ABSTRACT
The Helianthus annuus LEAFY COTYLEDON1-LIKE (HaL1L) gene encodes a HAP3 subunit of a CCAAT box-binding factor (NF-Y). Studies in Arabidopsis thaliana sharing a sequence omology of L1L e LEC1 (sequence identity 56%) with regard to the highly conserved central domain, and suggesting a functional conservation. The LEC1 gene confers typical embryonic characteristics and, when ectopically expressed, it induces the formation of somatic embryos from differentiated vegetative cells.
HaL1L transcripts are accumulated primarily at an early stage of embryogenesis. High levels of HaL1L mRNA have been detected in the developing embryos, endosperm, integument, and integumentary tapetum cells, while no or low transcript levels were detectable in organs such as the cotyledons, leaves, stem internodes and roots.
A genomic library from H. annuus was successfully screened to isolate the entire HaL1L gene. From databases analyses it has been suggested that the identified genomic DNA fragment is homologous to the A. thaliana chromosome V region carrying AtL1L and the immediately adjacent genes at the 5’ and 3’ sides, respectively. In the HaL1L 5’ flanking region, elements peculiar to a putative TATA-box promoter were identified.
In the 3’ region, in addition to the nuclear polyadenylation signal, a cytoplasmic polyadenylation signal which suggest a negative post-transcriptional regulation was also identified. Poly(A) tails, lengthened by cytoplasmatic poly(A)polymerases (PAPs), form complexes with regulative proteins which inactivate mRNAs. During embryo development PAPs act under hormonal control. The hypothesis of a translational control for HaL1L is also supported by the in situ hybridization analysis, performed in previous experiments, that demonstrate an accumulation of HaL1L transcripts in maternal tissues of developing embryos such as integument and integumentary tapetum cells.
L1L nucleotide sequence analyzed to individuate cis-regulatory sequences involved in trascription regulation by other transcription factors, show the presence of ARF and ABRE motifs. These motifs suggest that hormones, such as auxin and abscisic acid, could be involved in the expression control of this gene.
One of the most intriguing motifs, present in the 5’ flanking region as well in the HaL1L intron, is WUSATA, it represent the “target” sequence for the transcription factor WUSCHEL (WUS), which could by involved in the complex regulation system controlling the zygotic embryo development. A study on L1L/WUS interaction was started, which required the isolation of WUS homologous in H. annuus. The 5’3’ RACE and RAGE techniques were used to obtain a complete HaWUS sequence and for the identification of motifs in its promoter region. The amino acid sequence obtained, compared with the sequence of WUS and with WOX peptides of Arabidopsis, result to have the characteristic that identify the peptide as homologous of WUS.
The information obtained by the nucleotidic sequence will be use to make a probe for in situ hybridization experiments to verify that HaWUS expresses oneself with a pattern analogous to that of AtWUS. Moreover, analysis to ascertain the interplay between WUS and the regulative region of L1L.
