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Laporte et al. BMC Plant Biology (2020) 20:25 https://doi.org/10.1186/s12870-019-2229-5 RESEARCH ARTICLE Open Access Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta) Daniel Laporte1, Felipe Rodríguez1, Alberto González1, Antonio Zúñiga1,2, Eduardo Castro-Nallar3, Claudio A. Sáez2,4 and Alejandra Moenne1* Abstract Background: The marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 μ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance. Results: The levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3. Conclusions: The marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance. Keywords: Copper, Photosynthesis, Respiration, Transcriptomic analyses, Marine alga, Ulva compressa Background In photosynthetic organisms, copper is an essential heavy metal that it is required for the activity of several proteins and enzymes such as plastocyanin, cytochrome c oxidase, Cu/Zn superoxide dismutase (SOD), polyphenol oxidase, laccase and ascorbate oxidase, among others [1, 2]. Copper is required only in trace amount as in excess it produces an oxidative stress condition that becomes harmful for cellular macromolecules [2]. In photosynthetic * Correspondence: alejandra.moenne@usach.cl 1 Laboratory of Marine Biotechnology, Faculty of Chemistry and Biology, University of Santiago of Chile, Alameda, 3363 Santiago, Chile Full list of author information is available at the end of the article organisms, copper excess may lead to the replacement of magnesium in chlorophylls inhibiting the release of energy from chlorophylls to PSII under low light, or it can also directly inhibit the reaction center in PSII in under high light [3, 4]. Copper excess can also inactivate enzymes by replacing zinc or other heavy metals [5]. The marine green macroalgae Ulva pertusa and U. armoricana cultivated with increasing concentrations of copper, from 0 to 250 μg L− 1 (3.9 μM) for 3 days displayed a differential behavior regarding photosynthesis and N assimilation [6]. U. pertusa showed no inhibition of photosynthetic parameters until 100 μg L− 1 (1.5 μM) and an increase in the activity of nitrate reductase © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Laporte et al. BMC Plant Biology (2020) 20:25 activity, an enzyme involved in N assimilation [6]. In contrast, U. armoricana showed inhibition of photosynthetic parameters at 50–100 μg L− 1 of copper and an inhibition of nitrate reductase activity [6]. The green macroalga U. flexuosa cultivated with 0.8, 4 and 8 μM of copper for 5 days showed inhibition of photosynthesis when cultivated with 4 and 8 μM copper [7]. U. compressa L. Grev. (Cholorophyta) showed an increase in photosynthesis when cultivated with 10 μM of copper for 0 to 24 h [8]. Thus, green macroalgae from the same genus displayed differential responses to copper stress and it seems that, among Ulvaceae, U. compressa is the most tolerant species regarding copper stress. The red macroalga Gracilaria tenuistipitata cultivated with 16 nM of copper for 1 to 6 days showed an inhibition of photosynthesis from day 1 [9]. The red macroalga Porphyra haitiensis cultivated with 0.1 to 50 μM of copper for 3 days showed an increase in photosynthesis at concentrations of 0.1 to 1 μM and an increase in respiration with 0.1 to 50 μM suggesting that respiration is less sensitive to copper stress [10]. The brown macroalga Ectocarpus siliculosus showed an increase in photosynthesis when cultivated with 1.8 μM of copper for 8 h but a decrease in photosynthesis with 3.7 μM of copper [11]. Thus, marine macroalgae showed differential behaviors regarding photosynthesis and respiration in response to copper stress. The marine alga U. compressa is highly tolerant to copper excess since the alga cultivated with 50 μM copper for 7 days displayed cellular viability [12]. The alga exposed to a sub-lethal concentration of copper (10 μM) showed the accumulation of superoxide anions beginning at day 3 and increasing until day 7 and the production of superoxide anions occurred mainly in chloroplasts and mitochondria [12]. In addition, the alga cultivated with copper showed the activation of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase