caused premature leaf senescence. These data strongly suggest that RAV1 is sufficient to cause leaf senescence and it functions as a positive regulator in this process.

Fig. 1.

Differential expression of the RAV1 gene during maturation and leaf senescence. (A) For SSH, leaves at 10 DAE and 20 DAE were used as materials for driver and tester cDNA, respectively. Ten-DAE leaves are at the mature green stage (MG), and 20-DAE leaves are at the early senescence stage (S1), when the chlorophyll content is approximately 80% of initial values. Error bars indicate standard deviation (SD, n=25). (B) Age-dependent changes in RAV1 gene expression. Total RNA was extracted at 10, 12, 14, 18, 20, 22, and 26 DAE from the third and fourth rosette leaves of Col plants. Samples were subjected to RNA gel blot analysis using RAV1, CAB2, SAG12, and SEN4 cDNAs as hybridization probes. EtBr staining was used as a loading control.

Fig. 2.

Molecular analysis of RAV1. (A) Schematic of the domain organization of RAV1. (B) Nuclear localization of RAV1-GFP. Transient expression of 35S::RAV1-GFP in Arabidopsis protoplasts. Shown are a bright field image (a), a GFP fluorescence image for RAV1-GFP (b), an RFP fluorescence image for RFP-IAA3/SHY2 (c), and a merging of the two fluorescence images (d) of a representative Arabidopsis protoplast. IAA3/SHY2 was used as a control for nuclear localization. (C) Predicted amino acid sequence of RAV1 [NP_172784] and alignment with other sequences encoded by the RAV gene family (TEM1 [NP_173927], RAV2 [NP_564947], and RAV3 [NP_189201]). The nuclear localization signal (NLS) and two DNA binding motifs (AP2 and B3) are indicated by lines.

Fig. 3.

Expression of RAV homologues in response to senescence-inducing factors. Temporal expression of RAV genes during leaf development (A), and in response to ACC (B) and MJ (C). Total RNA was isolated from leaves harvested at the indicated days (A) or the indicated times after treatment (B and C). Transcripts were analysed by RT-PCR using primers specific to RAV1, TEM1, or RAV3. CAB2 and SAG12, ERF, and PDF were used as responsive genes for age-dependent senescence, ACC treatment, and MJ treatment, respectively. ACT2 was used as an internal control for cDNA. –, without treatment; +, with treatment.

Fig. 4.

Accelerated age-dependent leaf senescence phenotypes of constitutive RAV1 overexpressor lines. (A) Expression of RAV1 in three independent transgenic lines carrying a 35S::RAV1 construct, compared with wild-type Col plants. RAV1 expression was analysed by RNA gel blot analysis. (B) Age-dependent senescence phenotype of the fourth rosette leaves of wild-type (Col) plants and two RAV1 overexpressors. (C, D) Chlorophyll content (C) and photochemical efficiency of PSII (D) were examined in leaves of the indicated ages. Chlorophyll content and photochemical efficiency as compared with initial values of each line at 10 DAE are shown. F_v/F_m, maximum quantum yield of PSII electron transport (maximum variable fluorescence/maximum yield of fluorescence). Error bars, SD (n=25). (E) Age-dependent changes in gene expression. Total cellular RNA was isolated at the indicated DAE from wild-type leaves and RAV1 overexpressors, and RNA blots were hybridized with CAB2, SEN4, and SAG12.

Fig. 5.

Premature senescence symptoms in constitutive overexpressors of RAV1 during senescence accelerated by darkness or plant hormones. (A, B) The darkness-induced senescence phenotype of detached leaves of wild-type Col and RAV1-overexpressing plants. The third and fourth rosette leaves were detached at the age of 10 DAE and incubated in darkness. Chlorophyll content (A) and the photochemical efficiency of PSII (B) were monitored during the incubation in darkness. Error bars, SD (n=25). (C, D) Senescence symptoms of the RAV1 overexpressors during senescence induced by phytohormones (ACC, ABA, or MJ). The third and fourth rosette leaves were detached at the age of 10 DAE and incubated in continuous light with 50 μM ACC, 50 μM ABA, or 100 μM MJ for 2 d. Chlorophyll content (C) and photochemical efficiency (D) are shown as mean ±SD (n=25), relative to those of leaves incubated in light without hormones. Asterisks indicate values that are statistically different from the Col plants (Student's t test; P <0.05).

Fig. 6.

Inducible overexpression of RAV1 causes precocious age-dependent leaf senescence. (A) RT-PCR analysis with RAV1-specific primers of Col plants and two independent transgenic lines carrying the GVGpro::RAV1/35S::GVG construct 8 h after 15 μM DEX treatment. (B) Visible phenotypes of wild-type Col and transgenic plants harbouring GVGpro::RAV1/35S::GVG after treatment with MES or DEX. Pictures of representative plants were taken 6 d after DEX treatment. Chlorophyll content (C), photochemical efficiency (D), and ion leakage (E) were measured at the indicated time after DEX treatment. Error bars, SD (n=12). Asterisks indicate values that are statistically different from the Col plants (Student's t test; P <0.05). (F) Expression of SAG genes was observed 6 d after DEX treatment. –, Without DEX treatment; +, with DEX treatment.

Fig. 7.

Inducible RAV1 overexpression is sufficient to accelerate darkness-induced leaf senescence symptoms. Photochemical efficiency (A), chlorophyll content (B), and ion leakage (C) were measured 2 d or 4 d after DEX treatment. –, Without DEX treatment; +, with DEX treatment. Error bars, SD (n=12). Asterisks indicate values that are statistically different from the Col plants (Student's t test; P <0.05).