plants Article Eukaryotic and Prokaryotic Phytochelatin Synthases Differ Less in Functional Terms Than Previously Thought: A Comparative Analysis of Marchantia polymorpha and Geitlerinema sp. PCC 7407 Erika Bellini 1,4, Claudio Varotto 2 , Marco Borsò 3 , Lorenza Rugnini 4, Laura Bruno 4 and Luigi Sanità di Toppi 1,* 1 Department of Biology, University of Pisa, 56126 Pisa, Italy; [email protected] 2 Department of Biodiversity and Molecular Ecology, Research and Innovation Centre, Fondazione Edmund Mach, 38100 San Michele all’Adige (TN), Italy; [email protected] 3 Department of Surgery, Medical, Molecular, and Critical Area Pathology, University of Pisa, 56124 Pisa, Italy; [email protected] 4 Department of Biology, University of Rome “Tor Vergata”, 00133 Roma, Italy; [email protected] (L.R.); [email protected] (L.B.) * Correspondence: [email protected]; Tel.: + 39-0502211333 Received: 17 June 2020; Accepted: 16 July 2020; Published: 20 July 2020 Abstract: This paper reports functional studies on the enzyme phytochelatin synthase in the liverwort Marchantia polymorpha and the cyanobacterium Geitlerinema sp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 µM cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise, in vivo exposure to 10–36 µM cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels in M. polymorpha were constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels in Geitlerinema sp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as in M. polymorpha and other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought. Keywords: bryophytes; cadmium; cyanobacteria; glutathione; heavy metals; liverworts; phytochelatins; phytochelatin synthase 1. Introduction Phytochelatin synthase (PCS) is a γ-glutamylcysteine-dipeptidyl-(trans)peptidase (EC 2.3.2.15) belonging to the “Clan CA” of papain-like cysteine proteases [1,2]. In eukaryotes, PCSs are expressed in the vast majority of land plants [2–4] and algal groups [5–7], as well as in a number of Fungi (including lichens), Animalia, Amoebozoa, Stramenopila, Alveolata, Rhizaria, and Excavata [2,3,8–10]. Eukaryotic PCSs, particularly plant PCSs, display manifest transpeptidasic activity by catalyzing the cytosolic synthesis of phytochelatin (PCn) thiol-oligopeptides, whose general structure is (γ-glutamate–cysteine)n-glycine, with n ranging from 2 to 5; for this reason, PCn oligomers are named PC2, PC3, PC4, and PC5. The cysteine thiol-groups enable PCn to chelate several metal(loid)s Plants 2020, 9, 914; doi:10.3390/plants9070914 www.mdpi.com/journal/plants Plants 2020, 9, 914 2 of 12 and segregate them in the vacuo-lysosomal compartments [2,11], thus drastically reducing the metal(loid) toxicity. The transpeptidasic activity of PCS is manifested when the enzyme’s catalytic site tightly binds complexes between glutathione in reduced form (GSH) or its direct thiol-derivatives, and metal(loid)s such as cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As), copper (Cu), zinc (Zn), and iron (Fe) [3,11]. In prokaryotes, PCS enzymes with moderate sequence homologies to those of plants have been found in some cyanobacteria and β- and γ-proteobacteria ([3]; and references therein). So far, the very limited number of studies conducted on cyanobacterial PCS—in particular on Nostoc sp. PCC 7120 (sin.: Anabaena sp. PCC 7120)—suggest that this enzyme is a “half-size” protein with good homology to the N-terminal domain of eukaryotic PCS [12]. However, unlike PCS from plants and other eukaryotes, in cyanobacteria the enzyme only acts as a peptidase, thus regulating the catabolism of GSH-conjugates, by cleaving the glycine residue from GSH [13,14]. In fact, no differences in cyanobacterial PCS activity under metal stress have ever been detected when compared with untreated controls [12,14,15]. Further work showed that the PCS from Nostoc sp. PCC 7120 displays extremely weak transpeptidasic activity, regardless of whether Cd is present or not, resulting in the production of very low levels of PC2, but no PCn with higher degrees of oligomerization [12]. Interestingly, phylogenetic analysis supports a cyanobacterial ancestry of eukaryotic PCS genes; yet, phylogenetic relationships of PCS and prokaryotic PCS-like genes are not congruent with the topology of current taxonomic trees, thus suggesting multiple events of horizontal gene transfer [16]. Indeed, considering that cyanobacteria have