and glutathione reductase, and the synthesis of antioxidant compounds such as ascorbate (ASC) and glutathione (GSH) [12–14]. In addition, U. compressa accumulate copper in its tissue, reaching 620 μg g− 1 when cultivated with 10 μM copper for 12 days [15]. Copper accumulation correlates with the synthesis of GSH, and phytochelatins (PCs), which are peptides produced through condensation of GSH units, and with the increase in expression of metallothioneins (MTs), which are small size cysteine-rich proteins that bind monovalent and divalent metal ions [16]. Initial transcriptomic analyses using RNAseq performed in U. compressa cultivated with 10 μM copper for 0 and 24 h allowed the identification of 7 potential MTs as well as transcripts that encode antioxidant enzymes, and enzymes involved in ASC and GSH synthesis [16]. The levels of transcripts encoding MTs, antioxidant enzymes, and enzymes Page 2 of 16 involved in ASC and GSH synthesis, were increased in the alga exposed to copper excess [16]. Finally, transcripts encoding three MTs, MT1, MT2 and MT3, were cloned from U. compressa and their overexpression in bacteria allowed the accumulation of copper and zinc in vivo [17]. Thus, U. compressa may accumulate copper in its tissue through the binding of copper ions to GSH, PCs and MTs and this alga could represent a useful tool for phycoremediation of seawater contaminated with heavy metals. Photosynthetic organisms displaying an increase in photosynthesis may produce an enhanced level of NADPH that may increase C, N and S assimilation, since the latter are reductive processes that require NADPH [18–20]. The marine macroalga U. compressa cultivated with 10 μM copper for 0 to 24 h showed an increase in photosynthesis and in the level of transcripts encoding enzymes of the Calvin-Benson cycle, suggesting that C assimilation may be increased [8]. In this work, we investigate whether the initial increase in photosynthesis is maintained along days, and whether there is a concomitant increase in respiration, and in C, N and S assimilation. To this end, the alga U. compressa was cultivated with 10 μM copper for 0, 1, 3, and 5 days and the levels of transcripts encoding proteins involved in photosynthesis and respiration, and those encoding enzymes involved C, N and S assimilation, were determined. In addition, the level of produced and consumed oxygen, reflecting photosynthesis and respiration, respectively, were determined. The activities of key enzymes involved in C, N and S assimilation such as rubisco, glutamine synthase and cysteine synthase, respectively, were also analyzed. Results indicate that the alga tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance. Results Assembly, annotation and classification of transcripts The libraries obtained from samples of the alga cultivated in control condition (day 0) and treated with copper for 1, 3, and 5 days contained 785,500,000 M of reads, in total, and 98,187,500 reads, on average. The reads were subjected to quality control, they were trimmed and bacterial sequences were eliminated, resulting in 96,577,500 M of reads, on average, which correspond to 98.36% of the initial reads (Additional file 1: Table S1). Transcripts were assembled using Trinity software and resulted in 140,840 transcripts (contigs) of 300 to 5140 nucleotides in length, with an average length of 1575 nucleotides. The completeness of assembled transcriptomes was 93.4% (Additional file 1: Table S1). Transcripts were translated into Laporte et al. BMC Plant Biology (2020) 20:25 amino acids, and annotated proteins having an e value of 1e− 3, or lower, were 65,494 (Additional file 5: Table S2). The latter proteins are involved in different biological processes (Additional file 2: Figure S1). Of the 65,494 proteins, 23,692 (36%) showed higher amino acid similarity with plant and green algae (Plantae) proteins; 15,073 (23%) displayed a higher similarity with animal proteins; and 26,729 (41%) proteins showed similarity to other organisms (Fungi, Protista and Prokaryotes) (Additional file 3: Figure S2). Proteins having similarity to animal proteins showed homology with human, mouse, rat, and other