Reproducibility-Optimized Test Statistic (ROTS)

Reproducibility-Optimized Test Statistic (ROTS) was used as an ’R’ package as described by Pursiheimo et al., (2015). The number of bootstraps was set to 100 and the top list was set to 1

Chapter 3

Expression and Purification of

Rapid Alkalinisation Factor1

(RALF1)

3.1

Introduction

3.1.0.1 Rapid Alkalinisation Factor

Cell to cell messages are fundamental to many processes within the plant. The signalling peptide Rapid Alkalinisation Factor (RALF) was identified by Pearce et al. (2001) and is believed to be involved in growth and de- velopment. RALF was named after its ability to rapidly raise the pH of the media when applied to a cell culture (Pearce et al., 2001). The RALF family consists of 34 proteins in Arabidopsis, with high levels of homology with a hydrophobic region at the C-terminal end of the protein (Pearce et al., 2001). A review paper by Murphy and De Smet (2014) shows ten of the

RALF family proteins (including RALF1) share a conserved signal peptide, a propeptide with a dibasic Arg-Arg cleavage site just before the mature peptide. RALF23, one of the RALF family proteins that is highly homolog- ous with RALF1, has been shown to be cleaved by site-1 protease just before the junction between the propeptide and mature RALF1, leaving the mature RALF peptide containing four cysteines (Srivastava et al., 2009). Pearce et al mapped two disulfide bonds in the mature RALF1 peptide using Mass Spectrometry (MS) and showed a bridge between cys-18 and cys-28 and a bridge between cys-41 and cys-47 (Pearce et al., 2001). By reducing and alkylating the disulfide bonds, Pearce et al., (2001) showed the activity of RALF1 was lost, meaning that the structure of RALF1 is crucial for its func- tion. A hydrophobic conserved motif (YISY) at the N-terminal of RALF1 was also identified as being crucial for RALF function (Pearce et al., 2010). A landmark paper identified the peptide RALF1 as a ligand for the receptor- like kinase FERONIA (FER). RALF1 interacts with the extracellular do- main of the plasma membrane localised FER and the ’YISY’ motif at the N- terminal of the RALF1 peptide is essential for interaction with FER (Haruta et al., 2014). Exogenous RALF1 lead to the inhibition of the plasma mem- brane ATPase, AHA2, leading to the alkalinisation of the apoplast, which is known to cause cell elongation inhibition (Haruta et al., 2014). When RALF1 is applied exogenously to sugar cane, inhibition of primary root growth and the number of lateral roots was reduced, indicating a strong link with RALF1 inhibiting cell elongation in Arabidopsis (Mingossi et al., 2010). RALF1 was also shown to reduce hypocotyl length as well as root length but did not have any effect on pollen grain germination (Mingossi et al., 2010). In relation to FER signalling in roots, mutants of FER have

SP Propeptide RALF1 5’ 3’ RALF1 72 - 120 1 - 26 27 - 71 Sequence RR Cleavage site HHHHHH ATTKYISYQSLKRNSVPCSRRGASYYNCQNGAQANPYSRGCSKIARCRS 5’ 3’ Deleted in RALF-Null 6His-

RALF1 6 - His LinkerAIA

Disulfide bridge

A

B

RALF1

Disulfide bridge

Cys-18 Cys-28 Cys-41 Cys-47

Figure 3.1: Schematic of RALF1

expressed within the developing root, considerably higher than in any other tissue (Klepikova et al., 2016).

Many defence related peptides lead to mitogen activated protein kinase (MAPK) activation when applied to plants. The peptide PEP1 was shown to induce MAPK activation after 15 minutes (Bartels et al., 2013). RALF causes a peak MAPK activation after five minutes, which almost completely reduced by 30 minutes (Pearce et al. 2001). This is much quicker than a large number of known defence related peptides including systemins, which activate MAPKs after 15 minutes (Pearce et al., 2001).

To study the downstream signalling events of RALF1, two synthetic RALF1 (RALF1-Active and RALF1-Null) constructs were produced by IDT (Figure 3.1). RALF1-Active is the bioactive form of the peptide, with a N-terminal his-tag (HHHHHH) and linker (AIA) followed by the active peptide (AT- TKYISYQSLKRNSVPCSRRGASYYNCQNGAQANPYSRGCSKIARCRS) as used by Haruta et al (2014). The Null version was also used by Haruta et al. (2014) and contains the same his-tag and linker but with an eight amino acid deletion (ATTKYISY) from the beginning of the peptide sequence, which prevents RALF1 from interacting with FER.

brassinosteroid signalling. Plants over-expressing RALF1 were found to be less affected by exogenous brassinolide (a brassinosteroid (BR) involved in promoting plant growth) and exogenous RALF1 induced two negatively reg- ulated brassinosteroid-related genes, CONSTITUTIVE PHOTOMORPHO- GENIC DWARF (CPD) and DWARF 4 (Bergonci et al., 2014), suggesting that RALF1 may act antagonistically with brassinosteroid signalling. A review by Murphy and De Smet (2014) proposed that RALF1 and brassi- nolide may act through shared signalling components because the addition of RALF1 led to the upregulation of some cell wall-remodelling enzymes such as PROLINE RICH PEPTIDE 1 (PRP1) and TOUCH 4 (TCH4), which was greatly reduced when treated with RALF1 and Brassinolide simultan- eously (Bergonci et al., 2014). Furthermore, Haruta et al also showed that RALF1 caused the down regulation of some key genes involved in cell ex- pansion including brassinosteroid-6-oxidase 2 (BR6OX2) and that this down regulation was lost in the fer-4 mutant (Haruta et al., 2014).

3.1.0.2 Identifying X-linked peptides

Studying the structure and topology of proteins and complexes with mass spectrometry (MS) is becoming increasingly popular. StavroX is a leading software for the analysis of cross-linked peptides (Gotze et al., 2012). The software works by predicting tryptic digests from a protein FASTA sequence and calculates all possible theoretical cross-linked peptide candidates and then calculates all possible b and y ion series for a given cross-link site. The spectra are assigned by pattern matching and StavroX then scores how well the expected and observed fragmentation ions match up, while taking into

(Gotze et al., 2012).

3.1.0.3 Aims

The aim of this chapter was to purify large quantities of RALF1 to enable our research into the RALF1-FER signalling pathway by looking at root growth inhibition. Once RALF1 was purified the aim was to test the activity of the purified RALF1 and to test which RALF1 responses depend on FERONIA in our system.