Loss of function of Arabidopsis C-terminal domain phosphatase-like1 activates iron deficiency responses at the transcriptional level

Abstract

The expression of genes that control iron (Fe) uptake and distribution (i.e. Fe utilization-related genes) is tightly regulated. Fe deficiency strongly induces Fe utilization-related gene expression; however, little is known about the mechanisms that regulate this response in plants. Transcriptome analysis of an Arabidopsis (Arabidopsis thaliana) mutant defective in RNA polymerase II C-terminal domain-phosphatase-like1 (CPL1) revealed significant up-regulation of Fe utilization-related genes (e.g. IRON-REGULATED TRANSPORTER1), suggesting the importance of RNA metabolism in Fe signaling. An analysis using multiple cpl1 alleles established that cpl1 mutations enhanced specific transcriptional responses to low Fe availability. Changes in protein level were less prominent than those in transcript level, indicating that cpl1-2 mainly affects the Fe deficiency response at the transcriptional level. However, Fe content was significantly increased in the roots and decreased in the shoots of cpl1-2 plants, indicating that the cpl1 mutations do indeed affect Fe homeostasis. Furthermore, root growth of cpl1-2 showed improved tolerance to Fe deficiency and cadmium (Cd) toxicity. cpl1-2 plants accumulated more Cd in the shoots, suggesting that Cd toxicity in the roots of this mutant is averted by the transport of excess Cd to the shoots. Genetic data indicate that cpl1-2 likely activates Fe deficiency responses upstream of both FE-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent and -independent signaling pathways. Interestingly, various osmotic stress/abscisic acid (ABA)-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. We propose that the cpl1 mutations enhance Fe deficiency signaling and promote cross talk with a branch of the osmotic stress/ABA signaling pathway.

Figure 1.

Hierarchical clustering of CUTs based on their differential expression during abiotic stress. The expression levels of 109 CUTs in cpl1-2 plants and in wild-type plants subjected to Fe deficiency (Fe-free nutrient solution for 24 h; Gene Expression Omnibus accession GSE15189), ABA (1 µm ABA for 3 h; NASCArray accession 176), salt (130 mm NaCl for 6 h; NASCArray accession 139), or mannitol (300 mm mannitol for 12 h; NASCArray accession 140) were subjected to hierarchical clustering using the Pearson correlation distance and average linkage rule.

Figure 2.

Venn diagram representing the differential regulation of CUTs by abiotic stresses. The expression levels of CUTs were examined in public microarray data sets for Fe deficiency, ABA, and osmotic stress (mannitol or drought) treatments (see text and Supplemental Table S3).

Figure 3.

Time course of expression levels of Fe utilization-related genes in the roots of cpl1-2 and C24 under Fe deficiency. Plants were grown on basal medium for 7 d and then transferred to Fe-deficient basal medium containing 300 µm ferrozine (see “Materials and Methods”). Root samples were collected at the time of transfer (0) or at 12, 24, 48, or 72 h or 7 d after the transfer. The presented expression levels (relative to untreated C24 samples) are mean values of three biological replicates analyzed in duplicate. Error bars indicate se of biological replicates. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 4.

Basal expression levels of IRT1, FIT, FRO2, and LEA family proteins under different Fe concentrations. Plants were grown for 10 d on medium containing one-quarter-strength MS salts adjusted to the indicated concentrations of Fe-EDTA, 0.5% Suc, and 1.5% agar. 0_FZN indicates medium without Fe-EDTA but containing 300 µm ferrozine. Total RNA was extracted from whole plants. The presented expression levels (relative to C24 samples collected from medium containing 50 µm Fe-EDTA) are mean values of three biological replicates analyzed in duplicate. Error bars indicate the se of biological replicates. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 5.

FRO activity and IRT1 protein accumulation in roots of cpl1-2 and C24 under Fe-sufficient or -deficient conditions. Plants were grown on basal medium for 7 d and then transferred to Fe-sufficient (Fe+; 50 µm Fe-EDTA) or Fe-deficient (Fe−; 0 µm Fe-EDTA + 300 µm ferrozine) basal medium. A, FRO2 activity after 3 d of treatment. The presented values are means of three biological replicates, each consisting of six technical repeats. Bars indicate the se of biological replicates. FW, Fresh weight. B, IRT1 protein accumulation after 3 d of treatment. Twenty micrograms of total protein was analyzed by immunoblot using anti-IRT1 antibodies (αIRT1). An anti-actin antibody (αACT [A2066; Sigma]) was used as the loading control. Average band intensities of three experiments (±se) are given in arbitrary units. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 6.

