Characterisation of phloem-localised AtACBP1 and AtACBP3

Abstract

First reported from rat brains, acyl-CoA-binding proteins (ACBPs) were initially identified as a neuropeptide ligand that inhibits diazepam from binding to brain synaptic membranes. In Arabidopsis thaliana, there are six ACBPs (referred to AtACBPs hereafter), varying from 10.4 to 73.1 kDa in size. Two AtACBPs that are localised in the phloem, AtACBP1 and AtACBP3, were chosen for characterisation in this study. Subsequently, an understanding of AtACBP3 in wound response (Chapter 3), floral development (Chapter 4) and of AtACBP1 interacting with ABSCISIC ACID–RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (Chapter 5) were sought. The results showed that AtACBP3, but not its mRNA, was detected in wild-type Col-0 phloem exudates, and AtACBP3 is likely to be a phloem-mobile protein. AtACBP3 was immunolocalised to the companion cells, sieve elements and the extracellular space of the phloem. The mRNA transcript of AtACBP3 was wound-inducible in Arabidopsis Col-0, and acbp3 mutant was impaired in wound responsiveness from measurements in expression of wound-responsive jasmonate marker genes. More interestingly, the content of oxylipin-related fatty acids (FAs), including methyl-jasmonate, was lower in acbp3. The results implied that AtACBP3 participates in the maintenance of a pool of FA/acyl-CoA esters in the phloem. The expression profile of AtACBP3 in flowers was investigated using transgenic AtACBP3::GUS (AtACBP3 promoter-driving BETA-GLUCURONIDASE) flowers. GUS expression in stigma and transmitting tract was observed in transgenic flowers from stages 8 to 15, suggesting that AtACBP3 may play a role in reproduction. Further phenotyping analysis showed that Landsberg erecta acbp3 mutants displayed morphological defects in the stigmata of acbp3-Ds and ACBP3-RNAi-Ler, and pollen tube growth was impaired in acbp3-Ds and ACBP3-RNAi-Ler. Mature green siliques (Ler) dissected for scanning-electron microscopy analysis showed fully-developed seeds in siliques, while aborted seeds frequently occurred in acbp3-Ds and ACBP3-RNAi-Ler. Measurements of average dry seed weight per plant indicated lower seed yield in acbp3-Ds and ACBP3-RNAi-Ler which was presumed associated with impaired pollen tube growth and reduced levels of unsaturated FAs. The results support a role for AtACBP3 in floral and fruit development in Arabidopsis. Characterisation of AtACBP1 arising from previous studies of AtACBP1::GUS expression in the vascular tissues initially focused on its potential role in the phloem. However, AtACBP1 was subsequently not detected in the phloem exudates. As AtACBP1 had been previously reported to play a role in salt and drought stress during seed germination and seedling development, and preliminary data had indicated that AtACBP1 may interact with AREB1, pull-down assays between GST-AREB1 and (His)6-AtACBP1 were carried out to confirm AtACBP1/AREB1 interaction in vitro. Furthermore, stable transgenic plants co-expressing AtACBP1 and DsRed-AREB1 were generated herein, and the localization of DsRed-AREB1 appeared to be affected in ACBP1-overexpressing plants. In conclusion, AtACBP1/AREB1 interaction may promote ABA signalling under salt and osmotic stress during seed to seedling transition in Arabidopsis.