Arabidopsis ERF1 Mediates Cross-Talk between Ethylene and Auxin Biosynthesis during Primary Root Elongation by Regulating ASA1 Expression

Abstract

The gaseous phytohormone ethylene participates in the regulation of root growth and development in Arabidopsis. It is known that root growth inhibition by ethylene involves auxin, which is partially mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE α1 (WEI2/ASA1), encoding a rate-limiting enzyme in tryptophan (Trp) biosynthesis, from which auxin is derived. However, the molecular mechanism by which ethylene decreases root growth via ASA1 is not understood. Here we report that the ethylene-responsive AP2 transcription factor, ETHYLENE RESPONSE FACTOR1 (ERF1), plays an important role in primary root elongation of Arabidopsis. Using loss- and gain-of-function transgenic lines as well as biochemical analysis, we demonstrate that ERF1 can directly up-regulate ASA1 by binding to its promoter, leading to auxin accumulation and ethylene-induced inhibition of root growth. This discloses one mechanism linking ethylene signaling and auxin biosynthesis in Arabidopsis roots.

Fig 1. ERF1 expression is responsive to ethylene.

(a) Ethylene-induced ERF1 expression in wildtype. Seeds of Col-0 were germinated on MS medium for 5 d then treated with 10 μM ACC for 0, 0.5, 1, 3, 6, and 12 h. The transcriptional level of ERF1 was detected by quantitative RT-PCR (qRT-PCR). Values are mean ± SD of three replicates. ACC, 1-aminocyclopropane-1-carboxylic acid (precursor of ethylene biosynthesis). (b) Ethylene-induced expression of ERF1pro:GUS. Five-day-old seedlings of transgenic lines of were treated with 10 μM ACC for 0, 3, and 6 h before GUS staining. Upper DZ and lower DZ represent different primary root regions. Scale bar, 0.5 cm. (c) Ethylene-induced expression of ERF1 in wildtype. Seeds of Col-0 were germinated on MS medium with 0 or 0.8 μM ACC for 5 d, and relative ERF1 transcription levels measured by qRT-PCR. Values are mean ± SD of three replicates (***P<0.001). Asterisks indicate Student’s t-test significant differences. (d) Ethylene-activated expression in ERF1_pro:GUS lines. Transgenic plants were grown MS medium with either 0 or 0.8 μM ACC for 5 d before GUS staining assay. Scale bar, 0.5 cm. (e) The relative ERF1 expression level was determined in ethylene signaling related mutants ein2-5, ein3-1, ein3-1eil1and compared to wildtype (Col-0) seedlings. Seedlings carrying constructs for either constitutive (ctr1-1) or inducible EIN3-FLAG (iE/qm) (EIN3ox) expression were also examined. Seedlings were geminated on MS medium with either 0 or 0.8 μM ACC for 5 d. Seeds of EIN3ox were grown on medium containing 1 μM β-estradiol and 0 or 0.8 μM ACC. Roots of seedlings were used for qRT-PCR analysis. Values are mean ± SD of three replicas (***P<0.001). Asterisks indicate Student’s t-test significant differences.

Fig 2. Role of ERF1 in ethylene-mediated inhibition of primary root elongation.

