Article Figures & Data
Figures
- Figure 1.
Amino acid sequences alignment and phylogenetic analysis of maize Rpd3-like proteins. A, ZmRpd3/101, ZmRpd3/102, and ZmRpd3/108 sequences from maize and AtHDA19 sequence from Arabidopsis were aligned using ClustalW software (Thompson et al., 1994) and were subsequently edited with Genedoc software (http://www.psc.edu/biomed/genedoc/). The number of amino acids is indicated at the right site. A dash indicates gaps in the alignment, whereas asterisks represent amino acid identity. The white box indicates the HDAC domain (PF00805 in Pfam database) present in different Rpd3 proteins. Shaded boxes represent the N- and C-terminal regions of ZmRpd3/101 involved in binding of ZmRBR1 and ZmRbAp1; an internal domain that enhances ZmRpd3/101-ZmRbAp1interaction is also underlined (Rossi et al., 2003). Below the multiple sequence alignment, consensus motifs found in all Rpd3 class I HDACs are shown. These motifs represent highly conserved sequence positions in Rpd3-like sequences from 30 different eukaryotes (see below) identified by generating a logo sequence (http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi). Upper- and lowercase letters represent positions 98% and 60% conserved within class I HDACs, respectively; X indicates variable positions. B, Unrooted neighbor-joining tree of the HDAC domain of 30 Rpd3 class I HDAC proteins was obtained using ClustalW and Phylip software packages (Felsenstein, 1989; Thompson et al., 1994). Nomenclature of the proteins is referred to Pandey et al. (2002). Proteins and their accession numbers follow: AnRpd3A (AAF80489), AnHOS2A (AAF80490), AtHDA2 (AAD40129), AtHDA6 (BAB10553), AtHDA7 (BAB09994), AtHDA9 (CAB72470), AtHDA19 (AAB66486), CeHDA301 (CAB03984), CeHDA302 (Q09440), CeHDA303 (CAB03224), DmHDA401 (AAC61494), DmHDA402 (AAC83649), HsHDAC1 (Q13547), HsHDAC2 (NP_001518), HsHDAC3 (NP_003874), HsHDAC8 (NP_060956), LmHDA2 (CAC14522), McHdeac1 (AAF82385), OsHDA701 (BAB61857), OsHDA702 (AAK01712), PfHDA1 (AAD22407), ScRpd3 (P32561), ScHOS1 (Q12214), ScHOS2 (P53096), SpCLR6 (CAA19053), SpPHD1 (O13298), TtTHD1 (AAG00980), ZmRpd3/101 (AAK67142), ZmRpd3/102 (AAL33655), and ZmRpd3/108 (AAD10139). Abbreviations for species are: Aspergillus nidulans (An), Arabidopsis (At), Caenorhabditis elegans (Ce), fruit fly (Drosophila melanogaster; Dm), human (Homo sapiens; Hs), Leishmania major (Lm), common ice plant (Mesembryanthemum crystallinum; Mc), rice (Oryza sativa; Os), Plasmodium falciparum (Pf), Brewer's yeast (Saccharomyces cerevisiae; Sc), fission yeast (Schizosaccharomyces pombe; Sp), Tetrahymena thermophila (Tt), and maize (Zm). The class III HDACs AtHDA2 was used as out-group. Bootstrap values (100 replicates) are indicated at the nodes of the tree and are expressed as percentages; a 50% cut-off was applied. The ZmRpd3 proteins are highlighted in bold. Brackets indicate the three clusters containing ZmRpd3 sequences.
- Figure 2.
Northern-blot analysis of ZmRpd3 transcripts. Total RNA (20 μg) extracted from different maize organs (A), kernels/endosperms (B), and ears and tassels (C) harvested at different developmental stages was fractionated through an agarose/formaldehyde denaturing gel and blotted onto nylon membrane. The membranes were hybridized with DNA probes specific for each ZmRpd3 gene and with a probe specific for the histone H4 transcript. Ethidium bromide staining of the gel is shown as input control. The length of the transcripts (number of nucleotides) is reported on the right of each panel. The organs or developmental stages are reported according to indications from the Iowa State University of Science and Technology (1989; see “Materials and Methods”).
- Figure 3.