animal proteins (Additional file 3: Figure S2). Transcripts differentially expressed in response to copper excess Transcripts coding for 65,494 proteins previously mentioned that were differentially expressed were obtained considering times: 0 vs. 1, 0 vs. 3 and 0 vs. 5 (Additional file 4: Figure S3). At time point 0 vs. 1, the number of differentially expressed transcripts was 28,510, those up-regulated were 13,855 and those downregulated were 14,655, which represent 48.6 and 51.4%, respectively. At time point 0 vs. 3, the number of differentially expressed transcripts was 8174, those upregulated were 6047 and those down-regulated were 2127, which represent 74 and 26%, respectively. At time point 0 vs. 5, the number of differentially expressed transcripts was 30.589, those up-regulated were 17,218 and those down-regulated were 13,371, which represent 56.3 and 43.7%, respectively. Thus, the higher percentages of up-regulated transcripts were observed at days 3 and 5 in the alga exposed to copper excess. Transcripts with increased levels of encoded proteins involved in photosynthesis and respiration Regarding photosynthesis, the levels of transcripts encoding subunits of PSII corresponding to PsbA, PsbB, PsbC, PsbE, PsbH, PsbN, PsbO, PsbP, PsbR, PsbS, PsbW, PsbY and PsbZ were up-regulated in response to copper stress (Table 1). In addition, the levels of transcripts encoding subunits of Light Harvesting Complex II (LHCII), chlorophyll a/b-binding proteins LhcB1, LhcB4, LhcB5 and Cab1 as well as fucoxanthin-chlorophyll a/c-binding proteins, FcpA and FcpB, were increased. In addition, the subunits of the enzyme magnesium chelatase involved in chlorophyll synthesis, ChlD and ChlI, were upregulated (Table 1). The levels of transcripts encoding subunits of cytochrome b6f corresponding to cytochrome b6 (petB), iron-sulfur Rieske subunit (PetC), an essential protein for assembly of cytb6f (PetG), and a carrier of electrons from cytb6f to PSI (PetJ), were also increased (Table 1). The levels of transcripts encoding subunits of PSI corresponding to PsaA, PsaB, PsaD, Page 3 of 16 PsaF, PsaG and PsaL were also up-regulated (Table 1). Finally, the levels of transcript encoding a subunit of LHCI, LhcA, and the subunits of ATP synthase α, β, γ, δ, ε and γ subunits were also increased (Table 1). On the other hand, the levels of transcripts encoding the chaperone MET1, involved in the insertion of PsbA (D1) in PSII; the ATP-independent serine proteases Deg 1, 2 and 9, and the ATP-dependent metalloproteases FtsH 1, 2 and 11, involved in degradation of damaged PsbA, were increased (Table 2). The levels of transcripts encoding bc1 complex kinase 1 (ABC1K1), involved in the synthesis of quinones and tocopherol (vitamin E) and the xanthophyll lutein which protect PSII to oxidative damage; the enzyme 2-carboxy-1,4-naphtoquinone phytyl transferase (ABC4), involved in the synthesis of phylloquinone (vitamine K) required in PSI, were upregulated (Table 1). Moreover, transcripts encoding PGR5-1A, a protein involved in control of electron flow around PSI that protects PSI against photo-oxidation; YCF12, a protein involved in assembly and stabilization of PSI; the serine-threonine kinase STN8, involved in phosphorylation of LHCII allowing migration of subunits of PSII to PSI; and ATAB2, a light-regulated protein involved in the increased synthesis of photosystem proteins, were also increased (Table 1). Thus, the expression of a large number of proteins involved in photosynthesis and repair and protection of photosystems was increased in U. compressa exposed to copper excess (Additional file 6: Figure S4A). Regarding respiration, the levels of transcripts encoding subunit 1 and 2 of NADH dehydrogenase (complex I), subunit IV and V of cytochrome bc1 complex (complex III), and subunit γ of mitochondrial ATPase were increased (Table 1). Thus, the expression of a small number of proteins involved in respiration was increased in U. compressa compare to those involved in photosynthesis (Additional file 6: Figure S4B). Transcripts with increased levels of encoded enzymes involved in C, N and S assimilation The levels of transcripts encoding enzymes of the Calvin-Benson cycle