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. 2008 Jun 6;283(23):15932-45.
doi: 10.1074/jbc.M801406200. Epub 2008 Apr 11.

MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis

Salah E Abdel-Ghany  1 , Marinus Pilon
Affiliations

MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis

Salah E Abdel-Ghany et al. J Biol Chem. .

Abstract

In plants, copper is an essential micronutrient required for photosynthesis. Two of the most abundant copper proteins, plastocyanin and copper/zinc superoxide dismutase, are found in chloroplasts. Whereas plastocyanin is essential for photo-autotrophic growth, copper/zinc superoxide dismutase is dispensable and in plastids can be replaced by an iron superoxide dismutase when copper is limiting. The down-regulation of copper/zinc superoxide dismutase expression in response to low copper involves a microRNA, miR398. Interestingly, in Arabidopsis and other plants, three additional microRNA families, miR397, miR408, and miR857, are predicted to target the transcripts for the copper protein plantacyanin and members of the laccase copper protein family. We confirmed the predicted targets of miR397, miR408, and miR857 experimentally by cleavage site analysis. To study the spatial expression pattern of these microRNAs and the effect of copper on their expression, we analyzed Arabidopsis grown hydroponically on different copper regimes. On low amounts of copper the plants accumulated miR397, miR408, and miR857. The microRNA expression pattern was negatively correlated with the accumulation of transcripts for plantacyanin and laccases. Furthermore, the expression of other laccases that are not predicted targets for known microRNAs was similarly regulated in response to copper. For some of these laccases, the regulation was disrupted in a microRNA maturation mutant (hen1-1), suggesting the presence of other copper-regulated microRNAs. Thus, in Arabidopsis, microRNA-mediated down-regulation is a general mechanism to regulate nonessential copper proteins. We propose that this mechanism allows plants to save copper for the most essential functions during limited copper supply.

