Qiong Xiao | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES PhD Final Exam Seminar by Qiong Xiao. This seminar is open to the general public to attend.

PhD with Dr. Gavin Chen

Thesis Topic: Evolutionary, Biological and Physiological Characterization of Arabidopsis CTP: phosphocholine Cytidylyltransferase 1 (CCT1)

Abstract:

Phosphatidylcholine (PC) is the major phospholipid class in the non-plastidial membranes of plant cells. The de novo biosynthesis of PC via CDP-Choline pathway involves three sequential steps. Among these reactions, CTP:phosphocholine cytidylyltransferase (CCT1) catalyzes the conversion of phosphocholine and CTP into CDP-choline and pyrophosphate, a reaction considered the key regulatory step in certain plant species. The genome of Arabidopsis thaliana encodes two CCT isoforms, known as AthCCT1 and AthCCT2. The overall objective of this study is to advance our understanding of AthCCT1 through a combination of evolutionary, biochemical, and physiological approaches.

In the first study, a phylogenetic analysis of plant CCT genes was conducted to investigate the evolutionary history, genetic relationships, and structural variations among CCTs in the green lineage. To further explore the impact of selection pressure on the functional evolution of CCT genes, we conducted a selection pressure analysis on the representative gene AthCCT1 and investigated its biochemical properties through enzyme assays and protein structural analysis. The results revealed a widespread presence of CCT genes across green algae and land plants, with a notable expansion in eudicots. The phylogenetic division of the CCT gene family into eight primary clades was supported by the observed conservation and divergence in gene structures and motif patterns. The selection pressure analysis of AthCCT1, integrated with biochemical assays and three-dimensional structural investigation, has uncovered two amino acid sites under positive selection, emphasizing their important roles in modulating AthCCT1 enzyme activity and substrate affinity.

In the second study, the physiological roles of AthCCT1 in lipid biosynthesis and root development under osmotic stress were examined. Due to the lack of lipid profiling data in the cct1 cct2 knockout mutant, the precise role of AthCCT1 in PC biosynthesis is yet to be fully understood. Moreover, AthCCT1 contains a key phosphorylation site, Serine-187 (S187), which is regulated by Sucrose-nonfermentation1-related protein kinase1 (SnRK1). This SnRK1-mediated phosphorylation leads to approximately a 67% reduction in AthCCT1 enzyme activity. However, the effects of the phosphorylation at the S187 site on the dynamic and in vivo functions of AthCCT1 remain unclear. Accordingly, we generated Arabidopsis cct1 knockdown cct2 knockout lines and revealed their reduced PC intensity under normal conditions and impaired root growth in response to osmotic stress compared to the wild type, which could both be rescued by AthCCT1 overexpression. The S187D phosphomimetic mutant, where S187 is substituted by aspartic acid (D) to mimic the negative charge phosphorylation, displayed reduced enzymatic activity and altered structural properties, including reduced lipid-induced conformational changes and a more compact state compared to the native AthCCT1. Moreover, overexpression of the S187D was unable to restore the root growth phenotype under osmotic stress in the cct1 knockdown cct2 knockout lines, indicating that the mimicked phosphorylation state at S187 may influence AthCCT1’s enzyme function. Taken together, these findings highlight the role of AthCCT1 in PC biosynthesis and suggest that its phosphorylation possibly regulates both enzymatic activity and its physiological functions.

The third study aimed to further reveal the molecular mechanisms underlying the activity of AthCCT1, with an emphasis on its protein-protein interactions. The combination of yeast two-hybrid and bimolecular fluorescence complementation assays identified several interacting partners of AthCCT1, including AthCCT1 itself, AthCCT2, potential nuclear importin α and β subunits, and an Arabidopsis Sec14 family protein. These results shed light on the dimerization behavior of AthCCT1 and its role in forming potential protein complexes.

To summarize, this study investigates the roles of AthCCT1 in plant phospholipid metabolism, including its evolutionary history, structural dynamics, physiological functions, and protein-protein interactions. The research provides novel insights into AthCCT1’s involvement in PC biosynthesis, its important roles in plant development under stress conditions, and its participation in protein complexes, thereby contributing to a deeper understanding of its function in plant cellular processes.