involved in C assimilation, RbcS and RbcL corresponding to the small and large subunits of rubisco; phosphoglycerate kinase (PGK); glyceraldehyde 3-P dehydrogenase (G3PDH); fructose biphosphate aldolase (FBPA); fructose 1,6 biphosphatase (FBP), transketolase (TK); ribose 5-P isomerase (R5PI) and phosphoribulose kinase (PRK) were increased (Table 2). Thus, the expression of nine enzymes of the eleven enzymes of the Calvin-Benson cycle was increased in U. compressa exposed to copper excess. The levels of transcripts encoding enzymes involved in N assimilation, nitrate reductase and glutamine synthase, were increased (Table 2). In addition, the levels of Laporte et al. BMC Plant Biology (2020) 20:25 Page 4 of 16 Table 1 Up-regulated genes related to photosynthesis and mitochondrial electron transport chain Process ID Transcript Proteins Log2 Fold Change PSII Unigene17497_All PsbA 3.4 CL2571.Contig1_All PsbB 2.0 CL11753.Contig1_All PsbB 2.4 CL11753.Contig2_All PsbB 4.5 Unigene33413_All PsbC 3.3 CL4160.Contig3_All PsbE 2.6 Unigene34274_All PsbH 3.3 Unigene29880_All PsbN 3.3 Unigene13168_All PsbO 1.6 Unigene29944_All PsbP 2.1 CL7636.Contig1_All PsbP 1.2 CL699.Contig2_All PsbP 2.3 CL4419.Contig1_All PsbP 1.8 CL4419.Contig5_All PsbP 1.2 LHCII Cyt b6-f PSI CL3599.Contig1_All PsbR 2.0 CL10892.Contig2_All PsbS 3.6 Unigene33763_All PsbW 5.3 Unigene37851_All PsbY 1.6 Unigene1513_All PsbZ 2.5 Unigene596_All Chlorophyll a-b binding protein 1D (lhcB1) 2.6 CL4261.Contig2_All Chlorophyll a-b binding protein CP26 (lhcB5) 1.4 CL11211.Contig2_All Chlorophyll a-b binding protein CP26 (lhcB5) 1.9 CL1500.Contig3_All Chlorophyll a-b binding protein L1818 (lhcb4) 4.3 CL4399.Contig3_All Chlorophyll a-b binding protein L1818 (lhcb4) 6.8 CL9440.Contig1_All Chlorophyll a-b binding protein L1818 (lhcb4) 3.0 CL9440.Contig2_All Chlorophyll a-b binding protein L1818 (lhcb4) 4.7 Unigene29850_All Chlorophyll a-b binding protein L1818 (lhcb4) 1.5 CL1500.Contig2_All Chlorophyll a-b binding protein L1818 (lhcb4) 2.5 CL4399.Contig1_All Chlorophyll a-b binding protein L1818 (lhcb4) 1.4 CL12301.Contig27_All Chlorophyll a-b binding protein Type I (CabII-1) 2.9 CL12301.Contig28_All Chlorophyll a-b binding protein Type I (CabII-1) 2.5 Unigene22628_All Fucoxanthin-chlorophyll a-c binding protein (FcpA) 1.0 CL941.Contig8_All Fucoxanthin-chlorophyll a-c binding protein (FcpE) 2.4 CL2528.Contig4_All Magnesium-chelatase subunit ChlD 1.8 Unigene41646_All Iron-sulfur subunit (petB) 1.8 CL2893.Contig2_All Iron-sulfur subunit (petC) 1.5 CL2893.Contig4_All Iron-sulfur subunit (petC) 3.7 CL2893.Contig6_All Iron-sulfur subunit (petC) 5.7 CL2893.Contig9_All Iron-sulfur subunit (petC) 1.1 CL2893.Contig10_All Iron-sulfur subunit (petC) 4.9 Unigene174_All Iron-sulfur subunit (petC) 2.5 Unigene970_All Iron-sulfur subunit (petG) 2.5 Unigene33787_All Iron-sulfur subunit (petJ) 2.1 Unigene61010_All PsaA 7.7 Laporte et al. BMC Plant Biology (2020) 20:25 Page 5 of 16 Table 1 Up-regulated genes related to photosynthesis and mitochondrial electron transport chain (Continued) Process LHC I ATP synthase Assembly and Repair of PS II ID Transcript Proteins Unigene955_All PsaD Log2 Fold Change 1.6 Unigene21099_All PsaF 1.3 Unigene38056_All PsaG 3.3 Unigene25315_All PsaL 1.3 CL11079.Contig2_All Chlorophyll a-b binding protein 5 (LhcA1 like) 3.4 Unigene33050_All ATP synthase subunit alpha 2.8 CL10342.Contig2_All ATP synthase subunit alpha 2.6 Unigene28940_All ATP synthase subunit alpha 1.4 Unigene21967_All ATP synthase subunit alpha 2.7 CL5620.Contig2_All ATP synthase subunit alpha 2.8 CL10342.Contig6_All ATP synthase subunit alpha 1.4 CL2062.Contig2_All ATP synthase subunit beta 8.1 Unigene284_All ATP synthase subunit beta 1.3 Unigene283_All ATP synthase subunit beta 1.9 Unigene286_All ATP synthase subunit beta 4.1 CL2062.Contig1_All ATP synthase subunit beta 1.2 Unigene33306_All ATP synthase subunit beta 2.1 CL4160.Contig1_All ATP synthase subunit beta 3.9 Unigene19914_All ATP synthase subunit beta 2.4 CL5278.Contig5_All ATP synthase subunit gamma 1.4 Unigene16523_All ATP synthase subunit gamma 2.2 CL5278.Contig5_All ATP synthase subunit gamma 1.4 CL5278.Contig10_All ATP synthase subunit gamma 1.4 Unigene16523_All