SummaryPost-translational modi®cation of histones, in particular acetylation, is an important mechanism in the regulation of eukaryotic gene expression. Histone deacetylases are enzymes that remove acetyl groups from the core histones and play a key role in the repression of transcription. HD2 is a maize histone deacetylase, which shows no sequence homology to the histone deacetylases identi®ed from other eukaryotes. We have identi®ed two putative HD2-like histone deacetylase cDNA clones, AtHD2A and AtHD2B, from Arabidopsis thaliana by screening the expressed sequence tag database. AtHD2A and AtHD2B encode putative proteins of 246 and 305 amino acids, and share 44% and 46% amino acid identity to the maize HD2, respectively. Northern blot analysis indicated that AtHD2A was highly expressed in¯owers and young siliques of Arabidopsis plants, whereas AtHD2B was widely expressed in stems, leaves,¯owers and young siliques. AtHD2A repressed transcription when directed to a promoter containing GAL4-binding sites as a GAL4 fusion protein. Deletion of the extended acidic domain or the domain containing predicted catalytic residues of AtHD2A resulted in the loss of gene repression activity, revealing the importance of both domains to AtHD2A function. Arabidopsis plants were transformed with a gene construct comprising an AtHD2A cDNA in the antisense orientation driven by a strong constitutive promoter, ±394tCUP. Silencing of AtHD2A expression resulted in aborted seed development in transgenic Arabidopsis plants, suggesting that the AtHD2A gene product was important in the reproductive development of Arabidopsis thaliana.

ERFs (ethylene-responsive element binding factors) belong to a large family of plant transcription factors that are found exclusively in plants. A small subfamily of ERF proteins can act as transcriptional repressors. The Arabidopsis genome contains eight ERF repressors, namely AtERF3, AtERF4, and AtERF7 to AtERF12. Members of ERF repressors show differential expression, suggesting that they may have different function. Using a transient expression system, we demonstrated that AtERF4, AtERF7, AtERF10, AtERF11 and AtERF12 can function as transcriptional repressors. The expression of AtERF4 can be induced by ethylene, jasmonic acid, and abscisic acid (ABA). By using green fluorescent protein fusion, we demonstrated that AtEFR4 accumulated in the nuclear bodies of Arabidopsis cells. Expression of 35S:AtERF4-GFP in transgenic Arabidopsis plants conferred an ethylene-insensitive phenotype and repressed the expression of Basic Chitinase and beta-1,3-Glucanase, the GCC-box-containing genes. In comparison with wild-type plants, 35S:AtERF4-GFP transgenic plants had decreased sensitivity to ABA and were hypersensitive to sodium chloride. The expression of the ABA responsive genes, ABI2, rd29B and rab18, was decreased in the 35S:AtERF4-GFP transgenic plants. Our study provides evidence that AtERF4 is a negative regulator capable of modulating ethylene and abscisic acid responses.

SummaryHD2 (histone deacetylase) proteins are plant-speci®c histone deacetylases (HDACs). The Arabidopsis genome contains four HD2 genes, namely HD2A, HD2B, HD2C, and HD2D. We have previously demonstrated that HD2A, HD2B, and HD2C can repress transcription directly by targeting to promoters in planta. Here, we show that the N-terminal conserved motif (EFWG) and histidine 25 (H25), a potential catalytic residue, were important for the gene repression activity of HD2A. In situ hybridization indicated that HD2A, HD2B, and HD2C were expressed in ovules, embryos, shoot apical meristems, and primary leaves. Furthermore, all three genes were strongly induced during the process of somatic embryogenesis. HD2D mRNA was only detected in the stems and¯owers with young siliques and may have adopted different functions. Using green¯uorescent protein (GFP) fusions, we demonstrated that HD2A, HD2B, and HD2C accumulated in the nuclei of Arabidopsis cells. Overexpression of 35S::GFP±HD2A in transgenic Arabidopsis plants generated pleiotropic developmental abnormalities, including abnormal leaves, delayed¯owering, and aborted seed development. The data showed that normal pattern of HD2 expression was essential for normal plant development and that HD2A, HD2B, and HD2C may be needed for embryogenesis and embryo development. Reverse transcriptase (RT)-PCR analysis revealed that a number of genes involved in seed development and maturation were repressed in the 35S::GFP±HD2A plants, supporting a role of HD2A in the regulation of gene expression during seed development.

Seventy-six cultivars of alfalfa (Medicago sativa L., M. faleata L. and M. varia Martyn) were tested in vitro for their capacity to produce callus and somatic embryos. A three-step media protocol was used to survey the response of the cotyledons and hypocotyl of each genotype while the epicotyl region was conserved in order to recover highly responding genotypes. The best regeneration response was observed in creepingrooted cultivars which contained a strong genetic contribution of two landrace germplasm sources, defined as M. falcata and Ladak, in their ancestry. The callus and embryogenesis responses showed a high degree of variation both between cultivars and among the plants of many of the 76 cultivars tested. A higher number of plants produced somatic embryos in the high regenerating cultivars compared to the low regenerating cultivars regardless of the media protocol or explant.

SummaryThe four HD2 proteins of Arabidopsis thaliana (AtHD2A±D) belong to a unique class of histone deacetylases that is plant speci®c. Previously, we have demonstrated that one of the members, AtHD2A, can mediate transcriptional repression when targeted to the promoter of a reporter gene. Here, we report that AtHD2B and AtHD2C can also repress gene expression. AtHD2A and AtHD2C differ from AtHD2B and AtHD2D in the composition of their structural domains. Our data show that both structural types play a role in the repression of gene transcription. We demonstrate that AtHD2A can mediate gene repression through interactions with transcription factors in plants. By fusing AtHD2A with the DNA-binding domain of the plant transcriptional factor Pti4, the expression of a GCC box containing reporter gene was repressed. We also demonstrated repression of a GUS gene with GAL4 enhancers using transgenic plants that expressed a GAL4/AtHD2A fusion gene. Furthermore, the expression of the GAL4/AtHD2A protein using the seed-speci®c napin promoter (NAP2) and the constitutive tCUP promoter demonstrated that repression of transgenes could be achieved in a tissue-speci®c or unrestricted manner. Targeting of HD2 proteins to speci®c promoters using transcription factor DNA-binding domains may therefore provide a new technology for silencing target genes and pathways in plants as well as for assessing the function of unknown transcription factors.