Abstract
Key message
We herein demonstrated two of the Arabidopsis acyl-CoA-binding proteins (ACBPs), AtACBP4 and AtACBP5, both function in floral lipid metabolism and they may possibly play complementary roles in Arabidopsis microspore-to-pollen development. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the AtACBP4 and AtACBP5 promoters, as well as, qRTPCR analysis revealed that AtACBP4 was expressed at stages 11–14 in the mature pollen, while AtACBP5 was expressed at stages 7–10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatographyflame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds in comparison to the wild type (Col-0). Fatty acid profiling demonstrated a decline in stearic acid and an increase in linolenic acid in acbp4 and acbp4acbp5 buds, respectively, over Col-0. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5′-flanking regions indicated the minimal promoter activity for AtACBP4 (−145/+103) and AtACBP5 (−181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) mapped at AtACBP4 (−157/−153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting.
Abstract
In Arabidopsis thaliana, six acyl-CoA-binding proteins (ACBPs), designated as AtACBP1 to AtACBP6, have been identified to function in plant stress and development. AtACBP4 and AtACBP5 represent the two largest proteins in the AtACBP family. Despite having kelch-motifs and sharing a common cytosolic subcellular localization, AtACBP4 and AtACBP5 differ in spatial and temporal expression. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the respective AtACBP4 and AtACBP5 promoters, as well as, qRT-PCR analysis revealed that AtACBP4 was expressed at stages 11–14 in mature pollen, while AtACBP5 was expressed at stages 7–10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatography-flame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds, in comparison to the wild type. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5′-flanking regions indicated the minimal promoter region for AtACBP4 (−145/+103) and AtACBP5 (−181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) within AtACBP4 (−157/−153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. These results suggest that AtACBP4 and AtACBP5 both function in floral lipidic metabolism and they may play complementary roles in Arabidopsis microspore-to-pollen development.
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Abbreviations
- ACBP:
-
Acyl-CoA-binding protein
- DAG:
-
Diacylglycerol
- DFA:
-
Dicarboxylic fatty acid
- DGDG:
-
Digalactosyldiacylglycerol
- EMSAs:
-
Electrophoretic mobility shift assays
- FA:
-
Fatty acid
- FID:
-
Flame ionization detector
- GC-MS:
-
Gas chromatography-mass spectrometry
- GUS:
-
β-Glucuronidase
- HFA:
-
Hydroxy fatty acid
- MGDG:
-
Monogalactosyldiacylglycerol
- PC:
-
Phosphatidylcholine
- PE:
-
Phosphatidylethanolamine
- PI:
-
Phosphatidylinositol
- SEM:
-
Scanning electron microscopy
- TEM:
-
Transmission electron microscopy
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Acknowledgements
We thank Professor Wanqi Liang [Shanghai Jiao Tong University (SJTU)] for critical reading of the manuscript, Wing-Sung Lee [Electron Microscope Unit, the University of Hong Kong (HKU)] and Lu Zhu (SJTU) for help in electron microscopy, Dr. Guorun Qu (SJTU) for help in wax and cutin analysis, and Jonathan Tau for pollen collection. This work was supported by the Wilson and Amelia Wong Endowment Fund, the Research Grants Council of Hong Kong (HKU765813M) and a HKU CRCG award (104003169). ZWY was supported by a University Postgraduate Fellowship. JX and JS were supported by the Program of Introducing Talents of Discipline to Universities (111 Project, B14016) and the National Natural Science Foundation of China (31370026 & 31570312).
Author contributions
This study was designed, directed and coordinated by MLC and DZ. MLC provided the conceptual and technical guidance through the project. ZWY planned and performed the GUS-sectioning, qRT-PCR analysis, scanning and transmission electron microscopy (SEM; TEM), lipid analysis, pollen tube germination, electrophoretic mobility shift assays and DNase I footprinting. JX performed the analysis of SEM and TEM; JS contributed to the wax and cutin analysis. The manuscript was written by ZWY and MLC and commented by all authors.
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Ye, ZW., Xu, J., Shi, J. et al. Kelch-motif containing acyl-CoA binding proteins AtACBP4 and AtACBP5 are differentially expressed and function in floral lipid metabolism. Plant Mol Biol 93, 209–225 (2017). https://doi.org/10.1007/s11103-016-0557-5
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DOI: https://doi.org/10.1007/s11103-016-0557-5