Adaptive responses of marine diatoms to zinc scarcity and ecological implications

Identification of two Zn/Co responsive proteins in diatoms

Zn and Co growth rate experiments in which Zn or Co (omitting the other) were added to the growth media were conducted and harvested for proteomic analysis. Growth rates of the marine diatom species Thalassiosira pseudonana CCMP1335, Phaeodactylum tricornutum CCMP632, Pseudo-nitzschia delicatissima UNC1205 and Chaetoceros sp. RS19 (Chaetoceros RS19 herein) were conducted in a consistent media composition to allow for intercomparison among species (see “Methods”). The onset of growth limitation by Zn and Co was evident by decreased growth rates under low [Zn²⁺] and [Co²⁺], and the ability to use Co to restore Zn-limited growth was species-specific and consistent with prior results for the diatoms T. pseudonana, P. tricornutum and P. delicatissima (Fig. 1a, b)⁹ and for other eukaryotic algae^2,8,10. Growth rates of Chaetoceros RS19 were not stimulated by increasing [Co²⁺] up to 23.5 pM in the absence of added Zn. This inability to substitute Co for Zn in Chaetoceros RS19 was clearly distinct from that of other diatoms, but was consistent with previous observations in Chaetoceros calcitrans¹⁰, implying a genus-wide attribute.

The proteome as a function of Zn²⁺ and Co²⁺ was explored in the marine diatom T. pseudonana harvested during log phase growth. Global proteomic analysis comparing low (1.1 pM) versus high (10.2 pM) added [Zn²⁺] and low (2.3 pM) versus high (23.4 pM) added [Co²⁺] revealed two uncharacterized diatom proteins that greatly increased in abundance at low [Zn²⁺] or [Co²⁺] (Fig. 1c, d). These proteins were annotated as a CobW/HypB/UreG, nucleotide binding domain and a bacterial extra-cellular solute binding domain, respectively, within the manually curated JGI Thaps3 T. pseudonana genome¹⁷ and were identified in T. pseudonana cultures with high confidence (≥9 exclusive unique peptides, 100% protein probability; Supplementary Fig. 1). BLAST sequence alignments showed these proteins to be homologous with CobW-like proteins (with 31.69% identity relative to Pseudomonas denitrificans CobW) and with the bacterial nickel transport protein NikA (with 30.5% identity relative to E. coli NikA), respectively. Based on their clear response to Zn and Co in the proteomes of multiple diatom species (Fig. 2a–d), the lack of definitive annotations in diatoms, and their genetic distance from bacterial homologs, these proteins are referred to as ZCRP-A and ZCRP-B (Zn/Co Responsive Protein A and B) in this study. Abundance patterns of these proteins were also investigated in P. tricornutum, P. delicatissima and Chaetoceros RS19. ZCRP-A spectral abundance counts were significantly (Kendall correlation, p < 0.05) inversely related to the total amount of Zn or Co added to the culture media (Fig. 2a–d; Supplementary Fig. 2). A similar pattern was found for ZCRP-B in the diatoms T. pseudonana, P. tricornutum, and P. delicatissima, though ZCRP-B spectral counts were significantly inversely related only to Zn concentration (Supplementary Fig. 2, p = 0.034). Unlike the continuous decrease in ZCRP abundances with increasing metal concentrations observed in T. pseudonana and Chaetoceros RS19, ZCRP abundances in P. tricornutum and P. delicatissima at the lowest metal treatments were somewhat smaller compared to their abundances in the next highest metal treatments, possibly owing to increased cellular stress and reduction in overall metabolism at the lowest added metal treatment in these diatoms. Homologs of ZCRP-B were not detected in the proteome of Chaetoceros RS19 (Fig. 2d). We note that the transcriptome of Chaetoceros RS19 used for peptide-to-spectrum mapping of the proteome also lacked ZCRP-B, which implies that either the conditions the transcriptome were generated under (iron limitation) did not elicit a low Zn response and corresponding ZCRP-B transcripts, or alternatively, that Chaetoceros RS19 does not possess the ZCRP-B gene. As a result, proteomic nondetection of ZCRP-B in Chaetoceros RS19 in this study cannot currently be interpreted due to its potential absence from the genome.