(a-c) Primary root phenotypes of ERF1 knockdown (RNAi-1, RNAi-2) and overexpression (ERF1ox #2, ERF1ox #6, ERF1ox #12) transgenic lines. The expression level of ERF1 in 5 day-old-seedlings was tested by qRT-PCR (a). Values are mean ± SD of three replicas (*P<0.05, ***P<0.001). Asterisks indicate Student’s t-test significant differences. The representative seedlings were photographed (b). Scale bar, 1 cm. The primary root length of these related transgenic lines and Col-0 was measured from 4 to 8 d (c). Data shown are average and SD (Values are mean±SD, n = 20). (d-f) Epidermal cell length within the DZ was affected in ERF1 knockdown (RNAi-1, RNAi-2) and overexpression (ERF1ox #2, ERF1ox #6, ERF1ox #12) transgenic lines. The primary root MZ and DZ of the 5-day-old seedlings of indicated lines were photographed. Representative images are shown (d). Root meristem cell number (e) and epidermal cell length of the maturation zone (f) were measured. The root meristem cell number was counted from the QC to the first elongation cell in the cortex file. The mean ± SD (n = 20) is shown, **P<0.01, ***P<0.001). Asterisks indicate Student’s t-test significant differences. (g) Primary root length of ERF1 knockdown (RNAi-1, RNAi-2) and overexpression lines grown on medium with or without ACC treatment. Five-day-old seedlings grown on MS medium were transferred to MS medium containing 0 or 0.2 μM ACC and growth vertically for 3 d. The mean ± SD is shown (n = 50).

Fig 3. ERF1 enhances auxin accumulation in roots.

(a) DR5:GUS expression in 5-day-old seedlings of Col-0,ERF1 knockdown (RNAi-1, RNAi-2) and overexpression lines (ERF1ox #2, ERF1ox #6, ERF1ox #12). Three independent experiments were done, and each replica containing 15 plants for each line. Representative seedlings were photographed. Scale bar, 0.5 cm. (b) DR5:GUS expression in maturation region and root tip of primary root. Five-day-old seedlings of Col-0, ERF1 knock-down (RNAi-1, RNAi-2) and over-expression (ERF1ox #2, ERF1ox #6, ERF1ox #12) lines were treated with 0 or 0.8 μM ACC for 24 h before GUS activity was assayed. More than 20 plants were observed for each line. Representative photos were displayed. (c) Free IAA content. Seeds of ERF1 overexpression and RNAi lines were grown on MS plates for 5 d before root IAA content was measured. The mean ± SD of three replicas is shown (*P<0.05, **P<0.01). Asterisks indicate Student’s t-test significant differences. (d-e) The transcript level of IAA1 and IAA2. Seeds of ERF1 overexpression and RNAi lines were grown on MS plates for 5 d before RNA isolation from the roots. Transcript abundance of IAA1 and IAA2 was measured using qRT-PCR. The mean ± SD of three replicates is shown (*P<0.05, **P<0.01, ***P<0.001). Asterisks indicate Student’s t-test significant differences.

Fig 4. ERF1 directly binds to ASA1 promoter region in vitro and in vivo.

(a) EMSA assay for binding to GCC-box sequence in the promoter of ASA1 by ERF1 protein in vitro. Dig-labelled probes were incubated with ERF1-MBP protein. As indicated, unlabelled probes were used as competitors, unlabelled probes with mutated GCC-box sequence were used as non-competitors, and the ERF1-MBP protein bound probers were separated from free probes by an acrylamide gel. (b) Yeast-one-hybrid assay. pGADT7/ERF1 (AD-ERF1) and pHIS2/ASA1pro (BD-ASA1pro) constructs were co-transformed into yeast strain Y187. AD-empty and BD-empty, AD-empty and BD-ASA1pro, AD-ERF1 and BD-empty, AD-empty and BD-3*GCC-box were used as negative controls while AD-ERF1 and BD-3*GCC-box were used as a positive control. (c) Chromatin immunoprecipitation-PCR for ASA1 promoter. Roots of 5-day-old 35S:HA:ERF1 and Col-0 seedlings were used. Anti-HA antibodies were used for the enrichment of the DNA fragments containing GCC-box in the promoter of ASA1. The results were determined by real-time PCR. Tub8 was used as a negative control (NC). (d) Quantitative real-time PCR was performed using the same ChIP products and PCR primers flanking GCC-boxes in ASA1 promoter as in c. The region of ASA1 that do not contain GCC-box was used as negative control. Values are mean ± SD of three replicas (***P<0.001). Asterisks indicate Student’s t-test significant differences.

Fig 5. ERF1 up-regulates the expression of ASA1 in roots.

