The Pisum sativum psp54 gene requires ABI3 and a chromatin remodeller to switch from a poised to a transcriptionally active state.

Aspects of transcriptional regulation in plants, such as the order in which transcriptional factors and the preinitiation complex are assembled, are obscure because studies carried out under conditions in which native chromatin structure is preserved are still few in comparison with those carried out under other conditions. In vivo chromatin immunoprecipitation (ChIP) experiments were used here to study the regulation of Pisum sativum psp54, which codes for the precursor of a chromatin-associated protein in dry seeds. Antibodies against PsSNF5, a component of the SWI/SNF remodelling complex, and against the transcriptional factor Pisum sativum abscisic acid insensitive 3 (PsABI3) were raised and used for ChIP experiments, which showed that both factors are bound to the psp54 promoter only when the gene is actively expressed during seed maturation and germination. However, RNA polymerase II appeared to be bound to the inactive promoter, which was poised for transcription, before the assembly of factors. Micrococcal nuclease protection assays showed that chromatin conformation at the proximal psp54 promoter changes in shifting from the active to inactive state. The changes in the promoter chromatin of psp54 are discussed. Stalled polymerase is described for the first time at the promoter of a non-heat-shock plant gene.

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3-AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum.

Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNA-histone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723bp open reading frame encoding a 240 amino acid protein of 27.4kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with which it showed an overall amino acid identity of 78.5%. Two-hybrid experiments showed that PsSNF5 is functionally interchangeable with Arabidopsis BSH in the interactions with other components of the remodeling complex. Phylogenetic analysis demonstrated that PsSNF5 clustered with translated expressed sequence tags from other Leguminosae, hypothetically coding for new Snf5-like proteins. RT-PCR expression analysis demonstrated that the PsSNF5 gene is constitutively expressed in all the tissues examined, with minor differences in expression level in different tissues. Nevertheless, expression analysis revealed that PsSNF5 was up-regulated in the last stages of embryo development, when water content decreases. Moreover, abscisic acid and drought stress induced PsSNF5 accumulation in germinating embryos and vegetative tissues, suggesting that chromatin remodeling induced by PsSNF5-containing complexes might contribute to the response to that phytohormone.