Conclusions on potential functions of Physcomitrella PPOs

In recent literature, different functions were suggested for seed plant PPOs. PPO-mediated reactions are proposed to take part for example, in pest and pathogen defence, in strong light stress response or in the inhibition of proteolysis. It was further hypothesised that PPOs might generate electron acceptors for the re-oxidation of cytokinin oxidase (CKX).

A general function for PPOs is not known. From the phylogenetic metagenome analysis performed in this study, it is assumed, that o-diphenol oxidases are likely to have evolved with the water to land transition of plants, and since then evolved to perform diverse functions in different plant species. Even within a single species, different functions for different PPO gene family members are most likely, such as reported by Thipyapong and colleagues who observed that different tomato PPO gene family members were upregulated under different stress conditions (Thipyapong et al., 2004b; Thipyapong and Steffens, 1997).

Analysing PPOs and their substrates in the basal organism Physcomitrella, might give access to primal PPO involvements. With this aim, results obtained from this work are discussed with respect to functional aspects in the following sections.

4.8.1. Different functions of different PPO gene family members in Physcomitrella

* The basal land plant Physcomitrella possesses a large PPO gene family

Physcomitrella possesses a large PPO gene family with 12 members, sharing structural similarities, but also exhibiting differences to seed plant PPOs. Nearly all PPO genes were found to be expressed; besides, they were differentially regulated under different culture conditions.

* PPO1 knockout plants exhibit phenotypic changes

PPO1 knockout lines exhibit significant phenotypic changes, although the very closely related paralogous gene PPO2 is still present and transcribed.

Due to these findings, it is strongly assumed, that the different PPO gene family members hold different functions within the organism.

>>> The different members of the Physcomitrella PPO multigene family are likely to be involved in different processes (Fig. 4.5).

4.8.2. Possible involvement in establishment of proper environmental conditions

* Portion of total PPO activity is detectable in the culture medium

Physcomitrella possesses extracellular PPO activity. Analysis of PPO1_ko lines revealed that a large portion of the overall extracellular PPO activity was caused by PPO1 secretion.

* PPO1 and PPO8 gene expression is increased after incubation with caffeic acid

Thegene expression level of PPO1 was 3-fold increased in wild type tissue grown in the presence of caffeic acid. PPO8, not expressed under standard cultivation conditions, was transcribed after incubation with caffeic acid.

* PPO1 knockout plants exhibit a decreased tolerance towards 4-methyl catechol

Application of 4-methyl catechol to culture medium caused an enhanced susceptibility of plants lacking the secreted PPO1 protein.

Hence, the increased transcript level of PPO1 in the presence of caffeic acid as well as the decreased tolerance towards 4-MC of PPO1 knockout plants, suggests that PPO1 as a major extracellular PPO, is responsible for the conversion and detoxification of extracellulary occurring, growth inhibiting phenolic compounds.

>>> Secretion of Physcomitrella PPOs, more specifically PPO1 (and PPO8), suggests a role in establishment of appropriate extracellular conditions, like the removal of (growth inhibiting) phenolic compounds (Fig. 4.5).

4.8.3. Possible involvement in light stress adaptation

* PPOs occurred with the conquest of land

PPOs presumably occurred with the water to land transition, that was accompanied by the adaptation to harsh environmental conditions such as exposure to strong (UV) light.

* Upregulation of several PPO gene family members under strong light exposure

Irradiation with strong light caused an upregulation of PPO4 and PPO12 gene expression. PPO10, not expressed under standard cultivation conditions, was transcribed under strong light exposure.

>>> Transcriptional upregulation of Physcomitrella PPOs, more specifically PPO4, PPO12 (and PPO10), under strong light irradiation, suggests a role in adaptation mechanisms under strong light exposure (Fig. 4.5).

* PPO1 might not be involved in light stress adaptation

As PPO1 gene expression was decreased in wild type tissue irradiated with strong light, and PPO1_ko lines exposed to strong light exhibited only slight changes in PPO2 to PPO12 expression, as well as no differences in changes of photosynthetic activity compared to wild type, it can be assumed that PPO1 is not involved in light stress response.

4.8.4. Possible involvement in promotion of cytokinin degradation

* PPO1 knockout lines exhibit an enhanced differentiation

PPO1_ko plant produced significantly more gametophores than wild type under standard in vitro cultivation condition.

* PPO1 knockout plants possess reduced in vivo cytokinin degradation

Cytokinin feeding experiments revealed that plants lacking PPO1 had a reduced in vivo CKX activity. Consistent with the hypothesis of Galuszka et al. (2005), a correlation between the PPO-mediated oxidation of phenolic compounds promoting the re-oxidation of CKX enzyme can be explained with these results (Fig. 4.4).

* Putative phenolic compounds are secreted to the Physcomitrella culture medium

Physcomitrella secreted few putative phenolic-like compounds into the culture medium. In vivo these compounds might serve as substrates for an extracellular PPO(1)-mediated oxidation promoting the re-oxidation of extracellular CKX enzyme.

>>> Physcomitrella PPOs, more specifically PPO1, are likely to be involved in regulation or tuning of differentiation processes (Fig. 4.5).

Fig. 4.5 Schematic overview of hypothetical involvements of Physcomitrella PPOs concluded from the results of this work. Results obtained from this research propose Physcomitrella PPOs being involved in multiple processes, such as coping with strong light exposure (mainly PPO4 and PPO12) and metabolism of extracellular occurring phenolics (mainly PPO1) as well as involvement in differentiation processes (PPO1).

Elements marked in red are supported by results from this work and are discussed in section 4.4 to 4.6.