Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives

Abstract

In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of β-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation.

Keywords: Alternative promoter; Arabidopsis halleri; gene regulation; transcript stability; translation; zinc homeostasis..

Fig. 1.

Transcription initiation sites of FRD3 genes. (A, B) Organization of the FRD3 genomic loci and transcripts in A. thaliana (A) and A. halleri (B). For narrow rectangles denoting the 5′UTR, distinct white, grey shading, or black represent alternative segments of the FRD3 sequence. (C) FRD3 transcript variants (FRD3 _L and FRD3 _S) were detected with specific primers by qualitative RT-PCR in shoots of A. thaliana (At), A. lyrata (Al) and A. halleri (Ah). EF1α was used as a control.

Fig. 2.

Localization of FRD3 promoter activity in A. thaliana and A. halleri. Histochemical detection of GUS activity (blue) directed by a full (pAtFRD3_Full) (A–D) and a truncated (pAtFRD3_Trunc) (E–H) A. thaliana FRD3 promoter, or the A. halleri FRD3 (pAhFRD3) promoter (I–L) in whole mounts (A, C, E, G, I, K) and transverse sections (B, D, F, H, J, L) of roots (A, B, E, F, I, J) and leaves (C, D, G, H, K, L) of 3-week-old A. thaliana (top) and A. halleri (bottom) plants. Note that only weak GUS staining was observed for pAtFRD3_Trunc in A. halleri shoots. Bars, 1.5mm (A, E, I), 5mm (C, G, K) and 25 µm (B, D, F, H, J, L). 1, epidermis; 2, cortex; 3, endodermis; 4, pericycle; 5, xylem; 6, stomata; 7, mesophyll, 8, vascular bundle.

Fig. 3.

FRD3 promoter activity in A. thaliana. (A) Specific GUS activity was quantified in total protein extracts from roots and shoots of A. thaliana seedlings expressing the GUS reporter gene under the control of a full-length (pAtFRD3_Full) and a truncated (pAtFRD3_Trunc) A. thaliana FRD3 promoter, or the A. halleri FRD3 (pAhFRD3) promoter. Homozygous seedlings from four independent lines for each construct (T3 generation) were grown on solidified control Hoagland medium. Roots and shoots were harvested separately and pooled per plate (15 seedlings), with two replicate Petri plates per line. Values are mean± SEM with n=4 independent lines from one experiment representative of two independent experiments. MU, 4-methylumbelliferone. (B) Expression analysis of the endogenous AtFRD3 and GUS genes in A. thaliana GUS reporter lines. Steady-state levels of GUS, total AtFRD3 (AtFRD3 _tot), and short AtFRD3 (AtFRD3 _S) transcripts were determined by real-time RT-PCR in 12-d-old A. thaliana seedlings expressing the GUS reporter gene under the control of a full-length (pAtFRD3_Full) and a truncated (pAtFRD3_Trunc) A. thaliana FRD3 promoter, or the A. halleri FRD3 (pAhFRD3) promoter. Values are means of three technical replicates of one representative line for each construct grown on solid Hoagland control medium. RTL, relative transcript level.

Fig. 4.

Dependence of transcript abundance of FRD3 variants on zinc supply in A. thaliana and A. halleri. Steady-state transcript levels for total AtFRD3 (AtFRD3 _tot) (A), AtFRD3 _L (B), AtFRD3 _S (C), and AtFRD3 _S (D) expressed as a percentage of total AtFRD3 transcript levels and for AhFRD3 (E). Steady-state transcript levels were determined in the roots and shoots of A. thaliana and A. halleri cultivated under control conditions (Ctrl), upon zinc deficiency (0 µM Zn) and zinc excess (20 µM Zn for A. thaliana and 300 µM Zn for A. halleri). Values were normalized to EF1α and an inter-run calibrator. The inter-run calibrator differed for each species, and thus transcript levels could only be compared within species. Values are means±SEM of four (A–D) or two (E) independent experiments. Independent experiments included pools of at least 25 A. thaliana seedlings grown on Hoagland agar medium plates (A–D) or six A. halleri plants grown hydroponically in Hoagland medium (E) for each condition. *P<0.05, **P<0.01, and ***P<0.001 according to one-way analysis of variance, followed by Dunnett’s test for multiple comparisons of means. RTL, relative transcript level.

