Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles

Abstract

Copper is an essential cofactor for key processes in plants, but it exerts harmful effects when in excess. Previous work has shown that the Arabidopsis (Arabidopsis thaliana) COPT1 high-affinity copper transport protein participates in copper uptake through plant root tips. Here, we show that COPT1 protein localizes to the plasma membrane of Arabidopsis cells and the phenotypic effects of transgenic plants overexpressing either COPT1 or COPT3, the latter being another high-affinity copper transport protein family member. Both transgenic lines exhibit increased endogenous copper levels and are sensitive to the copper in the growth medium. Additional phenotypes include decreased hypocotyl growth in red light and differentially affected flowering times depending on the photoperiod. Furthermore, in the absence of environmental cycles, such as light and temperature, the survival of plants overexpressing COPT1 or COPT3 is compromised. Consistent with altered circadian rhythms, the expression of the nuclear circadian clock genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) is substantially reduced in either COPT1- or COPT3-overexpressing plants. Copper affects the amplitude and the phase, but not the period, of the CCA1 and LHY oscillations in wild-type plants. Copper also drives a reduction in the expression of circadian clock output genes. These results reveal that the spatiotemporal control of copper transport is a key aspect of metal homeostasis that is required for Arabidopsis fitness, especially in the absence of environmental cues.

Figure 1.

Arabidopsis COPT1 protein localizes to the plasma membrane. Arabidopsis protoplasts transiently transformed with the COPT1-GFP construct and analyzed by confocal microscopy 16 h after transformation are shown. The nontransformed protoplasts were used as a negative control, while the protoplasts transformed with a plasmid expressing GFP alone were used as a positive control. Green and magenta fluorescences are indicative of GFP protein and chlorophyll localization, respectively. Images show representative protoplasts at the same scale, including the merges and the light fields.

Figure 2.

Obtaining and selection of COPT1- and COPT3-overexpressing Arabidopsis plants. A, Complementation of the growth phenotype of the S. cerevisiae ctr1Δctr3Δ mutant by the COPT1-HA and COPT3-HA constructs. Yeast cells transformed with an empty vector (p426GPD; negative control), a vector containing yeast CTR1 or Arabidopsis COPT1 and COPT3 (positive controls), or the COPT1-HA and COPT3-HA constructs were assayed for growth on Glc, glycerol, or glycerol plus 100 μm CuSO₄ (Cu). Cells were spotted in 10-fold serial dilutions starting at A₆₀₀ = 0.1. B, Detection of COPT1-HA-fused protein in the transgenic plants. Western-blot analysis of the total protein extracts from leaves of the wild type (WT) and five independent transgenic C1^OE lines. C, Detection of COPT3-HA-fused protein in the transgenic plants. Western-blot analysis of the total protein extracts from leaves of the wild type and five independent transgenic C3^OE lines. The western blots shown in B and C were developed with commercial antibody against the HA epitope.

Figure 3.

Expression levels and endogenous Cu contents in COPT1- and COPT3-overexpressing Arabidopsis seedlings. A, Quantification by real-time RT-PCR of the COPT1 mRNA expression levels from the wild-type (WT; plain bars) and C1^OE (striped bars) seedlings grown on MS plates either without (MS; white bars) or with 10 μm CuSO₄ (Cu; gray bars) under 6 neutral days with photocycle and thermocycle. The UBQ10 gene was used as a loading control. Values are means ± sd of at least three technical replicates. a.u., Arbitrary units; R.E., relative expression. B, Quantification by real-time RT-PCR of the COPT3 mRNA expression levels, as indicated in A, except C3^OE (dotted bars). C, Cu content from the seedlings shown in A and B. Values are means ± sd of at least three technical replicates. D.W., Dry weight. Asterisks indicate statistically significant differences with respect to the wild type (* P < 0.05, ** P < 0.01).

Figure 4.

Cu sensitivity of COPT1- and COPT3-overexpressing Arabidopsis seedlings. A, Wild-type (WT), C1^OE, and C3^OE seedlings were grown vertically on MS plates either without (MS) or with 10 μm CuSO₄ (Cu) under 6 neutral days with photocycle and thermocycle. The inset shows an amplification of a representative C1^OE seedling in Cu to show the root morphology. B, Quantification of the root length of the wild-type (white circles), C1^OE (black squares), and C3^OE (black triangles) seedlings grown vertically on MS plates containing 0, 10, 20, or 50 μm CuSO₄, as indicated in A. Values are seedling means ± sd (n ≥ 7) from three independent experiments. C, Quantification of the fresh weight (F.W.) of the wild-type and C1^OE seedlings grown on MS plates containing 0 or 30 μm Cu, Fe, Zn, cadmium (Cd), or Ag sulfates, as indicated in A. Bars represent the percentage of fresh weight per seedling of the C1^OE line with respect to the wild type as means ± sd of at least three independent experiments. Asterisks indicate statistically significant differences with respect to MS (* P < 0.05, ** P < 0.01). [See online article for color version of this figure.]

Figure 5.