Among the four diatom species, both ZCRPs became undetectable at different threshold divalent metal cation concentrations. For T. pseudonana, both proteins became undetectable at 10.2 pM Zn²⁺, but both were still detected in the presence of up to 23.4 pM Co²⁺ (Fig. 2a). The threshold Zn²⁺ concentration for P. tricornutum was lower, as neither protein was detected in 3.2 pM Zn²⁺. A threshold Co²⁺ concentration also uniquely existed in P. tricornutum, with neither protein detected in 23.4 pM Co²⁺ (Fig. 2b). Similar to T. pseudonana, both proteins in P. delicatissima were detectable up to 23.4 pM Co²⁺, and a Zn threshold for ZCRP-B was observed at 10.2 pM Zn²⁺ (Fig. 2c). Overall, Zn²⁺ thresholds eliciting detectable ZCRP abundances among these diatoms were lower compared to Co²⁺ thresholds. ZCRP-B was not detected in Chaetoceros RS19, and ZCRP-A was still detectable up to 10.2 pM Zn²⁺ (Fig. 2d). No proteomic data is available for Chaetoceros RS19 grown in Co amended media as this diatom is incapable of substituting Co²⁺ for Zn²⁺, thus obtaining biomass under Co growth was not possible. An inverse relationship between spectral counts of both proteins and growth rate was apparent in P. tricornutum across +Zn and +Co treatments, with protein expression increasing as growth rate decreased (Fig. 2e, f; Supplementary Table 1).

Putative metal binding capability of ZCRP-A and cellular localization

Sequence alignment (BLASTp) of the ZCRP-A proteins detected in T. pseudonana, P. tricornutum, P. delicatissima, and Chaetoceros RS19 revealed the presence of four conserved motifs that classify these proteins into the COG0523 family of G3E GTPases. These include the G1/Walker A, G2/Switch I, putative metal-binding CXCC (C, cysteine; X, any amino acid, often hydrophobic), and G3/Walker B motifs^8,13 (Fig. 3a). Predicted tertiary structures of the ZCRP-A homologs in T. pseudonana, P. tricornutum, P. delicatissima, and Chaetoceros RS19 were modeled in SwissModel¹⁸ using E. coli YjiA as a template (PDB entry 4IXM.2.b) to investigate putative Zn²⁺ binding capacity. We included the E. coli COG0523 GTPase YjiA as it is the only member of the COG0523 subfamily to date with a determined crystal structure¹³. YjiA is currently thought to be a metal chaperone^8,19. We furthermore included as a template a COG0523 protein previously described in the freshwater marine alga Chlamydomonas reinhardtii that was observed to have increased transcript expression under Zn-deficient conditions⁸.

With the exception of the ZCRP-A homolog in Chaetoceros RS19, all ZCRP-A homologs were predicted to possess at least one Zn²⁺ binding site formed by coordination to two glutamate (E) and one cysteine (C) residue (Fig. 3b), the cysteine residue belonging to the conserved putative metal-binding CXCC motif (Fig. 3a, b). Previous work with bacterial CobW has shown that high affinity metal binding requires the association of the protein with magnesium (Mg²⁺) and GTP, with the thiol-containing Co²⁺ binding site in Mg²⁺GTP-CobW thought to be derived from the CxCC motif and a tetrahedral geometry¹⁹. While no model of a Mg²⁺GTP-bound COG0523 protein yet exists, the presence of GTP-binding domains in diatom ZCRP-A (Fig. 3a) and a Zn²⁺ coordination environment comparable to that of YjiA suggests that high affinity metal binding in diatom ZCRP-A also likely requires association with Mg²⁺ and GTP. Furthermore, while the model suggests that the Zn²⁺ coordination environment of ZCRP-A is comparable to that of YjiA, this does not mean that ZCRP-A and YjiA necessarily fulfill the same cellular function since the C-terminal domain, which is thought to be an indicator of protein–protein interaction specificity, is quite different in these two proteins.

The general cellular localization of ZCRP-A was also explored using overexpression (OE) lines of yellow fluorescent protein (YFP) tagged ZCRP-A in P. tricornutum (ZCRPA-OE). ZCRP-A appears to be an intracellular protein that localizes in proximity to the endoplasmic reticulum (ER) (Fig. 4a and Supplementary Fig. 3a). The intracellular distribution of YFP alone was distinctly different from the distribution of YFP-tagged ZCRP-A and YFP-tagged ZCRP-B (Supplementary Fig. 4). Diatoms possess complex plastids with four distinct membranes, the outermost being contiguous with the ER²⁰. Given that the P. tricornutum ZCRP-A sequence lacks a predicted plastid signal peptide (but rather has a predicted cytosolic signal peptide), we hypothesize that ZCRP-A localizes to the ER or the portion of the contiguous ER membrane directly adjacent to the plastids, the chloroplast ER (CER).