Fig. 5.

FRD3 transcript stability. (A–F) The half-life times of total AtFRD3 (AtFRD3 _tot) (A), AhFRD3 (B), AtFRD3 long (AtFRD3 _L) (C), and short (AtFRD3 _S) (D) transcripts were determined in the presence of the transcriptional inhibitor cordycepin, with AtFER1 (E) and AtSAND (F) included as controls (Ravet et al., 2012). Seedlings (A. thaliana) and root segments (A. halleri) were collected at several time points after the onset of cordycepin treatment. Transcript levels were determined by real-time RT-PCR. Values were normalized to time 0 and fitted by non-linear regression. (G) Transcript half-life (in min), confidence interval at 95%, regression correlation for the fit of the curve (r ²) and P values of an extra-sum-of-squares F comparison test for statistical differences between estimated half-life. Values are means±SEM of nine (A, C, D, E, F) or four (B) independent experiments. Each independent experiment included at least three technical replicates. RTL, relative transcript level.

Fig. 6.

Contributions of the 5′UTR of FRD3 transcript variants to steady-state transcript levels, transcript stability, and levels of the encoded protein. The AtFRD3 _L, AtFRD3 _S, and AhFRD3 5′UTR fused to the GFP coding sequence were transiently expressed in tobacco leaves by Agrobacterium infiltration under the control of a 35S promoter. A 35S:GFP control (Ctrl) lacking a 5′UTR was included in the experimental design. Leaf fragments were harvested 3–4 d post-infiltration. (A) Steady-state GFP transcript levels were normalized to HygB transcript levels generated in vivo from the introduced T-DNA and are given relative to AtFRD3 _S samples. (B) GFP transcript stability (cordycepin assay). Infiltrated leaf fragments were collected at several time points after the onset of cordycepin treatment. GFP transcript levels were determined by real-time RT-PCR. Values were normalized to time 0 and fitted by non-linear regression. (C) Quantification of GFP protein levels in total protein extracts by fluorimetric assay normalized to LUC activity. A 35S:LUC construct was co-infiltrated with all GFP constructs. Different letters above bars indicate significantly different values (P<0.05) according to a t-test. Values are means±SEM of two (A, C) or three (B) independent experiments, respectively. Independent experiments included at least three leaf fragments in three technical replicates. RTL, relative transcript level.

Fig. 7.

Differential transcriptional regulation of FRD3 in zinc-tolerant and zinc-sensitive A. thaliana genotypes. Steady-state transcript levels were determined for the AtFRD3 short (AtFRD3 _S) transcript in roots (A–C) and shoots (D–F) of 21-d-old A. thaliana seedlings grown under control conditions (Ctrl, 1 µM Zn) (B, E), under zinc deficiency (0 µM Zn) (A, D), and under zinc excess (20 µM Zn) (C, F) for 17 d. Bay-0 and NIL-Bay are zinc-tolerant genotypes, whereas Sha and NIL-Sha are zinc-sensitive genotypes (Pineau et al., 2012). Col-0 was included as reference genotype. AtFRD3 _S levels are expressed as a percentage of total AtFRD3 transcripts (see Materials and methods). Total steady-state AtFRD3 transcript levels are given in Supplementary Fig. S8. Values were normalized to EF1α and an inter-run calibrator. Values are means±SEM of two independent experiments. Each independent experiment included at least 25 pooled seedlings per treatment and genotype. Different letters above histogram bars indicate significantly different values (P<0.05) within treatments according to a one-way analysis of variance followed by Dunnett’s test for multiple comparison of means.