Phenotypes of COPT1-overexpressing Arabidopsis plants grown on soil. A, Four-week-old wild-type (WT) and C1^OE plants grown on soil. B, Two-week-old wild-type and C1^OE plants grown on soil. Arrows point to the first pair of rosette leaves of the C1^OE line. C, Fully expanded leaves from wild-type and C1^OE plants. D, Flowers of wild-type and C1^OE plants. Flowers are shown as either complete (left) or after eliminating frontal petals and sepals (right). Arrows point to the flower stamens. Images show representative plants, leaves, and flowers. [See online article for color version of this figure.]

Figure 6.

Effects of the cycling conditions on the COPT1- and COPT3-overexpressing Arabidopsis seedlings. A, Wild-type (WT), C1^OE, and C3^OE seedlings grown vertically on MS plates either without (MS) or with 10 μm CuSO₄ (Cu) under 6 neutral days with photocycle and thermocycle (LDHC) or in the absence of both (LLHH). B, Quantification of the fresh weight (F.W.) of the wild-type (plain bars), C1^OE (striped bars), and C3^OE (dotted bars) seedlings grown on MS (white bars) or Cu (gray bars), as indicated in A. Values indicate fresh weight per seedling as means ± sd (n ≥ 7) from three independent experiments. C, Quantification of the total chlorophyll content of the seedlings, as indicated in B. Values indicate the total chlorophyll content per fresh weight as means ± sd of at least three independent experiments. D, Quantification of the lipid peroxidation of the seedlings, as indicated in B. Values are MDA contents per fresh weight as means ± sd of at least two independent experiments. E, Quantification of total anthocyanin contents of the seedlings, as indicated in B. Values indicate total anthocyanin contents per fresh weight as means ± sd of at least two independent experiments. Asterisks indicate statistically significant differences with respect to the wild type (* P < 0.05, ** P < 0.01). [See online article for color version of this figure.]

Figure 7.

Regulation of the gene expression of circadian clock components and clock output genes in the COPT1- and COPT3-overexpressing Arabidopsis seedlings. Quantification by real-time RT-PCR of the CCA1, LHY, LHCB1.1, and COL1 mRNA expression levels from the wild-type (WT; plain bars), C1^OE (striped bars), and C3^OE (dotted bars) seedlings grown on MS plates either without (MS; white bars) or with 10 μm CuSO₄ (Cu; gray bars) under 6 neutral days with photocycle and thermocycle. Samples were taken at Zeitgeber time 0 h. The UBQ10 gene was used as a loading control. Values are means ± sd of three technical replicates. a.u., Arbitrary units; R.E., relative expression. Asterisks indicate statistically significant differences with respect to the wild type (* P < 0.05, ** P < 0.01).

Figure 8.

Cu regulation of the circadian clock and Cu homeostasis genes in Arabidopsis seedlings. A, RT-PCR products from total RNA of the wild-type seedlings grown on MS plates either without (MS; white circles) or with 10 μm CuSO₄ (Cu; black squares) under 6 neutral days with photocycle and thermocycle. Samples were taken every 4 h during the following 48 h. The relative intensity of the electrophoretic band for each gene versus the 18S band, used as a loading control, is represented. The experiments were repeated at least three times with similar results, and a representative experiment is shown. ZT, Zeitgeber time. The white and black bars at bottom indicate day and night, respectively. a.u., Arbitrary units; R.E., relative expression. B, RT-PCR products from total RNA of wild-type seedlings grown on MS plates with 0.2, 1, 10, or 50 μm CuSO₄ under 6 neutral days with photocycle and thermocycle. Samples were taken at Zeitgeber time 0 h. 18S rRNA was used as a loading control.

Figure 9.

Cu regulation of luciferase activity driven by the LHY promoter in Arabidopsis seedlings. A, Bioluminescence from the PLHY:luc seedlings grown on MS plates without (MS; white circles), with 5 μm CuSO₄ (gray circles), or with 10 μm CuSO₄ (black circles), entrained under 7 neutral days with photocycle and thermocycle, and transferred to continuous light. Bioluminiscence was measured every hour after transferring to continuous light. Values are seedling averages (n = 4–7). White and gray bars at bottom indicate subjective day and night, respectively. B, Bioluminescence from PLHY:luc seedlings grown on MS plates either without (MS; white circles) or with 10 μm CuSO₄ (Cu; black circles), entrained under 7 neutral days with photocycle and thermocycle, and transferred to continuous dark. Bioluminiscence was measured every hour after transferring to continuous dark. Values are seedling averages (n = 7–10). Gray and black bars at bottom indicate subjective day and night, respectively.

Figure 10.

Cu regulation of the gene expression of circadian clock components and clock output genes in the Arabidopsis seedlings. A, Quantification by real-time RT-PCR of the CCA1, LHY, LHCB1.1, and COL1 mRNA expression levels from wild-type seedlings grown on MS plates either without (MS; white bars) or with 10 μm CuSO₄ (Cu; gray bars), entrained under 6 neutral days with photocycle and thermocycle, and transferred to continuous light. Samples were taken at Zeitgeber time 0 h on the subjective day for continuous light cycle 2 (LC2) and cycle 3 (LC3) after transferring. The UBQ10 gene was used as a loading control. Values are technical replicate means ± sd (n ≥ 6) from two independent experiments. a.u., Arbitrary units; R.E., relative expression. Asterisks indicate statistically significant differences with respect to the wild type (* P < 0.05, ** P < 0.01).