(a) Transcript levels of ASA1 in 5-day-old Col-0, ERF1 knockdown (RNAi-1, RNAi-2), and overexpression (ERF1ox #2, ERF1ox #6, ERF1ox #12) seedlings. Values are mean ± SD of three replicas (*P<0.05, **P<0.01, ***P<0.001). Asterisks indicate Student’s t-test significant differences. (b) ASA1_pro:GUS expression in 5-day-old Col-0 and ERF1 knockdown (RNAi-1, RNAi-2) and over-expression (ERF1ox #2, ERF1ox #6, ERF1ox #12) seedlings. Three independent experiments were done, and each replica containing 15 plants for each genotype. Representative seedlings were photographed. Scale bar, 0.5 cm. (c) ASA1_pro:GUS expression in primary root maturation region and root tip of 5-day-old Col-0, ERF1 knockdown (RNAi-1, RNAi-2), and overexpression (ERF1ox #2, ERF1ox #6, ERF1ox #12) seedlings without or with ACC treatment. More than 20 plants were observed for each genotype. Representative photos were displayed.

Fig 6. Root elongation of asa1mutants in response to ACC treatment.

(a) Root elongation of Col-0 and asa1 without and with ACC treatment. CS16398 (asa1-1) and CS16397 (asa1-2) are two different loss-of-ASA1 mutants. Seeds of Col-0 and asa1 were grown on MS plates without or with ACC for 5 d. Three independent experiments were displayed with similar results. Representative seedlings were photographed. Scale bar, 0.5 cm. (b) The primary root length of 5-day-old Col-0 and asa1 mutants grown on medium without or with ACC. Values are mean ± SD of three replicas (*P<0.05, **P<0.01, ***P<0.001). Each replica contains 30 plants for each line. Asterisks indicate Student’s t-test significant differences.

Fig 7. The expression of ASA1 was regulated by ethylene signal pathway.

(a) The expression of ASA1 in ethylene signal pathway mutants. Ethylene signal pathway-related mutants ein2-5, ein3-1, ein3-1eil1, ctr1-1, and Col-0 were grown on MS medium for 5 d while the seeds of EIN3-FLAG (iE/qm) (EIN3ox) were grown on MS medium supplementing with 1 μM β-estradiol for 5 d. Total RNA was extracted from roots. The expression level of ASA1 was checked by qRT-PCR. Values are mean ± SD of three replicas (*P<0.05, **P<0.01, ***P<0.001. Asterisks indicate Student’s t-test significant differences). (b) ASA1_pro:GUS expression in 5-day-old seedlings. Three independent experiments were performed with each replica containing 15 plants for each line. Representative seedlings were photographed. Scale bar, 0.5 cm. (c) ASA1_pro:GUS expression in primary root maturation region and root tip of 5-day-old seedlings without or with ACC treatment. More than 20 plants were observed for each genotype. Representative photos were displayed.

Fig 8. ASA1 acts downstream of ERF1.

(a) The primary root phenotypes of Col-0, asa1-1, ERF1ox and ERF1ox asa1-1 seedlings grown on MS medium with either 0 or 0.1 μM estradiol for 5 d. ERF1ox is the transgenic plants expressing ERF1 protein under control of the estradiol-inducible promoter in Col-0 background. Scale bar, 1 cm. (b) qRT–PCR analysis of transcriptions of ERF1. The roots of 5-day-old Col-0, asa1-1, ERF1ox and ERF1ox asa1-1 seedlings grown on MS medium with either 0 or 0.1 μM estradiol were used. Values are mean ± SD of three replicas (***P<0.001. Asterisks indicate Student’s t-test significant differences). (c-d) Primary root length of Col-0, asa1-1, ERF1ox and ERF1ox asa1-1 seedlings grown on MS medium with either 0 or 0.1 μM estradiol were measured at the fifth days. Data shown are average and SD (Values are mean ± SD, n = 20).