Distinct expression profiles of acyl-CoA-binding proteins AtACBP4 and AtACBP5 during pollen development

Flower development is a process during which plants must progress from sexual immaturity to maturity. Throughout these phases, the differential expression of many genes and proteins is evident. In angiosperms, the pollen generates male sperms to facilitate pollination and fertilization but the molecular mechanisms leading to pollen development that are related to  lipid changes have remained largely elusive. Recently, we investigated the underlying molecular regulatory mechanisms of two kelch-motif-containing acyl-CoA-binding proteins, AtACBP4 and AtACBP5, during flower development. These two proteins, which shared 81.4% amino acid identity and contain an acyl-CoA-binding domain that binds lipids, were distinctly expressed, and exhibited diversified and complementary roles in lipid metabolism during pollen development.

Fig. 1. Analysis of GUS expression in transgenic Arabidopsis flowers transformed with AtACBP4pro::GUS and AtACBP5pro::GUS and their deletion derivatives.
Histochemical GUS stains of transgenic Arabidopsis 8-week-old flowers transformed with AtACBP4pro::GUS constructs (pAT698, pAT699 and pAT700) and AtACBP5pro::GUS constructs (pAT642, pAT668, pAT667, pAT640 and pAT669). Inflorescences from 8-week-old Arabidopsis transformants were stained with substrate X-gluc.

The earlier expression of AtACBP5 in anther development was detected in the microspores as well as the tapetum, endothecium and epidermis. AtACBP5 was most highly expressed before stage 9 when the petal primordia elongate. During stages 9-10 when the microspores vacuolate to expand their size, AtACBP5 was more highly expressed in the tapetal cells than the microspores. When the tapetal cells started to degrade at stage 10, AtACBP5 was no longer detected on immunoelectron microscopy using anti-AtACBP5 antibodies. On the other hand, AtACBP4 was expressed during the later stages (11-14) of anther development, in the pollen grains and the endothecium.

When the lipid-associated roles of AtACBP4 and AtACBP5 in pollen development were investigated, wax analysis of acbp4 and acbp4acbp5 mutant flower buds showed a significant increase in C29-alkanes in comparison to Col-0. Fatty acid profiling demonstrated a decrease in stearic acid and an increase in linolenic acid in the 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. Interestingly, ɑ-amylose content decreased in acbp5 but not acbp4. Given that our previous study had revealed that recombinant AtACBP4 and AtACBP5 could bind oleoyl-CoA in vitro and that oleoyl-CoA is known to passively regulate starch synthesis, it would be interesting to follow up on how AtACBP4 could affect starch synthesis.

To investigate the regulation of stage-specific expression for AtACBP4 and AtACBP5 in anthers, deletion analysis of their 5’-flanking regions was carried out (Fig. 1). Our results defined the minimal promoter regions for AtACBP4 (-145/+103) and AtACBP5 (-181/+81). Electrophoretic mobility shift assays (EMSAs) further identified a pollen-specific cis-acting element POLLEN1 (AGAAA) at AtACBP4 (-157/-153) which interacted with nuclear proteins from flower, and this was substantiated by DNase I footprinting assays. The POLLEN1 box, mapped in the AtACBP4 5’-flanking region, represents the first functional POLLEN1 to be reported in Arabidopsis. Thus far, specific elements that control the early-staged expression of AtACBP5 remain unindentified and warrants future investigations.

Zi-Wei Ye, Mee-Len Chye
School of Biological Sciences, The University of Hong Kong Pokfulam, Hong Kong, China

 

Publication

Kelch-motif containing acyl-CoA binding proteins AtACBP4 and AtACBP5 are differentially expressed and function in floral lipid metabolism.
Ye ZW, Xu J, Shi J, Zhang D, Chye ML
Plant Mol Biol. 2017 Jan

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