Physiological characterization of Chlamydomonas response to UV-C stress
Different physiological traits covering photosynthesis, biomass yield/composition and oxidative stress were measured along the experiment (Additional file 1: Table S1). The radiation had a negative effect over the microalgae photosynthetic parameters, with the reduction of Fv/Fm ratio and chlorophyll b concentration (ANOVA ⍺ 0.05) (Fig. 1a). Cultures cellular density and biomass (FW) kept increasing after the exposition to a low UV-C dose, despite cellular density had a transient drop 5 h after irradiation (Fig. 1b). Chlamydomonas cultures irradiated with low UV-C dosages show no reduction in their biomass yield (FW) compared to unstressed [22]. Although applied UV-C stress did not change yields, it modified Chlamydomonas biomass composition with the accumulation of starch and the fall of soluble sugars upon stress imposition (ANOVA ⍺ 0.05) (Fig. 1). This starch accumulation is also observed in UV-C irradiated crop species as sugar beet [19], potato [20], and lily bulb [21].
Integrated proteomic and metabolomic responses on Chlamydomonas exposed to UV-C stress
Proteomic analysis allowed the identification of 1441 protein species in whole cell protein extracts. After data pre-processing, 885 proteins were above the abundance threshold for confident quantitation (Additional file 1: Table S2). GC-MS allowed the unequivocal identification of 69 primary metabolites, out of these 68 were considered for quantitative purposes (Additional file 1: Table S3). Detected proteins and metabolites were classified according to MapMan V4 categories [23], and 703 proteins and 54 metabolites were assigned to functional bins. These bins comprised 27 pathways for proteins and 8 for metabolites, covering different cellular processes. Out of these, 398 proteins and 14 metabolites could be considered quantitatively differential in at least one sampling time ANOVA, ⍺ 0.05 (5% FDR for protein variables) (Additional file 1: Table S2, S3).
Heatmap clustering based on MapMan categories distinguished the different treatments with an adequate grouping of samples (Fig. 2). At protein level (Fig. 2a) UV-C stress induced a quick reduction on multi process regulation, RNA processing, Protein biosynthesis and Cell cycle related categories, all classified in the same meta-group. On the other hand, the abundance of Protein degradation, External stimuli response, Carbohydrate metabolism and Cellular respiration categories, within the same cluster, increased after 5 h. Interestingly, Redox homeostasis increased 24 h after stress start when Photosynthesis bin reached its maximum abundance. Metabolites (Fig. 2b) showed a divergent distribution to those of proteins, highlighting Amino acid and Carbohydrate metabolism categories time shifts with their protein counterparts. Within individual metabolites, amino acids and organic acids as serine, glutamine, and malic and citric acids down-accumulated upon stress imposition (Fig. 3a), while redox-related glycerol, arabinose and 4-hydroxybenzoic acid accumulated after 5 h (Fig. 3b). On the other hand, the increase of several unknown sugars after 24 h characterized acclimation (Fig. 3c, Additional file 1: Table S2, S3).
A protein-metabolite correlation network was also defined (Fig. 4b) through the integration of both datasets employing sPLS regression (Fig. 4a, Additional file 1: Table S6). This correlation network was overlapped to the STRING network of the correlation network nodes. Within the resulting network sPLS-STRING (Fig. 4b) the cluster centered on early depleting metabolites (citrate, malate, glutamate, dehydroascorbate) and early accumulated arabinose gathered multiple proteins depleting upon stress start and related to translation (ribosomal subunits); cellular respiration and carbon metabolism-related PYRUVATE KINASE (PYK2), ISOCITRATE LYASE (ICL2) and multiple mitochondrial respiratory chain subunits; and development/stress-related MYC INDUCED NUCLEAR ANTIGEN (MINA53) and PHOSPHATASE 2A (PP2A) homolog Cre03.g199983.t1.1. STRING respectively highlighted the interaction between the depleting mitochondrial and ribosomal subunits and the links of the later to the different and also early depleting translation initiation factors. Moreover, these translation-related proteins were predicted to be associated to the PP2A homolog.