a robust evolutionary link with plants—due to the fundamental event of endosymbiosis at the origin of the chloroplast [17]—a closer functional similarity between cyanobacterial and plant PCSs might be postulated and explored by more sensitive analytical techniques than those previously employed. If we are to delve into the past to understand the present, we feel that it is appropriate to focus not only on cyanobacteria, but also on early plants that can give primeval information, such as liverworts (Marchantiophyta), regarded as the oldest living land plants [18,19]. To this end, the relatively simple and primitive traits of liverworts can be extremely important for investigating ancient functional and molecular characters, which mark the evolution of more recent land plants. Therefore, in this paper, we have focused our attention on two photoautotrophic model-organisms, namely the cyanobacterium Geitlerinema sp. strain PCC 7407 and the liverwort Marchantia polymorpha L. Geitlerinema sp. PCC 7407 [20] is a filamentous freshwater/terrestrial cyanobacterium, of particular importance due to the broad response mechanisms that it may activate as a result of different stresses [21]. Geitlerinema sp. can grow in the same natural environments as Marchantia sp. [22] and also presents the considerable advantage of being relatively simply cultivated in axenic conditions, in a growth chamber. Most importantly, research on the entire genome of Geitlerinema sp. PCC 7407 has identified a PCS gene that codifies a putative protein of 401 amino acids, fairly similar to plant PCSs (https://www.ncbi.nlm.nih.gov/assembly/GCF_000317045.1/)[20]. However, the PCS in Geitlerinema sp. PCC 7407 has never been characterised from a functional standpoint. M. polymorpha is an extant thalloid liverwort with a key role in plant phylogeny [23,24]. In recent times, the development of transformation techniques and the sequencing of the entire genome [24,25] have rendered M. polymorpha a fundamental model-organism for functional, molecular, and evolutionary studies on land plants. In addition to the fact that M. polymorpha gametophytes can be axenically cultivated in a growth chamber, they also offer a number of other advantages, such as a short life cycle, a relatively small genome size (ca. 280 Mb), a haploid number of chromosome sets, and ease in propagation and crossing [24]. M. polymorpha currently includes three subspecies: (1) subsp. polymorpha; (2) subsp. montivagans; and (3) subsp. ruderalis. The latter corresponds to M. polymorpha sensu stricto [23], i.e., the taxon in which nuclear and organellar genomes have been entirely sequenced [24]. M. polymorpha has a high capacity for acquiring metals and may therefore provide a valuable tool for metal biomonitoring [26,27]. For all these reasons, in-depth investigations on M. Plants 2020, 9, 914 3 of 12 polymorpha PCS can provide relevant information on functional aspects of this enzyme in plants and allow useful comparisons with the cyanobacterial PCS. Therefore, in this work we performed a comparative functional characterization of the PCSs in the cyanobacterium Geitlerinema sp. strain PCC 7407 and in the gametophytes of the liverwort M. polymorpha subsp. ruderalis. The experimental approach was aimed at investigating the enzymatic activities of the respective PCSs in vitro and the production of PCn in vivo, in response to various Cd concentrations and exposure times. At the same time, we carried out a comparative quantification of the GSH levels in both organisms, taking into account that this fundamental thiol-tripeptide can exert direct and indirect effects in contrasting detrimental effects of Cd stress [28]. The experiments reported here will accordingly expand the current knowledge in the field of metal responses in cyanobacteria and early land plants. 2. Results 2.1. Phytochelatin Synthases from M. polymorpha and Geitlerinema sp. PCC 7407 are Constitutively Expressed and Produce Phytochelatins in vitro The in vitro assays of PCSs from Geitlerinema sp. PCC 7407 cultures and M. polymorpha gametophytes revealed that both prokaryotic and eukaryotic PCSs are constitutive enzymes able to produce, even in the complete absence of Cd, traces of PC2-4 (only PC2 in Geitlerinema sp. PCC 7407) (Table1). Table 1. In vitro activity assay of phytochelatin synthases (PCSs) from Marchantia polymorpha gametophytes and Geitlerinema