ATP synthase subunit gamma 2.2 Unigene32932_All ATP synthase subunit epsilon 1.0 Unigene43520_All ATP synthase subunit delta 2.4 Unigene29539_All Met1 5.6 CL1106.Contig1_All Deg/HtrA Protease Do-like 1 4.2 CL1106.Contig2_All Deg/HtrA Protease Do-like 1 2.6 CL1106.Contig5_All Deg/HtrA Protease Do-like 1 4.5 CL1106.Contig7_All Deg/HtrA Protease Do-like 1 2.1 CL1106.Contig8_All Deg/HtrA Protease Do-like 1 2.5 CL1106.Contig9_All Deg/HtrA Protease Do-like 1 2.4 CL1106.Contig13_All Deg/HtrA Protease Do-like 1 2.6 CL3921.Contig1_All Deg/HtrA Protease Do-like 1 3.6 CL3921.Contig3_All Deg/HtrA Protease Do-like 1 2.0 CL6287.Contig3_All Deg/HtrA Protease Do-like 2 1.4 CL6287.Contig4_All Deg/HtrA Protease Do-like 2 1.0 Unigene12439_All Deg/HtrA Protease Do-like 9 1.0 Unigene12443_All Deg/HtrA Protease Do-like 9 1.1 Unigene7791_All ATP-dependent zinc metalloprotease FtsH1 1.5 Unigene9549_All ATP-dependent zinc metalloprotease FtsH1 1.1 Unigene71521_All ATP-dependent zinc metalloprotease FtsH1 3.1 Unigene36205_All ATP-dependent zinc metalloprotease FtsH1 9.1 Laporte et al. BMC Plant Biology (2020) 20:25 Page 6 of 16 Table 1 Up-regulated genes related to photosynthesis and mitochondrial electron transport chain (Continued) Process Assembly and Protection PSI Mitochondrial ID Transcript Proteins CL8065.Contig1_All ATP-dependent zinc metalloprotease FtsH2 Log2 Fold Change 2.5 Unigene16464_All ATP-dependent zinc metalloprotease FtsH11 2.1 CL6915.Contig1_All ABC1K1 3.2 CL6915.Contig7_All ABC1K1 3.8 CL12141.Contig2_All 2-carboxy-1,4-naphthoquinone phytyltransferase 2.3 CL12141.Contig5_All 2-carboxy-1,4-naphthoquinone phytyltransferase 2.1 Unigene778_All PGR5 1A 6.4 CL653.Contig10_All Serine/threonine-protein kinase STN8 3.1 CL653.Contig13_All Serine/threonine-protein kinase STN8 3.2 CL653.Contig38_All Serine/threonine-protein kinase STN8 2.9 Unigene13129_All YCF12 3.1 CL5916.Contig1_All ATAB2 3.0 CL5916.Contig2_All ATAB2 3.6 CL2273.Contig1_All NADH dehydrogenase subunit 1 3.2 Electron Unigene75664_All NADH dehydrogenase subunit 2 3.1 Transport CL8189.Contig1_All cytochrome bc1 complex subunit V 2.4 Chain CL5196.Contig1_All Cytochrome bc1 complex subunit IV 2.8 Unigene965_All Cytochrome bc1 complex subunit IV 3.1 Unigene30266_All ATP synthase subunit gamma 3.1 transcripts encoding enzymes of the urea cycle allowing detoxification of ammonium excess such as argininosuccinate lyase and fumarate hydratase were upregulated (Table 2). Thus, the expression of two of the three enzymes involved in N assimilation was increased in U. compressa exposed to copper excess. The levels of transcripts encoding enzymes involved in S assimilation, adenylyl-sulfate transferase (ATP sulfurylase); adenosine 5′-phosphosulfate reductase (APS reductase); sulfite reductase, and cysteine synthase (O-acetylserine thiol lyase) were increased (Table 2). In addition, enzymes involved in the synthesis of amino acids methionine, serine and alanine were also increased (data not shown). Furthermore, the levels of transcripts encoding enzymes involved in glutathione synthesis, glutamate cysteine ligase (γ-glutamyl cysteinyl synthase) and glutathione synthase, were also upregulated (Table 2). Thus, the expression of the four enzymes involved in S assimilation was increased in U. compressa exposed to copper excess. Kinetics of normalized reads of transcripts involved in photosynthesis and respiration The levels of normalized reads of transcripts encoding proteins involved in photosynthesis that showed the highest increases were: subunit PetC of cytb6f that increased at day 1, decreased at day 3, and increased again at day 5; subunit PsbA of PSII that increased at day 1, decreased at day 3, and slightly increased at day 5; protease FtsH1 that increased from day 3 to day 5; proteases Deg1 that increased at day 1, decreased at day 3, and increased again at day 5; subunits LhB4 and LhB5 of LHCII that increased at day 3 and remained increased until day 5; subunit PetJ of cytb6f and the kinase ABC1K1 that increased at day 1, decreased at day 3 and remained decreased at day 5 (Fig. 1a). The levels of normalized transcripts encoding subunits of mitochondrial electron chain that showed the highest