STRING linked the early depleting PP2A and ribosome subunits cluster and also translation-related FLAGELLAR ASSOCIATED PROTEIN 204 (FAP204) to different early accumulated aminoacyl tRNA synthetases (TSM2, TSL1, SYK1) and the EUCARIOTIC TRANSLATION INITIATION FACTOR 1A (EIF1a). TSM2, TSL1, SYK1 and EIF1a were included along other early (t 5 h) accumulated elements into a cluster centered on early accumulated glycerol including redox and carbon fixation RIBOFLAVIN KINASE (RFK2), GLUTATHIONE S-TRANSFERASE homolog (CPLD58), AQUAPORIN (MIP1), NDA5, and MME5; translation modulation S-ADENOSYL-L-METHIONINE-DEPENDENT METHYTRANSFERASE (SAM MTase) and signaling (WD40 REPEAT PROTEIN) elements. STRING highlighted the possible interactions between the WD40 REPEAT PROTEIN and the early depleted development/stress-related MINA53. Glycerol cluster correlated negatively to another cluster with elements accumulated after 24 h centered in UNKNOWN SUGAR 5. Fatty acid (FA) synthesis Β-KETOACYL-[ACYL-CARRIER-PROTEIN] SYNTHASE III (KASIII) (Cre04.g216950.t1.2) and a DYRK like kinase (Cre01.g008550.t1.1 or au5.g1142_t1) were positively correlated to the late accumulated sugar. A uncharacterized FILAMENTOUS TEMPERATURE SENSITIVE H like (FTSH) like protease (Q32065) connected the cluster centered on early depleted elements with the clusters gathering elements accumulated after 5 and 24 h of UV-C irradiation.
Since protein-metabolite interactions in the sPLS-STRING network (Fig. 4b) were only based on mathematical models, STITCH analysis was applied to improve these interactions based on biological models (Additional file 2: Fig. S2). STITCH network highlighted the connections of early accumulated glycerol to protein biosynthesis, respiration and lipid metabolism along the mentioned STRING protein-protein interactions.
UV-C irradiation affected mitochondrial electron transport enhancing ROS production and inhibiting oxidative phosphorylation
Mitochondrial respiration and ATP synthesis downregulated under UV-C stress with the depletion of multiple subunits of respiratory and F1F0 ATP synthase complexes, excepting the early accumulated IV subunit (COX4) (Table 1, Additional file 1: Table S2). Interestingly, UV-C treatment increased the abundance of chaperones associated to respiratory complexes. NUOAF4, chaperone of the large complex I, was exclusively detected on early stress response and HSP70C, a subunit of the IV associated HSP70 complex, peaked at this stage (1-fold at t 5 h) (Table 1, Additional file 1: Table S2). HSP70 complex can aggregate with MGE1 and COX4, also up-accumulated under tested UV-C stress (Table 1, Additional file 1: Table S2), allowing the incorporation of COX4 into the IV complex [24]. Moreover, the accumulation of HSP70C has been described in response to heat stress induced protein misfolding in Chlamydomonas [25]. UV-C irradiation effect over mitochondrial proteins also triggers ROS bursts [8]. ROS scavenging catechin, catechin biosynthesis related CHALCONE ISOMERASE (Cre12.g517100.t1.1) and redox related TCA ISOCITRATE DEHYDROGENASE (IDH2) accumulated under UV-C (0.8-, 1.5-, 2.2-fold at t 24 h) (Table 1, Additional file 1: Table S2, S3). Moreover, also redox related RIBOFLAVIN KINASE (RFK2) was exclusively detected under early stress (Table 1, Additional file 1: Table S2). RFK overexpression protect human cells from oxidative stress enhancing GSH metabolism [26].