To date, connections between COG0523 proteins and utilization of Zn and Co have been explored primarily in prokaryotic organisms. For example, the COG0523 protein CobW has a role in vitamin B₁₂ biosynthesis and thus Co use^19,21. In contrast, a subgroup of other COG0523 proteins (YjiA, YeiR, ZigA, and ZagA) have been implicated in Zn²⁺ metabolism^{8,13,14,15,16}, and a client protein to the metallochaperone ZagA in Bacillus subtilis has been identified²².

Compared to bacteria, less is known about the function of COG0523 proteins in marine phytoplankton, though COG0523 protein family members are known to occur in all kingdoms^8,23. A recent study described the presence of COG0523 domain proteins upregulated under low Zn in the coccolithophore Emiliania huxleyi, but without further functional characterization²⁴, implying a potential Zn-related function of a COG0523 protein in a marine alga distinct from the marine diatoms included in this study.

Although various proteins belonging to the COG0523 subgroup share similar conserved domains, they possess different metal binding abilities and thus likely have different functions among the diverse organisms in which they are found. For example, recent work has established that CobW preferentially binds Co²⁺ as the cognate metal and acts as a Co²⁺ chaperone ultimately supplying vitamin B₁₂ in bacteria, whereas the closely related putative metal chaperones YeiR and YjiA (homologs of CobW) bind Zn²⁺¹⁹. We can infer from homology and the response to low Zn and low Co in the present study that Zn²⁺ and Co²⁺ are likely both cognate metals for diatom ZCRP-A. Further metal binding and affinity assays can confirm and characterize metal binding in this protein.

Frustule morphology

Phenotypic plasticity in P. tricornutum is well documented. Two basic cell morphotypes, fusiform and triradiate, are found in natural liquid environments. It is thought that by adopting the triradiate form, a cell increases its surface area and thus the area of membrane available for enzymatic activity or molecular diffusion of dissolved inorganic carbon (DIC) into the cell. The triradiate form is known to be more common under DIC limiting conditions, which supports this hypothesis²⁵. Distinct morphological differences resulted from the knockout (KO) of the ZCRP-A gene. In P. tricornutum, ZCRP-A knockout cells consistently adopted a triradiate shape while wild-type cells were fusiform (Fig. 4i). Normally, triradiate cells of P. tricornutum spontaneously revert to fusiform across generations²⁶, thus it is notable that ZCRP-A knockout cells have consistently maintained their triradiate shape for 4+ years in culture irrespective of media [Zn²⁺]. As Zn²⁺ is the predominant metal cofactor used in diatom CAs, the adoption of the triradiate form in knockout P. tricornutum cells may be a response to a disruption of the carbon concentrating mechanism caused by a reduction in Zn acquisition capability due to ZCRP-A knockout. This is consistent with the observed relative increase in Mn²⁺-utilizing CA (ι-CA) in the knockout line compared to the wild-type (Supplementary Fig. 5).

ZCRP-B sequence analysis and cellular localization

Unlike COG0523 proteins, the relationship of ZCRP-B abundance to environmental Zn and Co concentrations does not appear to have been previously described. Topology predictions of P. tricornutum ZCRP-B using TOPCONS^27,28 revealed a single predicted transmembrane domain near the N-terminus, with the majority of the protein predicted to be oriented outside the membrane (Fig. 4j). Overexpression and fluorescent tagging of ZCRP-B confirmed localization to the cell membrane (Fig. 4e–h; Supplementary Fig. 3b). A single predicted transmembrane domain contrasts with the Zrt/Irt-like divalent metal transporters (ZIPs) in eukaryotic algae, which have 7+ transmembrane domains and are key Zn transporters in many organisms^29,30. It is therefore most likely that ZCRP-B is not a transporter itself, but one part of a multi-protein membrane complex and potentially interacts with the ZIP system. A sequence database similarity search (BLASTp, NCBI) found the ZCRP-B protein to be homologous with NikA, a protein subunit of the bacterial ATP-binding cassette (ABC) type Ni transport system protein Nik (30.5% identity with E. coli NikA, E = 7e−49, Supplementary Fig. 6). This transporter is well characterized in bacteria and is comprised of five subunits NikA-E. NikB and NikC are two pore-forming integral inner membrane proteins, NikD and NikE are two inner membrane-associated proteins with ATPase activity, and NikA is the periplasmic component that functions as the initial metal receptor³¹. No proteins with homology to NikB nor NikC were detected in the P. tricornutum proteomes generated in this study. Two uncharacterized P. tricornutum proteins were homologous with NikD (28.8% identity, E = 1e−14) and NikE (34.9% identity, E = 1.33e−8), though neither had abundance trends similar to ZCRP-B, implying that their function and regulation are independent of ZCRP-B.

