doi: 10.1105/tpc.108.060137. Epub 2009 Jan 2.
SQUAMOSA Promoter Binding Protein-Like7 Is a Central Regulator for Copper Homeostasis in Arabidopsis
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- PMID:19122104
- PMCID: PMC2648088
- DOI: 10.1105/tpc.108.060137
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SQUAMOSA Promoter Binding Protein-Like7 Is a Central Regulator for Copper Homeostasis in Arabidopsis
Hiroaki Yamasaki et al. Plant Cell.2009 Jan.
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Abstract
Expression of miR398 is induced in response to copper deficiency and is involved in the degradation of mRNAs encoding copper/zinc superoxide dismutase in Arabidopsis thaliana. We found that SPL7 (for SQUAMOSA promoter binding protein-like7) is essential for this response of miR398. SPL7 is homologous to Copper response regulator1, the transcription factor that is required for switching between plastocyanin and cytochrome c(6) in response to copper deficiency in Chlamydomonas reinhardtii. SPL7 bound directly to GTAC motifs in the miR398 promoter in vitro, and these motifs were essential and sufficient for the response to copper deficiency in vivo. SPL7 is also required for the expression of multiple microRNAs, miR397, miR408, and miR857, involved in copper homeostasis and of genes encoding several copper transporters and a copper chaperone, indicating its central role in response to copper deficiency. Consistent with this idea, the growth of spl7 plants was severely impaired under low-copper conditions.
Figures
Identification ofcis Elements in the Promoter Region ofmiR398c.(A) Construction ofmiR398c pro full:LUC and various truncated derivatives,miR398c pro Δ1–4:LUC. The black boxes indicate the positions of GTAC motifs.(B) Luminescence images and relative luciferase activities in 2-week-old seedlings of transgenic lines. Each graph shows relative levels of luminescence in two independent transgenic lines (white and gray bars). Data are averages of four independent seedlings withsd . Note that the scales of they axes differ among panels.
The GTAC Motifs Located at −185 to −182 and −157 to −154 Are Sufficient for the Copper Response.(A) The −196 to −146 region of themiR398c promoter was fused with the minimal promoter of 46N. To adjust the distance from GTAC motifs to the transcriptional start site, the 100-bp unrelated sequence derived from pBI121 was inserted between them. The chimeric promoter was placed in front of theLUC gene. The GTAC motif was replaced by the mutant CATG as indicated.(B) Relative LUC activity in four independent T1 plants. The measurements are normalized so that luciferase activity at 5 μM copper (black) is set to 1. Note that the scales of they axes differ among panels.
Structure of SPL7, a Member of the SPL Family.(A) A phylogenetic tree of theArabidopsis SPL family members, includingChlamydomonas Crr1, based on their SBP domain sequences.(B) Exon–intron structure ofSPL7. The triangle labeledspl7 indicates the site of T-DNA insertion, and arrows (A and B) indicate primer sets for amplifying cDNAs in Figure 3C. The primer sequences are described in Supplemental Table 5 online.(C) RT-PCR analysis ofSPL7 mRNA with primer pairs A and B in 4-week old seedlings cultured on standard MS medium.(D) Diagram of the motif structures of Crr1 and SPL7. A triangle indicates the position from which the C-terminal part is truncated by the T-DNA insertion inspl7. The blue boxes indicate the SBP domain, and the pink boxes describe a putative nuclear localization signal. The yellow boxes show an unknown motif that is conserved between Crr1 and SPL7, and the green boxes indicate putative metal binding motifs.
SPL7 Activates Transcription ofmiR398.(A) RT-PCR analysis of genes responding to copper deficiency. The plants were grown for 3 weeks on MS agar medium containing 0.1 or 5 μM CuSO4. The number of PCR cycles was 30 formiR398a, 26 formiR398b, 26 formiR398c, 20 forCSD1 andCSD2, and 23 forActin (control). SPL7comp identifiesspl7 seedlings transformed with35S pro:SPL7 cDNA.(B) Immunoblot analysis of CSDs and PC in plants cultured on MS medium containing various concentrations of copper. Cytochromef was detected as a loading control.