increases were: cytochrome c1 (subunit IV of complex III); subunit γ of ATP synthase and subunit 1 of NADH dehydrogenase (complex I) that increased at day 3 and remained increased until day 5; cytochrome c1 (subunit IV of complex III) and subunit γ of ATP synthase that increased at day 3 and decreased at day 5; subunit V of complex III and subunit 2 of NADH dehydrogenase that increased at day 1, decreased at day 3, and remained decreased at day 5 (Fig. 1b). Kinetics of normalized reads of transcripts involved in C, N and S assimilation The levels of normalized reads of transcripts encoding enzymes involved in C assimilation that showed the highest increases were: G3PDH that increased mainly at day 3, and remained increased until day 5; rubisco small subunit RbcS, rubisco large subunit RbcL and enzymes PRK and FBPA that increased at day 3 and remained increased until day 5 (Fig. 2a). Laporte et al. BMC Plant Biology (2020) 20:25 Page 7 of 16 Table 2 Up-regulated genes related to Carbon, Nitrogen and Sulfur assimilation Calvin-Benson Cycle Nitrogen Assimilation Sulfur assimilation ID Transcript Proteins CL5634.Contig3_All Ribulose bisphosphate carboxylase small chain 1 Log2 Fold Change 1.2 CL5634.Contig2_All Ribulose bisphosphate carboxylase small chain 1 8.9 CL12005.Contig2_All Ribulose bisphosphate carboxylase small chain 1 4.7 CL12005.Contig1_All Ribulose bisphosphate carboxylase small chain 1 1.4 Unigene16805_All Ribulose bisphosphate carboxylase small chain 1 2.8 CL5634.Contig4_All Ribulose bisphosphate carboxylase small chain 1 1.3 CL12005.Contig3_All Ribulose bisphosphate carboxylase small chain 2 1.8 Unigene12933_All Ribulose bisphosphate carboxylase large chain 1.6 CL8954.Contig2_All Phosphoglycerate kinase 2.1 Unigene41371_All Phosphoglycerate kinase 3.4 CL1769.Contig1_All Phosphoglycerate kinase 1.0 CL1769.Contig2_All Phosphoglycerate kinase 1.5 Unigene32099_All Phosphoglycerate kinase 4.7 Unigene41512_All Phosphoglycerate kinase 5.1 CL1973.Contig6_All Glyceraldehyde-3-phosphate dehydrogenase 6.0 CL1973.Contig3_All Glyceraldehyde-3-phosphate dehydrogenase 1.3 CL8458.Contig1_All Glyceraldehyde-3-phosphate dehydrogenase 2.5 CL8458.Contig2_All Glyceraldehyde-3-phosphate dehydrogenase 3.0 CL1973.Contig7_All Glyceraldehyde-3-phosphate dehydrogenase 1.0 CL12146.Contig6_All Glyceraldehyde-3-phosphate dehydrogenase 1.6 Unigene26_All Glyceraldehyde-3-phosphate dehydrogenase 1.5 Unigene32957_All Fructose-bisphosphate aldolase 1 7.0 Unigene32956_All Fructose-bisphosphate aldolase 1 2.5 CL2940.Contig6_All Fructose-bisphosphate aldolase 1 1.3 Unigene13983_All Fructose-bisphosphate aldolase 5 3.8 CL2275.Contig2_All Fructose-bisphosphate aldolase 8 1.0 Unigene29154_All Fructose-1,6-bisphosphatase 1.3 Unigene38319_All Transketolase 4.3 Unigene29707_All Transketolase 1.8 CL11843.Contig2_All Phosphoribulokinase 1.9 Unigene42641_All Phosphoribulokinase 2.9 Unigene26010_All Nitrate reductase 2.4 CL10485.Contig1_All Nitrate reductase 3.0 Unigene32044_All Glutamine synthetase 5.3 Unigene79885_All Glutamine synthetase 1.2 Unigene10873_All Glutamine synthetase 2.3 CL1194.Contig12_All Argininosuccinate lyase 3.7 CL1194.Contig11_All Argininosuccinate lyase 9.2 CL1194.Contig13_All Argininosuccinate lyase 1.3 Unigene25622_All Fumarate hydratase 4.1 Unigene14757_All Fumarate hydratase 1.1 CL1413.Contig2_All ATP sulfurylase 1.8 CL1413.Contig5_All ATP sulfurylase 1.2 CL1413.Contig8_All ATP sulfurylase 1.9 Laporte et al. BMC Plant Biology (2020) 20:25 Page 8 of 16 Table 2 Up-regulated genes related to Carbon, Nitrogen and Sulfur assimilation (Continued) ID Transcript Proteins Log2 Fold Change CL1413.Contig9_All ATP sulfurylase 2.7 Unigene66570_All ATP sulfurylase 3.1 Unigene1609_All APS reductase 1.8 CL7094.Contig2_All APS reductase 3.3 Unigene803_All APS reductase 1.9 Unigene15137_All APS reductase 5.4 Unigene17508_All Sulfite reductase 1.7 CL9121.Contig1_All Sulfite reductase 2.4 CL9650.Contig2_All Sulfite reductase 2.8 Unigene823_All Cysteine synthase 2.1 CL10220.Contig15_All Cysteine synthase 3.1 CL10220.Contig39_All Cysteine synthase 2.2 CL10220.Contig51_All Cysteine synthase 1.9 CL10220.Contig56_All Cysteine synthase 1.0 Unigene33426_All Cysteine synthase 