Low intensity UV-C irradiation modulated thylakoid electron transport and fixed carbon allocation under inhibited respiration
Tested radiation damaged chloroplastic proteins, especially those of antenna and PSII as showed by the reduction of Fv/Fm ratio and chlorophyll b concentration (Fig. 1a, Additional file 1: Table S1). Enhanced damage was linked to an increase in protein turnover and protection. Central photosystem II (PSII) PsbD subunit (D2) peaked under early response along PSI P700 CHLOROPHYLL A APOPROTEIN A2 (psaB) (0.9-, 2.4-fold at t 5 h) and antennal CHLOROPHYLL a-b BINDING PROTEIN CP29 (LHCB4) up-accumulated under stress (1-fold at t 24 h) (Table 1, Additional file 1: Table S2). Chlamydomonas LHCB4 is a key modulator of non-photochemical quenching (NPQ) and state transitions [27]. The accumulation of D2 and LHCB4 proteins matched the accumulation of a FTSH-like chloroplastic protease (Q32065) (Table 1, Additional file 1: Table S2), working together as a photosynthetic protein protection and turnover mechanism [28–30]. These elements accumulated along others associated to the repair and assembly of photosynthetic complexes as the THYLAKOID LUMINAL FACTOR (TEF14), CYN38 and CHLOROPLASTIC DNAJ-LIKE PROTEIN (CDJ1) (2.7-, 2.4-, 1.2-fold respectively at t 24 h) (Table 1, Additional file 1: Table S2). Arabidopsis mutants on TEF14 and CYN38 orthologs fail to assembly and repair PSII [29,31]. Chlamydomonas CDJ1 organizes chloroplast HSPs under heat stress [32] and its accumulation under UV-C pointed to an enhanced protection of photosynthetic proteins and complexes. The accumulation of CDJ1 under stress matched the early accumulation of the mitochondrial HSP70C chaperone but no chloroplastic HSPs accumulated. Thus, these changes suggested that the tested low UV-C dosage damages PSII, triggering different measures to avoid and repair damage.
Response to chloroplastic UV-C damage also involved the accumulation of the PSI stabilizing PSI reaction center subunit V (psaG), FERREDOXIN NADP REDUCTASE (FNR1) and PLASTOCYANIN (PCY1) (2.7-, 1-, 1.6-fold at t 24 h) (Table 1, Additional file 1: Table S2). The accumulation of these proteins –involved in both Linear and Cyclic Electron Flow (LEF and CEF)– and the enhanced PSI/II turnover and protection are compatible with an enhancement on LEF and/or CEF on acclimation. In spite of this, CEF/LEF-related CYTOCHROME b6f RIESKE IRON-SULFUR CENTER SUBUNIT (PETC) down-accumulated on acclimation (-9.6-fold at t 24 h) and Fv/Fm ratio did not recover control values after UV-C irradiation (Table 1, Fig. 1a, Additional file 1: Table S1, S2). On the other hand, LEF and CEF happens as independent processes in Chlamydomonas, where supercomplexes of PSI, Cytb6f and FNR are exclusively dedicated to the later [33]. PSI subunits and FERREDOXIN NADP REDUCTASE (FNR1) accumulated under stress and other subunits associated to this complex as PETO, PGRL1, Cyt b6 and Cyt f maintained their abundance (Additional file 1: Table S2).
The fall of Fv/Fm ratio and chlorophyll b abundance, the accumulation of the NPQ/state transition-related antennal protein LHCB4, and the diverging changes in the abundance of LEF/CEF-related proteins described under tested stress matched early changes in the chloroplastic redox homeostasis. Redox modulates Chlamydomonas CEF rate, carbon metabolism and ROS scavenging mechanisms. NDA5 oxidoreductase, whose Arabidopsis homolog (NDC1) is a chloroplastic/mitochondrial NADPH-dependent quinone oxidoreductase [35,36], and tocopherol biosynthesis PHYTOL KINASE (CGL134) were exclusively detected at t 5 and t 24 h respectively (Table 1, Supplementary Table S2). Detected catalases and peroxidases, related to the detoxification of H2O2 and superoxide radicals did not change their abundance on stressed samples (Additional file 1: Table S2). Combined UV-A/B stress increases the production of superoxide radicals over singlet oxygen, which is the predominant reactive oxygen species under high light stress [37]. In spite of this, a protein containing a GLUTATHIONE-S-TRANSFERASE domain (CPLD58) was detected exclusively after 5 h of UV-C stress (Table 1, Additional file 1: Table S2). A glutathione peroxidase and a glutathione-s-transferase drive the Chlamydomonas detoxification response to the increased production of singlet oxygen under high light stress [38]. Applied UV-C dosage rapidly and transiently enhanced the tolerance of the treated cells to rose Bengal (RB)-induced singlet oxygen (1O2) oxidative stress. Cells from the t 5 h harvest were able to survive on 2 mM RB while cells from the t 24 h harvest showed an oxidative stress tolerance close to those of unstressed cells, unable to grow at that RB concentration (Fig. 5). D. salina and H. pluvialis accumulate singlet oxygen scavenging carotenoids and malondialdehyde (MDA) –a lipid peroxidation product– under UV-C stress [6]. Strawberry plants exposed to UV-C, experiment a transient increase in their antioxidant capacity [16]. Other redox regulators as THIOREDOXIN M (TRXm) accumulated under stress (2.5-fold at t 24 h). On the other hand, redox regulated chloroplastic protein NADP MALATE DEHYDROGENASE (MME5) accumulated exclusively under early stress (5-fold at t 5 h) (Table 1, Additional file: Table S2). This enzyme is part of the malate shuttle, transferring not only carbon but excess reducing power from chloroplast to cytoplasm and other organelles.
