Functional analysis of cytosolic acyl-coenzyme a-binding proteins in floral and seedling development

In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) are distributed across subcellular compartments: AtACBP1 and AtACBP2 in the endoplasmic reticulum and plasma membrane, AtACBP3 targeted to the extracellular space, and AtACBP4, AtACBP5, and AtACBP6 in the cytosol. Although some AtACBPs have demonstrated non-redundant biological functions in plant stress responses, AtACBP1 and AtACBP2 have been shown to play overlapping roles in embryogenesis. Herein, the roles of the cytosolic AtACBP4, AtACBP5, and AtACBP6 in floral and seedling development were addressed. To this end, a T-DNA insertional knockout mutant of acbp5 was first identified and characterized before crosses with the already available acbp4 and acbp6 mutants to examine their independent and combinatory functions in floral and seedling development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuoles accumulated in acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen with the most severe abnormalities occurring in the triple mutant. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant and its in vitro pollen germination rate was observed to reduced. Taken together, these results suggest the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development. To correlate the biological activities of AtACBP6, AtACBP4, and AtACBP5 with their lipid-binding abilities, their binding with acyl-CoA esters were investigated using isothermal titration calorimetry (ITC). Recombinant His-tagged AtACBP6 (rAtACBP6), rAtACBP4, and rAtACBP5, were shown to bind long-chain acyl-CoA esters in ITC and the binding kinetics (e.g. dissociation constant, stoichiometry, enthalpy change of ligand binding) were obtained. As data mining using the Arabidopsis Electronic Fluorescent Pictograph (eFP) browser showed that AtACBP4, AtACBP5, and AtACBP6 were highly expressed during seed development, the double and triple mutants of the cytosolic AtACBPs were again used to investigate their roles in seed and seedling development besides pollen development. The overlapping roles of cytosolic AtACBPs in seed development and germination were analyzed using seed germination assays on the double and triple mutants of these cytosolic AtACBPs under abscisic acid (ABA) treatment because ABA is a plant hormone with significant roles in (i) seed dormancy and germination, (ii) seedling establishment, (iii) plant development and (iv) responses to drought and salinity stresses. Among all these double and triple mutants, the acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to ABA during germination, suggesting their physiological functions in seeds. Given that clock regulation of AtACBPs has only been reported in 4-week-old rosettes but not seedlings, the expression of AtACBPs in 2- and 5-day-old seedlings of the cca1lhy mutant, the CCA1 over-expressing line (CCA1-OX), and the wild types (WS and Col-0) was studied. The expression patterns of all AtACBP mRNAs in the cca1lhy mutant were slightly deviated from the wild type. Although the diurnal expression pattern of AtACBPs in 2- and 5-day-old seedlings germinated under 12-h-light/12-h-dark cycles of wild-type Col-0 observed in this study showed some differences from results conducted on 4-week-old rosettes under 16-h-light/8-h-dark, similarities in AtACBP3 expression between seedlings and rosettes were noted. Indeed, AtACBP3 expression displayed the most significant changes between the cca1lhy mutant and WS and between CCA1-OX and Col-0, consistent with the previous reports demonstrating that AtACBP3 mRNA is most affected by light/dark cycling and the 5’-flanking region of AtACBP3 is responsive to dark/light. As to whether diurnal factors influence the mobilization of plant lipid storage reserves was further addressed in this study. An impaired germination phenotype accompanied by oil body retention was observed in freshly-harvested, after-ripening, and after-ripened seeds of the cca1lhy mutant in comparison to wild-type WS. Comparative quantitative real-time PCR (qRT-PCR) analysis on the expression of genes in lipid metabolism among 2- and 5-day-old seedlings from the cca1lhy mutant, CCA1-OX, and wild-type Arabidopsis (WS and Col-0) showed diurnal variations in lipid metabolism at transcription, suggesting an effect of CCA1. Lipid profiling further revealed differences in derivatives of acyl-CoA esters, fatty acid methyl esters, and triacylglycerols among cca1lhy, CCA1-OX, and wild-type Arabidopsis seedlings, suggesting that lipid metabolism including storage reserve mobilization is subject to diurnal regulation. In summary, this study shows the importance of AtACBP4, AtACBP5, and AtACBP6 in floral and seedling development. It also demonstrates that lipid metabolism in seedling development is a dynamic process and is subject to diurnal control.published_or_final_versionBiological SciencesDoctoralDoctor of Philosoph