The sequence of a functionally similar bacterial ABC transport complex, CntABCDF (cobalt nickel transporter, also known as Opp1) from Staphylococcus aureus was also compared to NikA and ZCRP-B (Supplementary Fig. 6). CntA shares 25.6% identity with ZCRP-B (E = 3e−28), and similar to NikA, is an extra-cytoplasmic solute-binding protein that transports Ni, Zn and Co. CntA functions as a Ni/Co acquisition system in Zn-limited S. aureus³². Although the Nik and Cnt systems serve Ni and Co transport in bacteria, ZCRP-B responds to Zn and Co in marine diatoms, which have a significant Zn demand. This may imply a recruitment and repurposing of this bacterial Ni transporter component as part of the Zn acquisition systems during the evolution of marine diatoms.

ZCRP-B as a putative high-affinity ligand

Sequence similarity to the extracellular transport components NikA and CntA (Supplementary Fig. 6), localization to the plasma membrane (Fig. 4b; Supplementary Fig. 3b), and increased abundance under low Zn and Co conditions (Fig. 2b) of P. tricornutum ZCRP-B suggests a metal-binding role as part of a high-affinity transport complex. The induction of ZCRP-B expression at low [Zn²⁺] (Fig. 2a–c) fits the description of a high-affinity Zn uptake system observed in marine algae that is known to be induced at low free [Zn²⁺]^33,34, suggesting that this protein is involved in an adaptive response to extremely scarce Zn availability. Furthermore, ZCRP-B could contribute to the pool of high-affinity organic ligands that complex dissolved Zn, either by dissociation from living cells or upon cell death by viral lysis and grazing, in the upper water column^12,35.

The identification of a membrane-associated Zn-Co responsive protein-containing putative metal-binding sites allows us to reconsider the mechanisms of cellular metal uptake in diatoms. Prior physiological experiments observed Zn uptake in marine diatoms to approach the limits of diffusion³³, and predicted kinetic control with fast cell surface metal binding and uptake relative to dissociation and release back to the seawater environment³⁶. To enable this transport capability, it was postulated that transporters might be so abundant that the membrane becomes crowded³⁷. Here, the observation of a putative Zn-binding, membrane-associated protein with only 1 predicted transmembrane domain instead implies a separation of the Zn concentrating function at the cell surface relative to its transport into the cell. In this scenario when Zn is scarce, biosynthesis of ZCRP-B increases and is tethered to the cell surface to compete Zn away from natural dissolved Zn ligands³⁵ and/or chelate Zn atoms that make it through the diffusive boundary layer to the membrane. In this manner, ZCRP-B would increase the surface Zn concentration in the vicinity of Zn transporters, and multiple ZCRP-B proteins could supply nearby surface ZIP transporters or be endocytosed, avoiding the predicted membrane crowding of transporters problem. Aristilde and colleagues have previously demonstrated that weak natural Zn-binding ligands containing cysteine do indeed enhance cellular Zn uptake within the diatom Thalassiosira weissflogii, with heightened effects in Zn-limited compared to Zn-replete cells³⁸. They proposed the formation of a transient tertiary complex between the Zn-bound ligand and Zn transporters (ZIPs and heavy metal P-type ATPases) at the cell surface, which could be mediated by a surface-tethered Zn binding ligand such as ZCRP-B. Future studies could examine the mechanism of Zn exchange between ZCRP-B and Zn/Co transporters such as the ZIPs in eukaryotic algae, which were also detected at lower Zn and Co abundances in P. tricornutum but with relatively lower spectral counts (Supplementary Fig. 7a, b), consistent with this model. Furthermore, the proposed mechanism of ZCRP-B binding is similar to that of the high-affinity Fe³⁺ binding protein ISIP2a, previously characterized in marine algae as an iron starvation-induced protein³⁹. ISIP2a has been characterized as a phytotransferrin involved in endocytosis-mediated high-affinity Fe uptake in P. tricornutum that acts to concentrate Fe at the cell surface and is an extracellular protein anchored to the membrane with one transmembrane domain³⁹. As the protein sequences of P. tricornutum ZCRP-B and ISIP2a share no significant similarity, it is possible that the uptake mechanism of ZCRP-B is similar to that of ISIP2a, but specific to high-affinity Zn and Co uptake rather than Fe. This suggests a common strategy of using extracellular membrane-anchored metal acquisition proteins in marine algae faced with metal limitation.