The SBP Domain of SPL7 Binds to a GTAC Motif.(A) Recombinant SBP domain of SPL7 was incubated with digoxigenin-labeledmiR398c promoter (−203 to −163; lane 2). The mixture was also incubated with various concentrations of nonlabeledmiR398c promoter (lanes 3 to 6) andAct12 promoter (lanes 7 to 10). Lane 1 contains only the probe. The molar ratio of protein to DNA is 125:1.(B) The mixture was incubated with an unlabeled mutant version of themiR398c promoter (lanes 3 to 6). The GTAC motifs were substituted with CATG. Lane 1 contains only the probe.(C) Recombinant SBP domain of SPL7 was incubated with the digoxigenin-labeled wild-typemiR398c promoter (−203 to −163; lane 2) and with the mutated probe that was used in(B) as a competitor (lane 4). Lanes 1 and 3 contain controls without SPL7-SBP. Arrowheads indicate the positions of two shift bands (SB) and the free probe (FP).
SPL7 Expression Patterns.(A) Quantitative RT-PCR analysis of tissue-specific expression. Plants were grown for 3 weeks on MS agar medium containing 5 μM CuSO4 for the preparation of roots (R) or for 5 weeks on soil for the preparation of rosette leaves (RL), cauline leaves (CL), stems (S), and flowers (F). Error bars indicatesd (n = 3).(B) Quantitative RT-PCR analysis of the response to copper deficiency. Roots (R) and rosette leaves (RL) were harvested from seedlings grown for 3 weeks on MS medium containing 0.1 or 5 μM CuSO4. Error bars indicatesd (n = 3). The primer sequences are described in Supplemental Table 6 online.
SPL7 Activates a Set of Genes Involved in Copper Homeostasis.(A) Quantitative RT-PCR analysis for microRNAs. Aerial parts of seedlings grown for 3 weeks on MS agar medium containing 0.1 or 5 μM CuSO4 were used for RNA extraction. Error bars indicatesd (n = 3). The primer sequences are described in Supplemental Table 6 online.(B) RT-PCR analysis of copper transporter genes. Roots were harvested from the seedlings used in(A) and RNA was extracted. The number of PCR cycles was 21 forCOPT1, 22 forCOPT2, 25 forZIP2, 27 forZIP4, 23 forFRO3, and 20 forActin (control).(C) RT-PCR analysis of copper chaperone genes. RNA samples extracted in(A) were used. The number of PCR cycles was 19 forCCH, 22 forATX1, and 23 forActin (control).
Genome-Wide Analysis of the Copper-Responsive Network Regulated by SPL7. The representative genes identified by transcriptome analysis (see Supplemental Tables 1 to 3 online) were analyzed by RT-PCR. Aerial parts of seedlings grown for 3 weeks on MS agar medium containing 0.1 or 5 μM CuSO4 were used for RNA extraction. The number of PCR cycles was 20 forFSD1, 25 forYSL2, 25 forbHLH, 20 forTAT3, 25 forAt1g14880, 26 forAt4g21840, and 23 forActin (control).
Excess Nickel Mimics Copper in Regulating the Expression ofmiR398.(A) RT-PCR analysis ofCSD mRNAs in seedlings grown for 3 weeks on MS medium containing the indicated concentrations of copper and nickel. The number of PCR cycles was 22 forCSD2, 25 formiR398b/c, and 23 forActin (control).(B) Immunoblot analysis of CSDs and cytochromef in the seedlings used in(A).
Thespl7 Phenotype in Various Concentrations of Copper.(A) Growth phenotypes of the wild type,spl7, and SPL7comp lines grown for 3 weeks on MS medium containing various concentrations of copper. Bar = 10 mm.(B) Fresh weights of seedlings. Data are averages of four independent seedlings. Error bars indicatesd (n = 4).(C) Copper contents in the wild type,spl7, and SPL7comp lines grown for 3 weeks on MS medium containing 0.1 or 5 μM CuSO4. Data are averages of three independent experiments. Error bars indicatesd (n = 3).
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