Effect of ABP1 Inactivation on the Transcriptome and Nuclear Proteome in Arabidopsis

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ABP1 and early auxin responses

Evidence for a role of ABP1 in auxin signalling was obtained from electrophysiological studies using protoplasts. These experiments showed that addition of ABP1, or peptides corresponding to the C-terminal region of ABP1, to protoplasts without addition of auxin, gives an auxin-like early response involving plasma membrane ion fluxes and protoplast swelling (Barbier-Brygoo et al., 1991; Thiel et al., 1993; Gehring et al., 1998; Leblanc et al., 1999a). Treatment of protoplasts with auxin causes hyperpolarisation of the plasma membrane, which is dependent on ABP1 and involves ion fluxes resulting from activation or deactivation of ion channels (K+ and anions) or transporters (H+); antibodies which either inhibit ABP1 or mimic auxin-binding to ABP1 can block or promote the hyperpolarisation response, respectively (Ephritikhine et al., 1987; Barbier-Brygoo et al., 1989; Leblanc et al., 1999b).
ABP1 is involved in auxin-induced swelling of protoplasts derived from maize coleoptiles and pea (Pisum sativum) internodes (Steffens et al., 2001; Yamagami et al., 2004). This swelling response is similar to the growth response: it depends on potassium (K+) channels (Keller and Van Volkenburgh, 1996) and the effect is observed with active auxins, but not the inactive analogue 2-NAA (Steffens and Lüthen, 2000).
Auxins differ in their ability to activate ABP1 responses, because they show different placement in the ABP1 binding pocket, which affects their affinity and activity (Napier, 2004; Dahlke et al., 2009). They differ in binding to box A and box C, and their placement in the binding site affects interaction with W151, which may cause differences in activity (Figure 1-5) (Dahlke et al., 2009). For example, IAA binds to both box A and C, whereas 2-NAA binds only to box A. Binding affinity is higher for 2-NAA than 1-NAA and in turn higher than IAA (Dahlke et al., 2009). However, 2-NAA exhibits very low activity, and does not induce protoplast swelling (Steffens and Lüthen, 2000), perhaps because it does not interact with W151 (Dahlke et al., 2009).
An antibody to ABP1, mAb12, binds an epitope containing residues from both boxes A and C. It inhibits the auxin-induced hyperpolarisation response in tobacco (N. tabacum) protoplasts (Leblanc et al., 1999b). When expressed in a plant as scFv12 (single chain Fragment variable) derived from mAb12, it inhibits ABP1 (Leblanc et al., 1999b; David et al., 2001; David et al., 2007). In contrast, apoplastic treatment with scFv12 was shown to induce protoplast swelling; this discrepancy could be because in a plant, scFv12 interacts with ABP1 in the ER which could lead to degradation of the protein (Dahlke et al., 2009).

ABP1 during plant development

As shown in the previous section, ABP1 is an auxin receptor involved in early auxin responses. Over the last 12 years progress has also been made to determine the role of ABP1 during plant development, including cell expansion and division at many stages of development. The ABP1 protein appears to be expressed throughout the plant, since it has been found in every tissue analysed (reviewed by Napier et al., 2002). The activity of the ABP1 promoter was investigated by Klode et al. (2011), and promoter expression was localised to regions of growth such as young leaves and the root meristem.
ABP1 function has been investigated using molecular genetics, and this analysis has revealed the importance of ABP1 in plant development. Chen et al. (2001a) identified a T-DNA insertion abp1 mutant of Arabidopsis. The homozygous null mutant exhibited embryonic lethality, with arrested development at the globular stage of embryogenesis, and defective cell elongation, division and patterning. This result was very interesting, showing that ABP1 is essential for plant development. It was later discovered that the heterozygous abp1 mutant also displays a phenotype, with changes in auxin transport and expression of auxin-regulated genes (Effendi et al., 2011). The embryonic lethality provided a challenge for investigating the function of ABP1 in subsequent developmental stages. To overcome this problem, a novel method to conditionally decrease the activity of ABP1 protein in tobacco (N. tabacum) BY2 cells was developed by David et al. (2007). These lines express an scFv „mini-antibody‟, which blocks ABP1 activity, and is controlled by the tetracycline de-repressible promoter. Braun et al. (2008) generated conditional abp1 mutants of Arabidopsis. They expressed SCFV12 or ABP1 antisense under control of an AlcA promoter, so that ABP1 is inactivated when ethanol is added. They showed that ABP1 is required for Arabidopsis postembryonic development, in addition to its essential embryonic role. They observed stunted shoots and changes in leaf size and shape.
Therefore, ABP1 is essential for shoot and leaf growth and development (Braun et al., 2008). A subsequent study by Tromas et al. (2009), which also used the conditional abp1 mutant, found that ABP1 is required for root development as well, since inactivation results in stunted roots. So it is clear that ABP1 plays a key role in various aspects of plant development.

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Chapter 1. Introduction
1.1. Auxin biosynthesis and transport .
1.2. Overview of auxin signalling .
1.3. TIR1/AFB signalling.
1.4. ABP1 signalling
1.5. Cell-wall growth and lignification
1.6. The importance of auxin signalling and ABP1 across the plant kingdom
1.7. Aims and significance of the project
Chapter 2. Materials and Methods
2.1. Enzymes, chemicals and consumables
2.2. Plant material and growth conditions
2.2.10. Statistical analyses
2.3. Molecular biology techniques
2.4. Cloning
2.5. Proteomics preparation and processing
2.6. Histology and microscopy
Chapter 3. Effect of ABP1 Inactivation on the Transcriptome and Nuclear Proteome in Arabidopsis .
3.1. Introduction
3.2. ABP1 inactivation trials for seedlings in liquid culture and on rockwool
3.3. RNA processing and quality control ..
3.4. Microarray processing and quality control .
3.5. Microarray results: an overview
3.6. Auxin- and other hormone-related gene
3.7. ABP1 inactivation compared to auxin treatment microarray data
3.8. Many signalling genes were affected by ABP1 inactivation
3.9. Cell cycle genes
3.10. Genes involved in cell-wall expansion and lignification .
3.11. Nuclear proteome
3.12. Discussion.
3.13. Conclusion .
Chapter 4. Signalling Candidate Gene Mutant Analysis
4.1. Introduction
4.2. Selection of candidate ABP1 pathway signalling genes
4.3. Shoot and root expression of selected signalling genes
4.4. Genotyping of candidate gene T-DNA mutants and confirmation of knocked-out expression of the gene
4.5. Phenotyping of candidate gene T-DNA mutants
4.5.1. Preliminary screen for auxin resistance
4.6. Epistatic analysis of the candidate gene mutants with the conditional abp1 mutant
4.7. Discussion
4.8. Conclusion
Chapter 5. Cell-Wall Histochemistry and Proteome of ABP1-Inactivated Stems
Chapter 6. Analysis of ABP1 Localisation Using Tagged ABP1
Chapter 7. Final Discussion
References
Appendices