Proteomics perspective on auxin biology

Summary

The plant hormone auxin profoundly affects many aspects of plant growth and development. Since its discovery, the pathway leading to alteration in gene expression has been well documented. Strikingly, this nuclear auxin pathway (NAP) is short and consist of only three dedicated components, the SCF TIR1/AFB auxin receptor complex, AUX/IAA co-repressors and the auxin transcription factors (ARFs). Due to the simplicity of this pathway, a major question in the auxin field is how specificity is determined. In Chapter 1, I provide an introduction of the NAP, other physiological effects elicited by auxin, and how proteomic approaches might shed more light on auxin signalling.

In Chapter 2, we provide a deeper insight into ARFs by dissecting and highlighting current views on ARF functioning. Since ARFs are the direct output of the NAP, we reason that the specificity within auxin signalling must be controlled by ARFs. Numerous aspects of ARF functioning may contribute to specificity. This can be deducted from specific cellular expression of ARFs, the promotor architecture where ARFs bind to, the combinatorial interactions amongst the NAP components and the functioning of ARF domains. We highlight that, although the DBD and PB1 domains of ARFs have been structurally and to some extent functionally resolved, the specificity in ARF functioning might reside in the middle region. From predictions, it appears that the middle region is intrinsically disordered. This might provide a signalling hub for ARF functioning. Intrinsically disordered regions have no structure but can provide platforms for co-factor interactions. 

That ARF co-factors are important for auxin output has been reported in a scattered fashion and is not clearly documented. We therefore, in Chapter 3, investigated which co-factors interact with ARFs. We utilized an unbiased quantitative affinity purification mass spectrometry approach to decipher the ARF interactome. Initial strategies utilizing conventional AP-MS/MS proved to be too cumbersome to retrieve ARF co-factors. We reasoned that the co-factors are probably too transient to survive the affinity purification procedure, and utilized crosslinkers to “freeze” and maintain the interactions during purification procedure. Optimisation of this strategy proved to be too cumbersome to be applied in a holistic ARF interactomic approach. Eventually, we integrated and optimized proximity labelling using BioID. This strategy tags neighbouring proteins with a biotin group within the cell allowing to capture “interacting” proteins of ARFs. Through this approach, we identified ARF-ARF and ARF-TPL interactions.

Besides the regulation of the NAP by auxin other non-NAP effects have been described. Auxin can for example elicit rapid membrane depolarisation and Ca2+ spiking. Other reports also implicated kinase cascades in auxin dependent processes. To dissect this, we employed (phospho)proteomic techniques. It can be generally noted that the field of plant proteomics is not as advanced as the field of animal proteomics. Therefore in Chapter 4, we first integrated and optimized sample preparation techniques for shotgun and phosphoproteomic techniques in plant. We interrogated single-vessel-based approaches and offline stagetip-based peptide fractionation techniques to gain deeper and reproducible proteomes. Our results show that simple and cost-effective strategies can be employed to generate high-quality proteome coverage for plant research. Further efforts were undertaken to compare phosphopeptide enrichment strategies. This revealed that metal based phosphopeptide methods outperform metal-oxide based methods.

In Chapter 5, we employed the optimized phosphopeptide enrichment procedure to dissect whether fast auxin response is mediated by phosphorylation dependent processes. Our results show that auxin can elicit rapid phosphorylation changes within 2 minutes and that these responses are TIR1-independent. Physiological responses to auxin are widespread in the plant kingdom, and can also be detected in algae that lack NAP components. We therefore further asked if the fast phosphorylation response may be part of a more ancient auxin response system. Our analysis revealed a deep evolutionarily conserved auxin response, and identified a PB1 domain-containing MAPKKK to mediate auxin-dependent regulation.

Eventually, in Chapter 6 we conclude this thesis and discuss the implications and context of our results. We further provide perspectives for future plant proteomic studies.