Cloning and characterization of a UV-B-inducible maize flavonol synthase
Article first published online: 6 JAN 2010
© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd
The Plant Journal
Volume 62, Issue 1, pages 77–91, April 2010
How to Cite
Falcone Ferreyra, M. L., Rius, S., Emiliani, J., Pourcel, L., Feller, A., Morohashi, K., Casati, P. and Grotewold, E. (2010), Cloning and characterization of a UV-B-inducible maize flavonol synthase. The Plant Journal, 62: 77–91. doi: 10.1111/j.1365-313X.2010.04133.x
- Issue published online: 29 MAR 2010
- Article first published online: 6 JAN 2010
- Received 13 October 2009; revised 11 December 2009; accepted 17 December 2009; published online 9 February 2010.
Figure S1. Phylogenetic tree of the amino acid sequences of enzymes that participate in flavonoid biosynthesis. The tree was constructed by aligning the sequences using ClustalW2 and visualizing with Treeview. Maize sequences with significant homology to OsFLS1 (NP_001048230) were also included.
Figure S2. Alignment of the predicted amino acid sequence of ZmFLS1 with maize sequences homologous to OsFLS1 (NM_001054765). Amino acids linking the ferrous iron and residues participating in 2-oxoglutarate binding are indicated in bold-underlined and bold letters, respectively. Similar amino acids are indicated by dots and identical amino acids by asterisks. Domains conserved in 2-oxoglutarate-dependent dioxygenases (2-ODDs) are highlighted in grey.
Figure S3. Alignment of the predicted amino acid sequence of ZmFLS1, AtFLS1, SbFLS1 and OsFLS1. Amino acids binding ferrous iron and residues participating in 2-oxoglutarate binding are in bold-underlined and bold letters, respectively. Similar amino acids are indicated by dots and identical amino acids by asterisks. The proposed residues involved in binding the DHQ substrate are in grey (Chua et al., 2008).
Figure S4. (a) Amplification of ZmFLS1 by RT-PCR. (b) Amplification of the ZmFLS1 promoter. –: negative control, without template.
Figure S5. Purification of recombinant ZmFLS1 analyzed by SDS-PAGE. The separation was accomplished in 5% (w/v) stacking and 12% (w/v) separation gels, and the proteins were stained with Coomassie Brillant Blue R-250. The purification steps correspond to: Lane 1, total soluble protein of Escherichia coli crude extract expressing ZmFLS1 loaded onto a HisTrap affinity column. Lane 2, wash fraction eluted with 20 mm imidazole (binding buffer). Lane 3, wash fraction eluted with 50 mm imidazole (wash buffer). Lane 4–7, elution fractions eluted with 200 mm imidazole (elution buffer). Lane 8, molecular weight size markers.
Figure S6. Presence of ZmFLS1 transgene in transformed A. thaliana fls1 mutant plants. Amplification of ZmFLS1 transgene by PCR on genomic DNA from Ws, fls1 mutant, and hygromycin-resistant plants transformed with p35::ZmFLS1 or p35::ZmFLS1-GFP. For PCR the following primer pairs were used: ZmFLS1-forward2 and ZmFLS1-reverse2 (a) and prom35S-forward and ZmFLS1-reverse1 (b), with PCR products of 152 and 1240 bp, respectively.
Figure S7. Flavonol accumulation in Arabidopsis transgenic plants expressing p35S::ZmFLS1. Three week-old seedlings (ZmFLS1 in fls1 mutant, T1-1) were grown on 2.5% sucrose MS vertical plates, and roots were stained with DPBA. Scale bar bottom right: 1 mm.
Figure S8. Flavonol accumulation in 2 week-old roots from Arabidopsis transgenic plants expressing p35S::ZmFLS1. (a) HPLC chromatographic profiles of hydrolyzed flavonols at 360 nm of 3 day-old roots of wild type (Col-0), fls1, and fls1 independent transformant expressing p35S::ZmFLS1. (b) HPLC profile of authentic standards K and Q. The standards eluted with retention times of 15.5 and 14.52 min, respectively.
Figure S9. Analysis of anthocyanins in wild type, fls1 mutant and complemented 3 day-old fls1 seedlings expressing p35S::ZmFLS1. Absorbance profiles of samples from wild type (Col-0, Ws), fls1 mutant and fls1 independent transformant expressing p35S::ZmFLS1. Inset: Extract of complemented plants do not exhibit red pigment accumulation as fls1 mutants.
Figure S10. ZmFLS1 expression level in B73, W23b pl and W23B PL lines under control and UV-B conditions analyzed by RT-qPCR. Each reaction was normalized using the Ct values corresponding to a thioredoxin-like transcript (AW927774). The means of the results obtained using three independent RNAs as a template are shown, the error bars indicate the SD. of the samples. The relative transcript levels between maize lines were compared in control and UV-B conditions. For each transcript, different letters indicate a significant difference at P < 0.05 (Student t-test).