Characterization of antibodies. A, IVT ZmRpd3 proteins were fractionated by SDS-PAGE, blotted, and immunodetected with anti-ZmRpd3 antibody (left panel) and anti-ZmRpd3/108 antibody (right panel). B, Recombinant full-length ZmRpd3/108 fused to the maltose-binding domain (pMAL ZmRpd3/108), C-terminal part of ZmRpd3/108 (c ZmRpd3/108), and full-length ZmRpd3/101(fl ZmRpd3/101) proteins were expressed in Escherichia coli, and the protein extracts were fractionated by SDS-PAGE followed by blotting onto nylon membranes and immunodetection with different dilutions of anti-ZmRpd3/108 antibody. Arrows mark the band specifically recognized by the antibody. The size of molecular markers is reported.
- Figure 4.
Western-blot analysis of ZmRpd3 protein. One hundred micrograms of crude protein extracts from various maize organs (A), from different developmental stages of kernel/endosperm (B), and ear (C) was fractionated by SDS-PAGE, blotted, and immunodetected with anti-ZmRpd3 antibody (top panels). The filters were subsequently stripped and reprobed with anti-ZmRpd3/108 antibody (middle panels). The size of molecular markers is indicated. Coomassie staining of SDS-PAGEs loaded with the protein extracts are shown as loading control (bottom panels).
- Figure 5.
Localization of ZmRpd3 transcripts in maize tissues. A through E, Longitudinal sections through a maize kernel at 6 (A) and 10 (B–E) DAP were hybridized with antisense probes specific for ZmRpd3/101 (A, B, and D) and ZmRpd3/102 (C) transcripts. E, Hybridization with ZmRpd3/101 sense probe is reported as an example of negative control. EN, Endosperm; NU, nucellus; TL, transfer layer; Ph, phloem. F through L, Cross (F–I) and longitudinal (L) sections through the shoot apex were hybridized with antisense probes specific for ZmRpd3/101 (G and L), ZmRpd3/102 (H), and ZmRpd3/108 (I). Hybridization with sense ZmRpd3/101 is shown as negative control (F). LP, Leaf primordium. M through Q, Cross sections of anthers during microsporogenesis at premeiotic (M–O) and tetrad stages (P and Q) were hybridized with sense (M) or antisense (N) probes specific for the histone H4 transcript and with antisense probes specific for ZmRpd3/101 (O), ZmRpd3/102 (P), and ZmRpd3/108 (Q). A, Anthers; T, tetrad; Tp, tapetum. The purple/blue staining represents the hybridization signal. Bars = 100 μm.
- Figure 6.
Localization of ZmRpd3/108 protein in maize tissues. A and B, Sections were incubated with anti-ZmRpd3/108 antibody. A, Cross section through a maize floret at a premeiotic stage during microsporogenesis. A, Anther; Tp, tapetum. B, Longitudinal section through maize endosperm at 10 DAP. EN, Endosperm; Al, aleurone. C and D, Sections were not treated with the secondary antibody. C, Cross section through a maize floret at a premeiotic stage. D, Longitudinal section through maize endosperm at 10 DAP. Bars = 100 μm.
- Figure 7.
Analysis of protein interactions among ZmRpd3s, ZmRBR1, and ZmRbAp1. A, Autoradiography of an SDS polyacrylamide gel loaded with 10% of the IVT ZmRpd3/101, ZmRpd3/102, and ZmRpd3/108 proteins used as input in GST pull-downs; equimolar amounts of the IVT products were used in the experiments. In vitro GST pull-down assays were performed using beads carrying recombinant GST-ZmRBR1 (B) or GST-ZmRbAp1 (C) incubated with IVT products of the different ZmRpd3 proteins. Beads with GST alone were used as negative control. D, GST pull-down assays were carried out as in B and C, but beads carrying either GST-ZmRBR1 (left panel) or GST-ZmRbAp1 (right panel) were first incubated with unlabeled IVT ZmRpd3 proteins and then IVT 35S radiolabeled products (35S IVT) were added (5:1 ratio unlabeled: radiolabeled) to the reaction mix for further incubation. All possible combinations of unlabeled and radiolabeled ZmRpd3/101, ZmRpd3/102, and ZmRpd3/108 were tested. Control indicates that only radiolabeled IVT and no unlabeled products were added to the reaction mix.