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Figures

FIGURE 1.
FIGURE 1.
Expression analyses of copper proteins in young plantlets. A, immunoblot analysis. Total proteins (20 μg/lane) were extracted from seedlings grown hydroponically for 2 weeks at the indicated copper concentrations and fractionated by 15% SDS-PAGE. Each protein was detected by immunoblotting using a specific antibody. B and C, Northern analysis. 10 μg of total RNA was separated by electrophoresis, transferred to Hybond N+ membranes, and probed with 32P-labeled gene-specific probes.
FIGURE 2.
FIGURE 2.
Effects of copper treatments in mature plants. A, visible symptoms in plants. Images of 5-week-old plants (upper) and of detached leaves at different developmental stages show the copper-deficiency symptoms (lower). B–D, content of copper, iron, and manganese. Metal ion accumulation (μg/g dry weight) in different tissues of Arabidopsis plants grown in hydroponic cultures at different copper concentrations. Data shown represent the mean ± S.D.
FIGURE 3.
FIGURE 3.
Chlorophyll fluorescence analysis. A, chlorophyll fluorescence traces from detached rosette leaves. Dark-adapted rosette leaves were exposed to a saturating light pulse (SP) (2050 μmol of photons m-2 s-1) to estimate Fv/Fm (PSII antennae efficiency). Light-adapted parameters were determined at the indicated actinic light intensities of 43, 350, and 770 μmol photons m-2 s-1 using saturating pulses (SP). A far-red illuminating pulse was initiated after each saturating pulse to establish initial fluorescence (Fo′). Actinic light was switched off, and relaxation of the photosystem was recorded. DA, dark adapted; AU, arbitrary units. B–D, light intensity dependence of chlorophyll fluorescence parameters. B, the relative electron transport rate (ETR) at the indicated light intensities (43, 350, and 770 μmol of photons m-2 s-1) was calculated according to Maxwell and Johnson (39). C, light intensity of non-photochemical quenching (NPQ). D, redox state level of the primary quinone-type electron acceptor, QA, of PSII was determined by a chlorophyll fluorescence parameter, 1-qP. Each point represents the mean ± S.D. (n = 5).
FIGURE 4.
FIGURE 4.
Expression analyses of plastocyanin and superoxide dismutases in hydroponically grown Arabidopsis plants at different copper concentration. A, immuno-detection of plastocyanin isoforms in wild type (WT) plants and in pc1 and pc2 mutants as well as a PC2 over-expressor (O/E). The mutants and over-producer were described before (10). Molecular masses are indicated on the left. B, Western blot analysis. Total soluble proteins (20 μg/lane) were extracted from different tissues (leaf, stem, root, and flower) of Arabidopsis plants grown hydroponically at different copper conditions and fractionated by 15% SDS-PAGE. Each protein was detected by immunoblotting using antibodies specific for the indicated proteins. C, Northern blot analysis. 10 μg of total RNA was separated by electrophoresis, transferred to Hybond N+ membranes, and probed with 32P-labeled gene-specific probes. -Cu, copper-deficient; 5 nm; copper-limited; 50 nm, copper-sufficient. D, immunoblot detection of components of the photosynthetic machinery in leaf tissue. The same leaf samples as in panel B were used and probed with the indicated specific antibodies. Antibodies for cytochrome f and PsaA/B (66), cytochrome b6 (67), PsaD (68), small subunit of Rubisco (69), and the loading control chloroplastic RRF (70) have been described. Specific antibodies for the D1 subunit of PSII and subunit A of ATP synthase were generous gifts from Alice Barkan (University of Oregon, Eugene, OR) and Anna Sokolenko (Ludwig-Maximilians University, München, Germany), respectively. Antibody for the large subunit of Rubisco was purchased from Agrisera (Agrisera, Vannas, Sweden).
FIGURE 5.
FIGURE 5.
Experimental validation of the predicted mRNA targets for miRNAs (miR408, miR397, and miR857). The mRNA cleavage sites were determined by modified 5′ RNA ligase-mediated RACE. The gray boxes represent the exons, and the horizontal lines represent introns. White boxes represent 5′-UTRs (if known), and white arrows represent 3′-UTRs (if known). The mRNA sequences and complementary miRNA sequences are shown. Vertical arrows indicate the 5′ termini of miRNA-guided cleavage products, as identified by 5′-RACE, with the frequency of clones shown. Watson-Crick pairing (vertical dashes) and G:U wobble pairing are indicated. Horizontal arrows indicate primers used for qRT-PCR.
FIGURE 6.
FIGURE 6.
Expression analysis of miR408 and predicted targets. A, Northern blot analysis of miR408 and U6snRNA. 20 μg of total RNA from different tissues was separated by electrophoresis, transferred to Hybond N+ membrane, and probed with complementary sequences of miR408 that were end-labeled with [γ-32P]ATP. The U6snRNA probe was used as an internal control for Normalization. B, Northern blot analysis of plantacyanin. 10 μg of total RNA was separated by electrophoresis, transferred to Hybond N+ membranes, and probed with 32P-labeled plantacyanin PCR product. A 18 S rRNA Northern blot is shown as an internal control for loading. The intensity of bands was quantified and normalized with the internal control. C–E, quantitative real-time PCR expression levels of LAC3, LAC12, and LAC13. DNase-treated total RNA was reverse-transcribed into cDNA and used as a template for qRT-PCR. Expression levels are expressed as 40-ΔCT, where ΔCT is the difference in quantitative real-time PCR threshold cycle number (CT value) between the studied gene and the reference gene (ACT2). The number 40 was chosen because the PCR run stops after 40 cycles. A value of 40 indicates that the expression is equal to that of ACT2. The average CT value for ACT2 in all runs is 17.55 (±1.39). The results are the mean ± S.D. for technical triplicates of two independent experiments. On the right side of the graphs a log scale is given to allow visual comparison of relative expression levels.
FIGURE 7.
FIGURE 7.
Quantitative real-time PCR expression levels of miR397 precursor (A), LAC2 (B), LAC4 (C), and LAC17 (D) in different tissues grown at deficient (-Cu), limited (5 nm), and sufficient (50 nm) copper conditions. Expression levels are given as described in the legends to Fig. 6, C–E.
FIGURE 8.
FIGURE 8.
Quantitative real-time PCR expression levels of miR857 precursor (A) and its target LAC7 (B) in different tissues grown at deficient (-Cu), limited (5 nm), and sufficient (50 nm) copper conditions. Expression levels are given as described in the legends to Fig. 6, C–E.
FIGURE 9.
FIGURE 9.
Quantitative real-time PCR expression levels of LAC5 (A), LAC8 (B), LAC11 (C), and LAC15 (D)in different tissues grown at deficient (-Cu), limited (5 nm), and sufficient (50 nm) copper conditions. Expression levels are given as described in the legends to Fig. 6, C–E.
FIGURE 10.
FIGURE 10.
Effect of the hen1-1 mutation. Upper panel, immunoblot analysis of PC, CSD1, CSD2, and FeSOD. Hsp70 was use as a loading control. Lower panel, quantitative real-time PCR expression levels of LAC5, LAC8, LAC11, and LAC15 in wild type (WT) and miRNA maturation mutant (hen1-1) seedlings grown on synthetic MS agar plates with no added copper (-Cu) or with sufficient copper (5 μm Cu) for 3 weeks. The whole seedling (root and shoot) was collected and used for total RNA preparation. 1 μg of total RNA was treated with DNase and reverse-transcribed into cDNA. The cDNA was used as a template for qRT-PCR directly after dilution. Expression levels are given as described in the legends to Fig. 6, C–E. The average CT value for ACT2 in all PCR runs is 16.5 ± 0.24. The results are the mean ± S.D. for technical triplicates of two independent experiments.
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