Table S1. Identity percentage at the nucleotide level between maize sequences with significant coverage and identity to OsFLS1 (NM_001054765) obtained by BLASTn.
Appendix S1. Experimental Procedures
Maize growth conditions and UV treatments
Maize plants were grown in greenhouse with supplemental visible lighting to 1000 mE m−2 sec−1 with 15 h of light and 9 h of dark without UV-B for 28 day. Greenhouse UV-B treatments were done illuminating plants with UV-B lamps (Bio-Rad) for 8 h using fixtures mounted 30 cm above the plants (F40UVB 40 W and TL 20 W/12; Phillips) at a UV-B intensity of 2 W m−3 and a UV-A intensity of 0.65 W m−3. The bulbs were covered with cellulose acetate filters (100 mm extra clear cellulose acetate plastic, Tap Plastics, Mountain View, CA, USA); the cellulose acetate sheeting does not remove any UV-B radiation from the spectrum but excludes wavelengths lower than 280 nm. Control, no UV-B-treated plants were exposed for the same period of time under the same lamps covered with polyester filters (100 mm clear polyester plastic; Tap Plastics, 0.04 W m−3, UV-A, 0.4 W m−3). This polyester filter absorbs both UV-B and wavelengths lower than 280 nm. Lamp outputs were recorded using a UV-B/UV-A radiometer (UV203 AþB radiometer; Macam Photometrics) to ensure that both the bulbs and filters provided the designated UV light dosage in all the treatments. Samples were collected immediately after irradiation and stored at −80°C. The UV-B treatment experiments were repeated at least three times.
Cloning of cDNAs
Total RNA from B73 maize tissues was isolated with a Qiagen plant total RNA isolation kit and cDNA was synthesized using Superscript II Reverse Transcriptase enzyme (Invitrogen) with oligo-dT as a primer. Full-length ORFs were amplified from RNA obtained from anther and leaf tissues. PCR reactions were performed with Platinum Pfx Polymerase (Invitrogen) under the following conditions: 1× Pfx buffer, 1× enhancer, 1.5 mm MgSO4, 0.5 mm of each dNTP, 0.5 mm of each primer, 0.3 U Platinum Pfx Polymerase, and sterile water added to obtain a volume of 20 μl. Cycling conditions were as follows: 30 sec denaturation at 95°C, 30 sec annealing at 64°C, 90 sec amplification at 68°C, with a 1°C decrement of annealing temperature in each cycle until it reached 54°C, followed by 25 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 54°C, 90 sec amplification at 68°C. Primers for cDNA were designed based on the sequence provided by GenBank accession number BT039956; the forward primer (ZmFLS1-forward1) included the start codon, and the stop codon was not included in the reverse primer (ZmFLS1-reverse1) (see Table S3 for the sequences). PCR products were cloned into pENTR-D-TOPO generating the plasmid pENTR-ZmFLS1, sequenced and recombined into the Gateway site of the pGWB2 and pGWB5 binary vectors (Karimi et al., 2002), resulting in p35S::ZmFLS and p35S::ZmFLS1-GFP, respectively.
Expression and purification of ZmFLS1 protein
Full-length ZmFLS1 cDNA was amplified by PCR using the pENTR-ZmFLS1 vector as a template. The primers ZmFLS1-BamHI-forward and ZmFLS1-HindIII-reverse with the BamHI and HindIII restriction sites, respectively, were used for further cloning (see Table S3). PCR was performed with GoTaq (Promega) and Pfu Polymerases (Invitrogen) (10:1) under the following conditions: 1X GoTaq buffer, 1.5 mm MgCl2, 0.5 μm of each primer, and 0.5 mm of each dNTP, in 25 μl of final volume. The amplified product was purified, cloned into pGEM®-T-Easy vector (Promega) and sequenced. The BamHI-HindIII fragment was subcloned into pET32-a(+) (Novagen) generating the vector pET-ZmFLS1. The expression of a His6-tag sequence in the N-terminal region of the protein facilitated the protein purification using a one-step process for purification on Ni2+-agarose column (GE Healthcare).