Co uptake in wild-type and mutant diatom strains

As ZCRP-A and ZCRP-B abundance is related to media [Co²⁺] (Fig. 2a–d), we investigated differences in the extent of Co uptake after 24 h among Zn/Co-limited wild-type, ZCRP-A knockout, ZCRP-A overexpression, and ZCRP-B overexpression lines of P. tricornutum via addition of the radiotracer ⁵⁷Co (see methods). The extent of Co uptake among genetically modified P. tricornutum lines was observed to be significantly different via one-way ANOVA (f(3) = 23.16, p = 0.000268). A Dunnet post hoc test revealed that uptake was significantly greater (2.6× larger) in the ZCRP-A overexpression line compared to wild-type (p = 0.00048, Fig. 4k). We interpret this result as the overexpression of ZCRP-A creating a larger intracellular binding capacity for Co, thus protecting it from intracellular sensor or regulatory systems and/or efflux pumps. In contrast, no significant difference in Co uptake rates was observed when comparing ZCRP-A knockout, ZCRP-B overexpression, and wild-type lines, suggesting that P. tricornutum ZCRP-A knockout cells are capable of compensating for knockout to maintain Co metabolism, perhaps through the use of low-affinity transporters³³. This is consistent with these uptake experiments being conducted using seawater media with a relatively abundant concentration of Zn (background of 7.7 pM Co and 4.0 nM Zn in the absence of EDTA), thus the use of low-affinity transporters was likely sufficient to acquire Zn and Co for growth, and neither ZCRP-A knockout nor ZCRP-B overexpression would be expected to add any metabolic benefit (Fig. 4k). Moreover, if ZCRP-B is only one part of a multi-protein acquisition and transport complex as hypothesized, overexpression of the single protein may not result in enhanced functionality.

Abundance patterns of CAs in two diatoms

Carbonic anhydrase enzymes constitute a major reservoir of Zn and Co within marine diatoms⁷. Within the stroma, intracellular chloroplastic CAs are essential in supplying CO₂ to RUBISCO as they convert HCO₃⁻, the predominant species of inorganic carbon in the pyrenoid, into CO₂^40,41. Seven subclasses of CAs have been identified in marine diatoms to date and are designated as alpha, beta, gamma, delta, zeta, theta, and iota (α, β, γ, δ, ζ, θ, and ι). While Zn²⁺ is the cofactor most commonly used in algal CAs, utilization of both cadmium (Cd²⁺) and cobalt (Co²⁺) in place of Zn²⁺ at the active site of ζ-CA (CDCA) and a δ-CA, respectively, has been previously documented^2,5,42. Overall, Zn-utilizing CAs increased in abundance with increasing Zn, consistent with the need for rapid HCO₃⁻ conversion at faster growth rates (Fig. 5; Supplementary Fig. 7). Specifically, spectral abundance counts of two β-type CAs, PtCA1 and PtCA2, became abundant in high [Co²⁺] (23.4 pM) and [Zn²⁺] (> 1.1 pM) and were inversely related to ZCRP-A abundance (Supplementary Fig. 7). Both PtCA1 and PtCA2 are known to localize to the chloroplast pyrenoid^41,43. Moreover, the increasing abundance trends of the Zn-utilizing α-CAs (CA-II and CA-VI) and the θ-CA Pt43233, which localize to the periplastidial compartment, chloroplast endoplasmic reticulum, and thylakoid lumen, respectively, at higher and Zn/Co provide further evidence for this strategy of increasing CA use under Zn-replete and higher growth rate conditions (Fig. 5; Supplementary Fig. 7)^43,44.