The early accumulation of redox/carbon-related MME5 after UV-C irradiation concurred with the accumulation of glycerol (0.7-fold at t 5 h) (Fig. 3, Additional file 1: Table S3). Glycerol synthesis is an ATP producing and NADH consuming fermentative process. Moreover, glycolate/glycerate (TEF24), triose phosphate:Pi (APE2) antiporters accumulated under stress (2.9-, 4-fold respectively at t 5 h) and glycerol transporter (MIP1) was exclusively detected at t 5 h (Table 1, Additional file 1: Table S2). On the other side, OPP 6-PHOSPHOGLUCONATE DEHYDROGENASE (GND1), a NADPH producing enzyme, also accumulated on t 5 h (1.3-fold). Arabidopsis GND1 homolog (PGD) accumulate under ROS stress providing the required NADPH for the ROS scavenging mechanisms [39]. RuBisCO large subunit (RBCL) also accumulated 5 h after UV-C irradiation (1-fold at t 5 h) (Additional file 1: Table S2). UV-B and other ROS producing stresses as O3 induce a decrease in the abundance of rbcL transcripts, its translation, and in the activity of RBCL [40]. The damage of RBCL can inhibit its translation, through the exposure of its N-terminal regions, a known inhibitor of rbcL translation, after the disassembly of RuBisCO holoenzyme [41]. However, low intensity UV-B or UV-C exposure can enhance RuBisCO activity as in D. salina [42] and cyanobacteria [43], respectively. In Chlamydomonas, UV-C radiation depleted RuBisCO small subunit isoform 1 (RBCS1), while isoform 2 (RBCS2) abundance was not affected (Additional file 1: Table S2). In this algae both RBCS isoforms have a large equivalence [44] and, contrary to RBCL, its combined abundance is stable during cell cycle [45]. However its particular functional differences need to be characterized, as different ratios of RBCS isoforms differentially modulate the holoenzyme carboxylation activity in plants [46]. Starch accumulated under stress (Fig. 1b) along many enzymes related to their synthesis as GRANULE BOUND STARCH SYNTHASE (STA2) (2.1-fold at t 5 h) and STARCH BRANCHING ENZYME (SBE3), accumulated at t 5 h (Table 1, Additional file 1: Table S1, S2). UV-C enhanced starch synthesis matched the down-accumulation of soluble sugars (Fig. 1b), the late accumulation of Glucose and unknown sugars as UNKNOWN SUGAR, UNKNOWN SUGAR 4 and UNKNOWN SUGAR 5 (1-, 1.2-, 1.2-, 1-fold at t 24 h), and the depletion of TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) (Table 1, Fig. 3c, Additional file 1: Table S1, S2, S3). TPS produces trehalose-6-phosphate (T6P), an inhibitor of the SnRK1 kinase, which was a key node in plant carbon metabolism also influenced by the cellular redox status [47,48]. Sugars and starch also accumulate in UV-C treated lily bulb [21] and sugar beet [19].
Accumulation of proteases as FTSH and photosystems subunits under tested UV-C irradiation (Table 1, Additional file 1: Table S2) suggested an increased protein turnover which could act as both a source and a sink of free amino acids. Serine and Glutamate down-accumulated 5 h after irradiation (-2.65-, -3.83-fold respectively) (Fig. 3a, Additional file 1: Table S3). Many enzymes related to amino-acid biosynthesis as SERINE ACETYLTRANSFERASE (SAT3), SERINE and ALANINE GLYOXILATE TRANSAMINASES (SGA1, AGT2), ISOPROPYLMALATE DEHYDROGENASE (LEU3) accumulated (2.3-, 1.5-, 1.8-, 3.6-fold at t 5 h) and nitrogen fixation proteins as GLUTAMINE SYNTHETASE and Fd-DEPENDENT GLUTAMATE DEHYDROGENASE remained unchanged. Other amino acid-related enzymes as OXOPROLINASE (Cre07.g325748.t1.1) were exclusively detected on t 5 h or as CYSTATHIONINE BETA LYASE (METC) and METHYLCROTONYL CoA CARBOXYLASE alpha subunit exclusively on stressed samples (Table 1, Additional file 1: Table S2).