Recombinant N6H-ZmFLS1 was expressed in Escherichia coli BL21-(DE3)-pLys cells transformed with the plasmid pET-ZmFLS1 and grown on 500 ml LB medium containing 100 mg l−1 ampicillin and 35 mg l−1 chloramphenicol, at 37°C. Induction with 0.5 mm IPTG was performed at mid log phase (OD600 0.5–0.6). After incubation for 4 h at 30°C, cells were harvested by centrifugation at 3000 g for 20 min at 4°C. Pellet were washed twice and resuspended in 2 ml of binding buffer (0.5 m NaCl, 0.02 m imidazole, 0.02 m NaH2PO4 pH 7.4) per gram of cells. Cells were disrupted by sonication and then centrifuged at 12 000 g for 20 min at 4°C to obtain soluble cell extracts. The crude extract was filtered through a 0.2 μm cellulose acetate membrane and loaded onto a HisTrap FF affinity columns (GE) equilibrated with binding buffer. The column was washed with five volumes of binding buffer and ten volumes of washing buffer (0.5 m NaCl, 0.05 m imidazole, 0.02 m NaH2PO4 pH 7.4). The protein was then eluted with 0.2 m imidazole in binding buffer. Fractions containing ZmFLS1 were pooled, and desalted using buffer 0.1 m NaH2PO4 pH 6.8 plus 10% v/v glycerol, concentrated by centrifugation in Amicon Ultra-15 tubes (Millipore) and immediately used for activity assays. Total protein concentration was determined by the Bradford method (Bradford, 1976).
The p35S::ZmFLS1 and p35S::ZmFLS1-GFP constructs were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation and the transformation of A. thaliana by the resulting bacteria was performed by the floral dip method (Clough and Bent, 1998). Transformed seedlings (T1) were identified by selection on solid MS medium (Murashige and Skoog plant salt mixture, Gamborg’s B5 vitamin salt mixture, 1% sucrose, 0.05% MES buffer, pH 5.7, 0.8% agar) containing hygromycin (30 mg l−1) and then the plants were transferred to soil. The presence of the ZmFLS1 transgene in transformed plants was analyzed by PCR on the genomic DNA using the primers ZmFLS1-forward2 and ZmFLS1-reverse2 (product size of 152 bp) and prom35-forward and ZmFLS1-reverse2 (product size of 1.24 kbp). The expression of the ZmFLS1 transgene in transformed plants was analyzed by RT-qPCR, each reaction was normalized using the Ct values corresponding to the EF1αA4 transcript (see Table S3 for the corresponding primer sequences).
Cloning of the ZmFLS1 promoter
To amplify the promoter of ZmFLS1, primers were designed to amplify a 1.5 kbp fragment upstream of the start codon, as predicted from www.maizesequence.com. Restriction sites, NotI and KpnI, were included in the forward and reverse primers, respectively (NotI-ZmFLS1-prom-forward and KpnI-ZmFLS1-prom-reverse, Table S3). Genomic DNA was isolated from leaf tissue using a DNA isolation kit (Qiagen). PCR reactions were performed with Phusion Taq Polymerase (BioLab) in the following condition: 1X HF or GC buffer, 0.3 mm DMSO, 1.5 mm MgCl2, 0.5 mm of each primer, 0.5 mm of each dNTP, 100 ng genomic DNA, and 0.3 U Phusion Taq Polymerase in a volume of 50 μl. Cycling conditions were as follows: 30 sec denaturation at 95°C, 20 sec annealing at 68°C, 90 sec amplification at 72°C, with a 1°C decrement of annealing temperature in each cycle until it reached 58°C, followed by 25 cycles of 30 sec denaturation at 95°C, 20 sec annealing at 58°C, 90 sec amplification at 72°C. The PCR products were purified from the gels, cut with the corresponding restriction enzymes and purified. The pA1::Luc construct (pMSZ011) (Sainz et al., 1997) was restricted with NotI and KpnI and the A1 promoter was replaced by the ZmFLS1 promoter, resulting in pZmFLS1::Luc.
Chromatin immunoprecipitation (ChIP) experiments
Three hundred mg of BMS and BMSR+C1 cultured cells were used for ChIP experiments performed with a modified method previously described (Morohashi et al., 2007; Morohashi and Grotewold, 2009). Briefly, after fixation with buffer A (0.4 m sucrose, 10 mm Tris–HCl, pH 8.0, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride) containing 1% formaldehyde, high quality nuclei were isolated by using CelLytic™ PN (Sigma). The isolated nuclei were sonicated by Bioruptor™ (Diagnode) for 30 min and subjected to preclearing. In order to enrichment of binding regions, real-time PCR with technical triplicates to quantify enriched DNA regions were performed. For ChIP experiments of P1, pericarps were dissected from kernels at different developmental stages, 14 and 25 days after pollination (DAP), of P-ww and P-rr, and immediately soaked in liquid nitrogen. Sixty miligram of dissected pericaps were immersed in buffer A with 0.1% formaldehyde, and incubate for 10 min in vacuum. Then, nuclei were isolated by using CelLytic™ PN (Sigma), and sonicated as described above. The antibody, #344, which specifically recognized the carboxy-terminus of P1 was used for immunoprecipitation.
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