In contrast, abundance trends of the recently discovered ι-CA were inversely related to Zn²⁺ (Fig. 5). Originally identified in T. pseudonana, ι-CA was found to localize to the inner chloroplast membrane surrounding the stroma and is unusual in that it prefers Mn²⁺ to Zn²⁺ as a cofactor⁴⁵. In the present study, spectral counts of P. tricornutum ι-CA decreased as metal concentrations increased, similar to that observed for ZCRP-A and ZCRP-B (Fig. 5). This ι-CA response was consistent with a Zn sparing strategy under low [Zn²⁺] and [Co²⁺] used to prioritize the use of Zn²⁺ for other metalloenzyme functions.

Due to the inverse relationship between the abundances of ZCRP-A and chloroplastic Zn²⁺-requiring CAs in P. tricornutum (that is, all CAs detected with the exception of ι-CA) and the various types of CAs in T. pseudonana (Supplementary Fig. 7), it seems unlikely ZCRP-A directly interacts with CAs. These results are instead consistent with the hypothesis that ZCRP-A functions as a Zn²⁺ allocation and prioritization mechanism during Zn limitation. The role of Zn²⁺ in key transcriptional and translational proteins such as RNA polymerase and ribosomal proteins is well known, and major reservoirs of Zn are associated with these transcription and translation systems in the fast-growing copiotrophic bacterium Pseudoalteromonas⁶. The availability of Zn in ribosomes and the ER is therefore likely also a cellular priority in diatoms, and could benefit from utilizing the putative chaperone and trafficking capability of ZCRP-A when Zn is scarce. We, therefore, posit that ZCRP-A may serve as a Zn²⁺ trafficking or storage protein that contributes to the prioritization and movement of Zn²⁺ to the ER or CER, while the Mn-utilizing Mn ι-CA compensates for the lowered Zn availability in the chloroplast. The increased biosynthesis of ZCRP-A may be an important function to shift Zn homeostasis, competing for intracellular Zn and trafficking it towards the ER or CER.

Distribution of putative ZCRP homologs among oceanic taxa

Putative ZCRP homologs among eukaryotic oceanic taxa were identified by BLAST searching the P. tricornutum ZCRP-A and ZCRP-B protein sequences against all available transcriptomes in the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) database, which includes over 650 assembled and annotated transcriptomes of oceanic microbial eukaryotes⁴⁶. Phylogenetic analysis revealed the presence of putative ZCRP-A and ZCRP-B homologs in a wide variety of organisms belonging to the Chromista kingdom that could be further categorized into Bacillariophyceae, Dinophyceae, and Prymnesiophyceae classes (Supplementary Figs. 8 and  9). Notably, the Chaetoceros RS-19 ZCRP-A homolog did not phylogenetically cluster with the other diatoms (Bacillariophyceae), but instead appears to be more closely related to E. coli YjiA (Supplementary Fig. 8). Furthermore, the lack of the conserved G2/Switch I region in the Chaetoceros RS-19 homolog (Fig. 3) is anomalous in comparison to other putative homologs identified within the MMETSP database. Overall, ZCRPs are not exclusive to oceanic diatoms, but rather are widely distributed amongst oceanic taxa.

Metaproteomic detection of ZCRP-A and ZCRP-B

To investigate the use of ZCRP-A and ZCRP-B in the natural environment, we searched metaproteomic data collected during the KM1128 METZYME (Metals and Enzymes in the Pacific) research expedition on the R/V Kilo Moana October 1–25, 2011 from Oʻahu, Hawaiʻi, to Apia, Samoa (Fig. 6a). dZn followed a nutrient-like distribution as described previously, with an average surface (40 m) dZn concentration of 1.21 nM and average deep water (3000 m) concentration of 10.37 nM⁴⁷ (Fig. 6b). dCo was highly depleted in the upper photic zone as the result of biological uptake^48,49 (Fig. 6c). Eukaryotic homologs of ZCRP-A and ZCRP-B were detected at multiple stations at surface (<200 m) depths with increased abundances moving southward. This was coincident with a deepening of the upper water column layer in which dZn and dCo were depleted (a “zincocline”), with depleted surface concentrations of dZn (and dCo) observed at greater depths south of the equator (Fig. 6b, c). The phytoplankton pigments chlorophyll a, fucoxanthin, and19′-hexanoyloxyfucoxanthin (19′-Hex) are used as chemotaxonomic proxies for diatom and prymnesiophyte abundances⁵⁰. Distinct differences in the distribution of pigments compared to that of ZCRP proteins over this transect demonstrated that increased ZCRP protein abundances cannot be simply ascribed to an increase in overall biomass (Figs. 6; 7).