3.9 Unigene1587_All Cysteine synthase 4.6 CL1730.Contig3_All Glutamate-cysteine ligase 2.1 CL2777.Contig3_All Glutamate-cysteine ligase 1.8 Unigene6218_All Glutamate-cysteine ligase 4.2 Unigene6958_All Glutamate-cysteine ligase 2.6 Unigene5542_All Glutamate-cysteine ligase 3.6 Unigene25762_All Glutamate-cysteine ligase 1.2 Unigene80578_All Glutamate-cysteine ligase 5.4 Unigene28997_All Glutathione synthetase 3.6 Unigene28998_All Glutathione synthetase 1.3 Unigene28999_All Glutathione synthetase 1.8 Unigene42348_All Glutathione synthetase 1.3 The levels of normalized reads of transcripts encoding enzymes involved in N assimilation that showed the highest increases were: nitrate reductase that increased day 1, decrease at day 3, and increased again at day 5; glutamine synthase that increased at day 1, decreased at day 3, and remained decreased at day 5; fumarate hydratase and argino-succinate lyase that increased at day 3 and remained increased until day 5 (Fig. 2b). The levels of normalized reads of transcripts encoding enzymes involved in S assimilation showing the highest increases were: ATP sulfurylase that increased at day 1, decreased at day 3, and slightly increased at day 5; APS reductase that increased at day 3 and continued to increase until day 5; glutamine cysteine ligase and APR reductase that increased at day 3 and remained increased until day 5; sulfite reductase that increased at day 1 and remained increased at days 3 and 5; and cysteine synthase that increased at day 3 and remained increased at day 5 (Fig. 2c). It is important to mention that the number of normalized reads of transcripts encoding enzymes of C and S assimilation showed higher levels (20 to 300 reads) compare to those of enzymes involved in N assimilation (1–12 reads). Transcripts with decreased levels encode proteins involved in protein synthesis and degradation, signal transduction, and replication and DNA repair The most down-regulated transcripts at time points 0 vs. 1, 0 vs. 3 and 0 vs. 5 were involved in protein synthesis and degradation, signal transduction and, replication and DNA repair. Transcripts encoding proteins involved in protein synthesis and degradation were: ribosomal proteins L2, L15, L16, S12 and S17, ribosomal protein S6 kinase, eukaryotic translation factor 5B, tRNA dehydrouridine synthase, elongation factor 2a kinase, cysteine tRNA ligase, glu-tRNA amidotransferase, peptidyl prolyl trans-isomerase CYP61, RING finger protein 32, protease ESD4, neurotrypsin, E3 ubiquitin protein ligase, Laporte et al. BMC Plant Biology (2020) 20:25 Page 9 of 16 Fig. 1 Level of the highest increased transcripts encoding proteins involved in photosynthesis (a) and respiration (b) in U. compressa cultivated with 10 μM copper for 0, 1, 3 and 5 days. Transcripts encoding proteins involved in photosynthesis corresponding to subunit PetC of cytb6f complex (PetC), subunit PsbA of PSII (PsbA), the protease FstH1 (FstH1), the protease Deg1 (Deg1), subunit 4 of LHCII (Lhcb4), the kinase ABC1K1, subunit 5 of LHCII (Lhcb1) and subunit PetJ of cytb6f complex (PetJ) are indicated with an arrow (a). Transcripts encoding proteins of the mitochondrial electron transport chain (respiration) corresponding to subunit IV of bc1 complex (complex III), subunit γ of ATP synthase (ATP-γ), subunit 1 of NADH dehydrogenase (complex I), subunit V of bc1 complex (complex III) and subunit 2 of NADH dehydrogenase (complex I) are indicated with an arrow (b). The level of transcripts is expressed as the number of normalized reads and time in days signal peptide peptidase, prefoldin subunit 6, Hsp40 (DNAJ) and hsp70, among others (Additional file 5: Table S2). Transcripts encoding proteins involved in signal transduction were: calreticulin, the serine/threonine protein kinases PKWA, PRP4, SAPK8 and SAPK38, the MAPKKK11, myb-related protein 1, tyrosine protein kinase SRK3, the protein phosphatases 1 regulator subunit 7 and the bifunctional phophatase IMPL2, among others (Additional file 5: Table S2). Transcripts encoding proteins involved in replication and DNA repair were: histone deacetylase HD11, DNA polymerase ε subunit subunit B, DNA topoisomerase 6 subunit A, DNA helicase II, RNA helicase