PaRBOH1 and its activation by phosphorylation and Ca 2+

studied, as noted by Pinard and Mizrachi (2018). The authors propose that a term

´xyloplasts´ be used for these organelles. The results in study II show that eight genes encoding enzymes of the shikimate pathway are co-expressed with the monolignol biosynthesis baits across different datasets. However, only one of these was present in the proteomic data, phosphor-2-dehydro-3-deoxyheptonate aldolase 1 (MA_10436001g0020).

To test the hypothesis that PaRBOH1 plays a role in developmental lignification, a hairpin construct of parboh1 was created to silence this gene in Norway spruce (III).

Embryogenic cultures of spruce were transformed, and plants were regenerated by somatic embryogensis. The silencing, however, was not complete, as revealed by a real- time quantitative polymerase chain reaction (RT-qPCR) (III). This was quite unfortunate, as the aim was to resolve the role of PaRBOH1 in developmental lignification. As such, this remains an open question awaiting future studies.

However, many other aspects of PaRBOH1 were successfully studied: a sequence analysis of the cloned cDNA revealed that the N-terminus of the protein contains a four-times-repeated sequence that, in many other RBOHs studied so far, is present only once. We hypothesised that this repetitive part may be a target for regulation of enzyme activity. The enzyme was then heterologously expressed in human embryonic kidney (HEK) cell line HEK293T. These cells do not have NADPH oxidases and thus only generate low amounts of endogenous apoplastic ROS (Ogasawara et al., 2008).

The measurable ROS production in transformed cells was thus due to PaRBOH1 activity. As with many other RBOH enzymes that have been studied, Ca²⁺ activated ROS production in HEK cells expressing PaRBOH1. In addition, phosphorylation activated ROS production, as indicated by an increase in activity upon inhibition of phosphatases by calyculin A, and by a decrease in activity when a kinase inhibitor, K252a, was added. The highest enzyme activity was achieved by the combination of supplementation with 1 mM Ca²⁺ and inhibition of phosphatases. These results are in line with the regulation of a plethora of RBOHs that are activated by elevated Ca²⁺ and by phosphorylation (Hu et al., 2020). As PaRBOH1 is the first gymnosperm RBOH whose activity has been studied, the results suggest that the regulation of RBOHs is evolutionarily conserved across diverse plant taxa.

Additional insights into regulation could be achieved with a better understanding of the exact phospho-sites in this enzyme. In silico predictions and kinase assays with synthetic, small peptides representing short segments of PaRBOH1 N-terminus suggested specific residues in the amino acid sequence that could be the targets of phosphorylation. Next, the PaRBOH1 coding sequence was point-mutated to create either alanine or aspartate residues at the putative sites of phosphorylation. Alanine served as a kinase-inactive residue and aspartate chemically resembles serine residues which are phosphorylated, thereby mimicking phosphorylation (Dean et al., 1989).

Several point-mutated versions of PaRBOH1 were heterologously expressed in HEK cells and the ROS production was assessed. Multiple serine (S150, S160, S374) and threonine residues (T174, T190, T300) seemed to have a role in enzyme activation. In particular, the observation that phosphomimicking of T174 induced high ROS production suggested that this is an important regulatory residue. Also, relatively high ROS production was caused by phosphomimicking T300 and S374. However, phosphomimicking in the repetitive sequence gave inconsistent results, suggesting that this particular area of the protein does not control enzyme activity, at least if the sites are phosphomimicked one at a time.

As the kinase assay was done with short synthetic peptides, we repeated this assay using the full-length 400-amino-acid-long N-terminus of PaRBOH1, which was heterologously produced in E. coli. In vitro kinase assays with fresh MF and soluble protein preparations from the developing xylem of Norway spruce were used, and the N-terminus was then purified and analysed by LC-MS^E to detect phosphorylations.

Several serine and threonine residues were phosphorylated in vitro. In the region of the repetitive sequence, S32, S45, S58, and S71 (serine residues in each of the four repetative units) were phosphorylated at low levels. In Arabidopsis, Botrytis-induced kinase 1 (BIK1) phosphorylates the S39 residue of RBOHD, which corresponds to S71 in PaRBOH1. In Arabidopsis, this phosphorylation activates the enzyme, however Ca²⁺

is needed for full activity (Li et al., 2014) and this activation is part of immunity signalling (Kadota et al., 2014, 2015). It is possible that phosphorylating several serine residues in the repetitive sequence of PaRBOH1 would have led to increased enzyme activity. However, it is not known which kinase(s) in spruce might be responsible for phosphorylation of these residues. We made an effort to study the natural phosphorylation levels of PaRBOH1 in developing xylem. However, the abundance of PaRBOH1 protein was too low to study phosphorylation in vivo (III), so it remains a mystery whether the sites are phosphorylated during normal development. As this region of the protein was not clearly responsible for enzyme activation in the HEK cell assays, it is possible that the region serves in some other regulatory functions.

Outside of the repetitive region, mild phosphorylation without a sign of enzyme activation was detected in the LC-MS^E analysis at T75, T149, S154, S175, Y342, and S366. T16 and S188 were phosphorylated more highly. T16 was phosphorylated mainly by soluble kinases and S188 was phosphorylated by membrane kinases – but no

phosphomimicking nor kinase-inactivation assay in HEK cells were performed with this residue.

However, in an in vitro assay, T174 was highly phosphorylated by membrane kinases, and mildly by soluble kinases. The peptide containing this serine was also phosphorylated in in vitro peptide kinase assays. Phosphomimicking suggested that phosphorylation of T174 greatly activates PaRBOH1 enzyme activity. As MF membranes from developing xylem were used as a source of kinases, this suggests that such phosphorylation can occur during normal xylem development. These results suggest that kinases in the MF membranes could be interesting candidates for PaRBOH1 activation. There were 50 kinases detected altogether in the proteomic analysis (II), of which 46 were detected in Norway spruce developing xylem samples.

Among these kinases, a probable receptor-like protein kinase (MA_118589g0010) was co-expressed with monolignol biosynthesis genes in all the datasets (II). It is tempting to speculate that this kinase may be responsible for regulating enzymes involved in lignification. However, any discussion about a kinase for T174 without any evidence is extremely hypothetical. In the future, further experiments would be required to identify a kinase for PaRBOH1 activity regulation.