Alternative signalers modulate cell proliferation, development and metabolism under UV-C
The observed changes in protein turnover/protection carbon metabolism and ROS/redox-related elements induced by UV-C were coupled to rapid changes on the abundance of many development and translation-related proteins. sPLS-STRING network (Fig. 4b) clustered many of these and linked the WD40 REPEAT PROTEIN (Cre12.g495650.t1.2) –accumulated exclusively at t 5 h, to the development related MINA53 (Cre07.g356600.t1.2) –down-accumulated under stress (Table 1, Additional file 1: Table S2). The mutation of the human homolog of MINA53, MINA53/RIOX2, inhibit DNA replication/repair mechanisms and cell proliferation in human cell lines [49]. MINA53 was also linked in the sPLS-STRING network (Fig. 4b) through C/N metabolism to JmjC protein JMJC DOMAIN CONTAINING PROTEIN 7 (Cre03.g175750.t1.2), also down-accumulated under UV-C stress. Arabidopsis homolog to JMJC DOMAIN CONTAINING PROTEIN 7 (JMJ32) is a HISTONE H3 lysine 27 (H3K27) demethylase, while its human homolog (JMJD7) is a lysyl hydroxylase. HISTONE-ARGININE N-METHYLTRANSFERASE (PRMT2) also depleted upon stress imposition (Table 1, Additional file 1: Table S2). The fluctuations in the abundance of these histone related proteins under UV-C suggested the importance of the epigenetic- and/or amino acid residue hydroxylation-based modulation mechanisms in this stress response. sPLS-STRING and STICH networks also highlighted the link of C/N metabolism and protein synthesis to early accumulated translation modulation-related elements as EIF1a, FAP204 and aminoacyl tRNA synthases, and to a signaling PP2A like protein (Cre03.g199983.t1.1), which were exclusively detected in control samples (Fig. 4b, Table 1, Additional file 2: Fig. S2, Additional file 1: Table S2). This PP2A like phosphatase is homolog to several Arabidopsis PP2A which are key nodes in plant immunity integrating pathogen perception at membrane level with pathogen response at multiple levels including SA, ABA and TOR signaling, and C/N metabolism modulation [50,51]. PP2As link to TOR allow these proteins to regulate cell growth in response to the environment [52]. Thus, the connection of PP2A to protein synthesis and C/N metabolism in the sPLS-STRING and STITCH networks (Fig. 4b, Additional file 2: Fig. S2) is suggestive of the PP2A like mediated tuning of TOR pathway in UV-C stressed Chlamydomonas. More directly related to the modulation of carbon metabolism under stress, sPLS-STRING network (Fig. 4b) highlighted a correlation between DYRK kinase (Cre01.g008550.t1.1 or au5.g1142_t1) and the accumulation of sugars and glycerol. This DYRK kinase, registered as CMGC_DYRK-PRP4 in the iTAK database [53], accumulated 24 h after UV-C irradiation start (Table 1, Additional file 1: Table S2). Other Chlamydomonas DYRK as TAR1 and STD1 are known for their roles in carbon storage under nutrient stress [54,55].
Results showed an early (t 5 h) Chlamydomonas response to UV-C focused on damage avoidance through enhanced singlet oxygen scavenging (Fig. 5) and protein protection/turnover (Table 1, Additional file 1: Table S2). In plants UV-C enhances ROS production and triggers ROS scavenging mechanisms [4,8]. This early response also involved rapid changes in the cell carbon allocation with the accumulation of starch and glycerol (Fig. 1, 3). Starch accumulation remained increased 24 h after UV-C irradiation (Fig. 1) and carbon metabolism was further modulated as specific sugars were also accumulated (Fig. 3). Late accumulated sugars and starch matched the up-accumulation of a DYRK kinase (Cre01.g008550.t1.1) (Table 1) whose homologs regulate carbon fluxes in Chlamydomonas [54,55]. Moreover, these responses were linked to protein expression/epigenetic modulation elements as MINA53 and PRMT2 that might be driving the proteogenomic changes after the observed UV-C adaptation and enhanced oxidative stress resistance in Chlamydomonas. All these elements would help for further UV-C stress characterization or exploitation towards the generation of enhanced strains.