Characterisation of the GAAP (Golgi anti-apoptotic protein) gene family in Arabidopsis thaliana

Abstract

Programmed cell death (PCD) plays an essential role in eukaryotes during growth and development and in response to stress signals. GAAPs (Golgi anti-apoptotic protein) are a novel, evolutionarily conserved group of anti-apoptotic proteins. Human and viral GAAPs have been shown to inhibit apoptosis and modulate intracellular calcium fluxes. There is an apparent expansion of the GAAP gene family in plants, with five paralogous genes present in the Arabidopsis thaliana genome (AtGAAP1-5). AtGAAPs share the UPF0005 signature motif with animal and plant proteins that have been shown to function as inhibitors of cell death, including Bax inhibitor-1 and Lifeguard. AtGAAP genes show distinct expression patterns with AtGAAP4 and AtGAAP2 showing the highest overall transcript abundance based on publicly available microarray data and RT-PCR analysis. AtGAAP gene expression analysis using promoter-GUS fusions revealed overlapping expression patterns for AtGAAP1, AtGAAP2 and AtGAAP4 in floral organs, with AtGAAP2 and AtGAAP4 also highly expressed in leaf tissue. AtGAAP5 however showed floral-specific expression that was mostly distinct from the expression pattern of AtGAAP1, AtGAAP2 and AtGAAP4 in the flowers. AtGAAP3 expression was undetectable by GUS staining. Intracellular localisation of fluorescent protein-tagged AtGAAPs was studied using stable or transient expression in Arabidopsis and Nicotiana benthamiana, respectively. All AtGAAPs were confirmed to localise to the Golgi at low expression levels and AtGAAP1 and AtGAAP2 additionally localised to the tonoplast at higher expression levels. Analysis of single knock-out mutants of AtGAAPs revealed no obvious developmental or PCD-related phenotypes. Measurement of cytosolic Ca2+ rises following H2O2 or mannitol treatment in atgaap null mutants, transgenically expressing proaequorin, indicated a potential role for AtGAAPs in Ca2+ signalling, however, these data are preliminary. Several double and triple atgaap mutants have been generated, all of which display a wild-type growth habit suggesting either redundancy within the AtGAAP gene family or the existence of a subtle phenotype that is not apparent under the conditions used. Phenotypes have however been uncovered in plants overexpressing AtGAAP-YFP fusion proteins. AtGAAP1 overexpressors display a slight dwarf phenotype whereas AtGAAP2 overexpressors show severely twisted branches. AtGAAP5 overexpressors display a severe dwarf phenotype, enhanced senescence and development of spontaneous lesions in both rosette and cauline leaves. Moderate to high expression of AtGAAP5 presumably leads to lethality, as no transgenic plants that express AtGAAP5-YFP at these levels have been recovered.