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

Abstract

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.

Fig. 1.

Structure of the PsABI3 gene. (A) Nucleotide and deduced amino acid sequence of PsABI3-1. The two purines (G) located at –3 and +4 around the initiation codon are labelled with stars. The location of the five canonical introns is indicated with black arrows marked I with the subscripts 1 to 5. The sites of alternative splicing are identified by black arrows pointing to their 5′ (arrows labelled A) and 3′ (arrows B) sites. When a second splicing process occurs, the arrows are marked A′ and B′. The subscripts 2 to 7 refer to the different isoforms. The basic domains B1, B2, and B3 are shadowed in grey. The two putative amphipathic α-helical within domain B1, α₁ and α₂ are underlined with dots. Bold letters represent two putative nuclear localization signals in the protein sequence. The PST domain is boxed. (B) Schematic diagram of modular structure of PsABI3-1 polypeptide.

Fig. 2.

Southern blot analysis of the PsABI3 gene. Genomic DNA from leaves (30 μg) was digested with restriction enzymes EcoRI, DraI, BclI, and BamHI, blotted and hybridized to a probe containing the PsABI3-1 coding sequence. The migration of size markers is shown at the left.

Fig. 3.

Structure of cDNAs derived from PsABI3. The Nakako and Mori sequence is depicted in the first row and the numbers on the left of the remaining rows refer to the isoforms found in this work. The left column represents the processed mRNA between the initiation and termination codons as well as the length of the coding region. When applicable, the position of premature stop codons originated by frame shift is indicated. The exon sequences excised from the complete isoform (PsABI3-1) are shown as folded lines. Numbers above the lines stand for the size of the removed or conserved sequences in nt. The arrow heads in Nakako and Mori's sequence indicate the position of insertions relative to PsABI3-1 cDNA. The dinucleotides at the borders of the splicing sites are shown. In both columns, the numbering of the nucleotides is relative to the first translation start site in PSABI3-1.

Fig. 4.

Expression of the PsABI3 gene as determined by RT-PCR. Total RNA was extracted and used for the generation of the corresponding cDNAs. The presence of PsABI3 mRNAs was detected by amplification with primers P1f and P3r (see Table S1 at JXB online), which include the first canonical intron. A 185 bp fragment from actin gene was amplified as an internal control using the primers Act1f and Act2r. The RT-PCR analysis was performed in: (A) different plant organs, (B) during seed development, at different DAF, (C) in embryonic axes, and (D) in cotyledons at different HAI.

Fig. 5.

Yeast one-hybrid analysis of the transactivating potential of the different isoforms and artificial constructs. (A) Functional analysis of the full-length PsABI3 isoforms. The β-galactosidase activity was analysed by lift filter assays and the His3 gene expression analysis was performed by growing on SC medium without His. pCL1 and pGBKT7 vectors were used as positive and negative controls, respectively (data not shown). (B) Functional analysis of different constructs derived from PsABI3 isoforms. The primers (see Supplementry Table S1 at JXB online) used for each construct are shown. The numbers over the constructs indicate the position of amino acids in the sequence of PsABI3-1. In both panels, thin blue lines represent the segments missing in every isoform or construct. (This figure is available in colour at JXB online.)

Fig. 6.

Yeast two-hybrid analysis of the interaction between several constructs from PsABI3 proteins and AtABI5. The transformants were selected on plates containing SC medium lacking tryptophan and leucine. Positive interactions were confirmed with β-galactosidase filter assays. The thin blue lines represent segments missing in the different isoforms or constructs. The primers (see Supplementary Table S1 at JXB online) used for each construct are shown. Negative controls with empty plasmids are shown. The PsABI3 binding domains in the constructs used as bait were identified by the N- and C-terminal amino acids (one-letter code and sequence number in PsABI3-1). (This figure is available in colour at JXB online.)

Fig. 7.

Subcellular localization of PsABI3-1 and PsABI3-2 in onion epidermal cells. Confocal microscopy images of onion cells transformed with (A) GFP alone, (B) construct GFP-PsABI3-1, and (C) construct GFP-PsABI3-2. The fluorescent signal, confined to nuclei in the case of both constructs, is observed in the left panels, while the bright-field images of the same cells is shown in the right panels. Nuclei are arrowed. (This figure is available in colour at JXB online.)