DExH10, RNA polymerase I subunit RPA12, DNA repair protein RAD45, and DNA mismatch repair protein MSH13, among others (Additional file 5: Table S2). Copper-induced increases in net photosynthesis and respiration The level of produced oxygen under light (photosynthesis) decreased in control alga from 8.5 to 6.1 nmoles μL− 1 min− 1 whereas in treated alga it increased from 8.5 to 9.6 nmoles μL− 1 min− 1 at day 2, which represent a 32% of increase compare to control, and then decreased to 7.5 nmoles μL− 1 min− 1 at day 5, which represent a 23% of increase compare to control (Fig. 3a). The level of consumed oxygen in dark (respiration) in control algae was 1.7 nmoles μL− 1 min− 1 and did not change at day 1, but increased to 3.8 nmoles μL− 1 min− 1 at day 5 whereas in treated alga it increased from 1.7 to 3.5 nmoles μL− 1 min− 1 at day 1 (106% of increase), and to 4.5 nmoles μL− 1 min− 1 at day 5 (18% of increase) (Fig. 3b). Thus, net photosynthesis and respiration increased in U. compressa exposed to copper excess. It is important to mention that oxygen produced in high light was higher than oxygen consumed in dark, which is in accord with level of normalized reads observed in kinetic analyses. Copper-induced increases in activities of enzymes involved in C, N and S assimilation The activity of the enzyme rubisco in control algae was 2 μmol min− 1 mg− 1 of protein and it remained at this level until day 5 whereas in treated alga it increased to 4 μmol min− 1 mg− 1 of protein at day 3 and decreased to 3 μmol min− 1 mg− 1 of protein at day 5 (Fig. 4a). The activity of glutamine synthase increased in control alga from 0.15 to 0.22 nmol min− 1 mg− 1 of protein, and in treated alga it increased to 0.61 nmol min− 1 mg− 1 of protein at day 3 and then decreased to 0.1 nmol min− 1 mg− 1 of protein at day 5 (Fig. 4b). The activity of cysteine synthase in control algae increased from 1.8 to 2.3 μmol min− 1 mg− 1 of protein whereas in treated algae it increased to 3.3 μmol min− 1 mg− 1 of protein at days 3 and remained at this level at day 5 (Fig. 4c). Thus, the activity of key enzymes involved in C, N and S assimilation increased in U. compressa in response to copper excess. The activities of enzymes rubisco and cysteine Laporte et al. BMC Plant Biology (2020) 20:25 Page 10 of 16 Fig. 2 Level of the highest increased transcripts encoding enzymes involved in C, N and S assimilation in U. compressa cultivated with 10 μM copper for 0, 1, 3 and 5 days. Transcripts encoding enzymes involved in C assimuilation corresponding to glyceraldehyde 3-P dehydrogenase (G3PDH), small subunit of rubisco (RbcL), phosphorribulo kinase (PRK), large subunit of rubisco (RbcL) and fructose biphosphate aldolase (FBPA) are indicated with an arrow (a). Transcripts encoding enzymes involved in N assimilation corresponding to nitrate reductase (NitrateR), fumarase (Fum), glutamine synthase (GlnS) and arginine-succinate lyase (ArgSL) are indicate with an arrow (b). Transcripts encoding enzymes involved in S assimilation corresponding to ATP sulfurylase (ATPS), APS reductase (APSR) glutamine cysteine ligase (GCL), sulfite reductase (SulfiteR) and cysteine synthase are indicated with an arrow (c). The level of transcripts is expressed as the number of normalized reads and time in days synthase were higher than the activity of glutamine synthase, which is in accord with the level of normalized reads. Discussion Copper-induced increased expression of proteins involved in photosynthesis and in repair and protection of photosystems In this work, we showed that the marine alga U. compressa cultivated with 10 μM copper for 5 days displayed an increased expression of transcripts encoding subunits of LHCII, PSII, cytb6f, LHCI, PSI and ATP synthase as well as proteins involved in repair of PSII and protection of PSI. It is important to mention that the level of transcripts encoding subunits of PSII, PSI, and proteins involved in PS repair and protection, showed higher increases compared with those observed in the alga cultivated with 10 μM copper for 0 to 24 h [8]. In this sense, the level of transcripts encoding subunits of PSII increased 2 Log2 FC in the alga cultivated with copper for