Putative ZCRP-A homologs corresponded to nearest taxonomic matches to the diatom Helicotheca tamensis, the dinoflagellate Azadinium spinosum, and the prymnesiophytes Emiliania huxleyi and Phaeocystis sp. (Fig. 6d, f; Supplementary Table 2). Putative ZCRP-B homologs were identified in various eukaryotes corresponding to nearest taxonomic matches for the diatoms Pseudo-nitzschia fraudulenta and Leptocylindrus danicus and to the dinoflagellates Azadinium spinosum, Gonyaulax spinifera, and Symbiodinium sp. (Fig. 6e, g; Supplementary Table 2). Increased expression of these homologs occurred at low [dZn] (0–1.5 nM) at surface depths in the Equatorial and South Pacific (Fig. 6f, g; Supplementary Fig. 10), a region where Fe limitation was also documented^47,48. The preponderance of eukaryotic ZCRP-A and ZCRP-B coupled to observations of Zn scarcity in the South Pacific implies the existence of marine eukaryotic protists responding to Zn stress in this area.

Deployment of ZCRP-A and ZCRP-B as bioindicators of oceanic Zn stress

While nitrogen, phosphate, and iron are widely recognized to be nutritionally important to phytoplankton⁵¹, other elements such as Zn are rarely examined, likely due to methodological difficulties arising from pervasive Zn contamination, yet Zn is present in extreme scarcity in large tracts of the surface ocean⁵². Given the demonstrated use of Zn by eukaryotic phytoplankton, it has been hypothesized that Zn may limit oceanic production rates and influence the global carbon cycle⁵. Laboratory-based studies with phytoplankton isolates maintained in culture have shown that Zn²⁺ may act as a limiting nutrient for some species at concentrations of ~1 pM^2,4,33, and we have shown in the present study that expression of ZCRP-A and ZCRP-B occurs at Zn²⁺ and Co²⁺ concentrations ~1 pM (Fig. 2a–d). We explored the utility of field detection of these proteins and suggest that ZCRP-A and ZCRP-B may be useful candidates as biomarkers of Zn stress in the field, just as the detection of other protein biomarkers has been successfully applied to diagnose regions of nutrient stress⁴⁸. Our observations of homologous proteins among multiple eukaryotic protists throughout the South Pacific imply that Zn scarcity may be far more prevalent than previously recognized, and that the role of Zn in influencing marine primary productivity may therefore be underestimated.

We have identified and characterized two Zn and Co responsive proteins, ZCRP-A and ZCRP-B, in the marine diatoms T. pseudonana, P. tricornutum, P. delicatissima, and Chaetoceros RS-19. These findings describe previously uncharacterized proteins that are involved in the metabolic response to Zn/Co metal limitation in marine diatoms, and are widespread in marine protists. A putative Zn²⁺ metallochaperone role for ZCRP-A in the allocation and reallocation of Zn under low Zn conditions and characterization of ZCRP-B as a membrane-associated putative metal-binding protein belonging to a multi-protein transport complex best fit our observations. Increased abundance of ZCRP-B at low [Zn²⁺] and [Co²⁺] with nondetection at higher metal concentrations fits the description of the high-affinity uptake mechanism in marine algae previously observed in physiology experiments³³. We have also shown that metaproteomic detection of both ZCRP-A and ZCRP-B in the field is possible and that protein abundances are highest at the surface where dZn and dCo are scarcest, corroborating our findings in culture and demonstrating their potential for use in detecting Zn stress. Based on our characterization of these two Zn and Co responsive proteins in multiple marine diatoms in culture and the detection of homologs in the field, we suggest that Zn nutritional stress in the surface ocean could be more prevalent than previously thought.

Source: Ecology - nature.com