Metal contents of cpl1-2 and C24 roots and shoots under Fe-sufficient or -deficient conditions. Plants were grown on basal medium for 7 d and then transferred to Fe-sufficient (Fe+; 50 µm Fe-EDTA) or Fe-deficient (Fe−; 0 µm Fe-EDTA + 300 µm ferrozine) basal medium. After 3 d, root and shoot tissues were collected separately and dried at 65°C for 48 h, and elemental levels were determined from 100 mg of dried tissues by ICP-MS analysis. The presented elemental levels are mean values of three biological replicates analyzed in triplicate. Error bars indicate the se of biological replicates. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 7.

Primary root growth of C24 and cpl1 mutants under different levels of available Fe. Four-day-old seedlings of C24, cpl1-1, and cpl1-2 were subjected to Fe deficiency as described in “Materials and Methods.” A, Root growth after 5 d of treatment. Root tip positions are marked by arrowheads. Dashed lines show the positions of the roots at the time of transfer. Bars = 10 mm. B, Root length was quantified to calculate relative root growth of the wild type and the cpl1 mutants. Error bars indicate the se of three biological replicates, each consisting of 20 seedling measurements. *P < 0.05 by Student’s t test between mean values of C24 and each mutant under Fe deficiency conditions. Fe+, 50 µm Fe-EDTA; Fe−, 0 µm Fe-EDTA + 300 µm ferrozine.

Figure 8.

Cd resistance of cpl1-2. Primary root growth (A) and Cd levels in the roots (B) and shoots (C) are shown for C24 and cpl1-2 growing on medium containing various levels of Fe and Cd. Seeds were germinated and grown for 10 d on one-quarter-strength MS medium adjusted to the indicated concentrations of Fe-EDTA and CdCl₂. The presented root lengths are means of three biological replicates, each consisting of 20 seedlings. The presented Cd levels are means of three biological replicates analyzed in triplicate. Error bars indicate the se of biological replicates. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 9.

Expression levels of FIT-LUC, FIT, IRT1, FRO2, and group Ib bHLH transcription factors in Col-0, cpl1-6, fit-2, and fit-2 cpl1-6 plants containing the FIT-LUC reporter gene. Plants were grown on basal medium for 7 d and transferred to Fe-sufficient (Fe+; 50 µm Fe-EDTA) or Fe-deficient (Fe−; 0 µm Fe-EDTA + 300 µm ferrozine) basal medium. Total RNA was extracted from root tissue after 3 d of treatment. The presented expression levels (relative to untreated Col-0 samples) are mean values of biological duplicates analyzed in duplicate. Error bars indicate the se of biological replicates. Different letters show significant differences between genotypes under Fe+ and Fe− conditions (P < 0.05 by one-way ANOVA followed by Tukey’s HSD post hoc test). *P < 0.05 by Student’s t test between mean values of Fe+ and Fe− for the same genotype.

Figure 10.

CPL1 was expressed in the root tip and stele. Plants expressing the CPL1-GUS fusion protein were grown on basal medium for 7 d and transferred to Fe-sufficient (50 µm Fe-EDTA; A–C) or Fe-deficient (0 µm Fe-EDTA + 300 µm ferrozine; D–F) basal medium. After 3 d, GUS activity was visualized and documented. Bars = 10 mm in A and D; 2 mm in B, C, E, and F; and 0.05 mm in C, inset.

Figure 11.

The expression of LEA family transcripts in response to ABA or Fe deficiency treatments. Plants were grown on basal medium for 7 d and transferred to basal medium containing 1 µm ABA or to Fe-deficient basal medium (0_FZN; 0 µm Fe-EDTA + 300 µm ferrozine). The duration of treatment was 1 h for ABA and 72 h for Fe deficiency. Total RNA was extracted from roots. The presented expression levels (relative to untreated C24 samples) are mean values of biological triplicates analyzed in duplicate. Error bars indicate the se of biological replicates. *P < 0.05 by Student’s t test between mean values of cpl1-2 and C24 for the same conditions.

Figure 12.

Model for the role of CPL1. A, Role of CPL1 in root-shoot Fe distribution. CPL1 in the stele likely promotes the root-to-shoot transport of Fe and attenuates the Fe deficiency signal in shoots. Communication between different cell types in shoot tissues is omitted in this model. B, Coregulation of a subset of osmotic stress/ABA response genes by ABA and Fe deficiency signals and its attenuation by CPL1. Solid and broken arrows indicate pathways that operate during Fe deficiency and in the presence of ABA, respectively. White bars indicate the repression activity of CPL1, which is absent in cpl1 mutants. CPL1 attenuates the Fe deficiency signaling pathway upstream of FIT and group Ib bHLH. Direct targets of CPL1 in the pathways are currently unknown. B3 family transcription factors up-regulate the expression of a subset of osmotic stress/ABA-responsive genes through the RY motif in response to Fe deficiency and ABA signaling. ABA also promotes gene expression via the ABA-responsive element. When ABA levels are elevated, the enhanced expression of ABA-responsive genes causes slow growth, which inhibits the expression of Fe utilization genes.