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Flower development in angiosperms is regulated by the family of MADS-box transcription factors. MADS-box genes have also been reported from gymnosperms, another major group of seed plants. AGAMOUS (AG) is the class C MADS-box floral organ identity gene controlling the stamen and carpel development in Arabidopsis. We report the characterization of an ortholog of the AG gene, named Cycas AGAMOUS (CyAG), from the primitive gymnosperm Cycas edentata. The expression pattern of CyAG in Cycas parallels that of AG in Arabidopsis. Additionally, the gene structure, including the number and location of the introns, is conserved in CyAG and other AG orthologs known. Most importantly, functional analysis shows that CyAG driven by the AG promoter can rescue the loss-of-function ag mutant of Arabidopsis. However, the ectopic expression of CyAG in ag mutant Arabidopsis cannot produce the carpeloid and stamenoid organs in the first and second whorls, although the stamen and carpel are rescued in the third and fourth whorls of the transformants. These observations show that the molecular mechanism of class C function controlling reproductive organ identity (stamen and carpel of angiosperms or microsporophyll and megasporophyll of gymnosperms) arose before the divergence of angiosperms and gymnosperms, and has been conserved during 300 million years of evolution thereafter.

Wheat HMGa protein is a typical member of the plant HMGA family. It has four AT hooks and a histone H1-like region. A panel of deletion mutants of HMGa was generated to study the role of different regions of HMGa in its binding to 4H (a synthetic DNA that mimics the in vivo structure of intermediates of homologous recombination and DNA repair) and linear DNAs. Although the histone H1-like region of HMGa does not bind to 4H or linear DNAs, it does enhance the binding. Mutants with any two adjacent AT hooks show specific binding to both 4H and linear P268 (and P31) with different binding affinities, which is partly due to the flanking regions between AT hooks. Conformational studies indicate that the alpha-helical content of HMGa increases significantly when it binds to 4H compared to that after binding to P31, linear DNA. In contrast, linear DNA, but not 4H, undergoes substantial conformational change when it binds to HMGa, indicating that linear DNA is relatively more flexible than 4H. A more significant difference in the affinities of binding of the mutants of HMGa to 4H was observed compared to their affinities of binding to linear DNA, P31. These differences could be due to the rigidity of the DNA and the characters of the AT hook regions in the mutants.

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.

To better understand the molecular mechanisms controlling development of sexual characters in Cycas edentata, we attempted to clone genes expressed differentially in male or female reproductive organs. We report a novel gene, named Fortune-1 (Ft-1), with enhanced expression in male reproductive organs. The 593-base-pair Ft-1 cDNA is predicted to encode a 77-amino-acid protein, and exists as a single copy gene in the C. edentata genome. Ft-1 expression is enhanced in male cones, including the cone axis, microsporophylls and microsporangia, but is reduced in ovules and undetectable in megasporophylls. Ft-1 is also weakly detectable in leaves. In roots and stems of C. edentata, Ft-1 transcripts are undetectable. The secondary structure prediction and homology search of Ft-1 protein show that it has a helix-loop-helix motif, and is without any homologue in the database., (Copyright 2002 Elsevier Science Ireland Ltd.)

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.