ABSTRACT

   This study explored the contrasting physiological and transcriptional
   responses to iron (Fe) and warming temperature interactions in two
   South China Sea Synechococcus isolates belonging to clade II from the
   open ocean and CB5 from the coastal ocean. The two picocyanobacterial
   strains utilised contrasting photosynthesis, Fe uptake, and nutrient
   acquisition strategies to cope with Fe limitation. In the oceanic
   strain, moderate warming under Fe limitation upregulated expression of
   photosynthesis and nutrient and Fe transport genes, increasing its
   growth and photosynthesis. In contrast, gene expression under low Fe in
   the coastal strain was less affected by warming. The oceanic isolate
   exhibited substrate regulation of Fe acquisition and preferred organic
   nutrient sources. The coastal strain had a much higher Fe quota, faster
   turnover of the D1 gene in photosystem II, and was optimised for
   inorganic nitrogen sources. Both strains showed multi‐tiered Fe uptake
   strategies and general stress responses to heat shock and oxidative
   stress. In general, gene regulation in the oceanic strain responded
   more effectively to both stressors than in the coastal isolate.
   Fe‐temperature interactions in both strains are complex and may lead to
   synergistic and antagonistic responses, potentially influencing global
   biogeochemical cycles in warmer oceans.

   Keywords: Fe limitation, ocean warming, picocyanobacteria,
   Synechococcus, transcriptome
     __________________________________________________________________

   Oceanic Synechococcus respond to the interactive effects of iron (Fe)
   limitation and ocean warming by regulating photosynthesis, nutrient
   metabolism, and heat shock gene expression, while a coastal strain has
   fewer regulatory mechanisms. The oceanic strain may be a superior
   competitor with more comprehensive and flexible responses in Fe‐poor
   subtropical open ocean regimes. Fe‐temperature interactions may lead to
   complex synergistic and antagonistic responses, with significant
   implications for future plankton community dynamics and marine
   biogeochemistry.

   graphic file with name EMI4-17-e70158-g005.jpg

1. Introduction

   The Sixth Assessment Report (AR6) of the Intergovernmental Panel on
   Climate Change (IPCC) has stated that global temperatures have risen by
   ~1.5°C due to increased CO[2] emissions since the Industrial
   Revolution. In the ocean, warming can intensify stratification,
   especially in subtropical and tropical regions. This hinders the
   exchange of nutrients between surface and deep waters, reducing
   supplies to primary producers (Hutchins and Fu [39]2017). This includes
   vertical advective fluxes of iron (Fe), the most important trace
   element limiting primary production in the ocean (Tagliabue
   et al. [40]2017). At the cellular level, Fe plays a role in many
   crucial metabolic pathways, including photosynthesis, carbon fixation,
   and nitrogen assimilation (Kranzler et al. [41]2013). Warmer
   temperatures and Fe availability can either individually or
   interactively affect biological enzymatic activity and central
   metabolism (Hutchins and Boyd [42]2016).

   The marine Synechococcus genus is one of the most dominant
   picocyanobacterial groups, as it is widely distributed across various
   ocean regions and contributes around 17% of marine net primary
   production (Lee et al. [43]2019). Previous studies have investigated
   the evolutionary adaptations of Synechococcus clades and ecotypes to
   environmental factors (Lee et al. [44]2019; Ahlgren et al. [45]2020).
   The key drivers of niche specialisation in this genus are Fe,
   temperature, light, and nutrients (Lee et al. [46]2019; Sohm
   et al. [47]2016; Kling et al. [48]2023).

   Open ocean and coastal areas typically have different environmental
   conditions, including variations in Fe and temperature. The main source
   of Fe in oceanic areas is dust from the atmosphere, and surface
   concentrations are very low, usually less than 0.2 nM (Johnson
   et al. [49]1997). Coastal areas receive Fe from multiple sources, such
   as river input and upwelling. Thus ambient Fe concentrations are higher
   than in the open ocean, although phytoplankton iron limitation can
   still occur in some nearshore regions (Capone and Hutchins [50]2013).
   The temperature in oceanic areas is also more stable compared to
   coastal waters because the open ocean has a greater heat capacity,
   which helps moderate temperature changes, while coastal waters are more
   affected by seasonal temperature fluctuations, wind patterns, and ocean
   currents (Wang et al. [51]2020). Specifically, the warming rate in the
   South China Sea is about twice the global ocean mean rate (Fang
   et al. [52]2006).

   Previous studies have explored differences in growth, photophysiology,
   and proteomic allocation for photosynthesis and Fe acquisition in
   Synechococcus isolated from oceanic and coastal areas of the North
   Atlantic, which have varying concentrations of key nutrients like
   nitrogen (N) and phosphorus (P) (Mackey et al. [53]2015). Considering
   the complexity of natural environments, we performed a multi‐stressor
   experiment in the laboratory using Synechococcus isolates from adjacent
   coastal and oceanic areas of the South China Sea to explore how
   different ecotypes respond to environmental changes; proteomic
   responses in this experiment were presented in Schiksnis
   et al. ([54]2024). Here, we investigated differences in transcriptional
   networks in both nearshore and oceanic Synechococcus clades in response
   to iron and warming interactions in these contrasting ocean regimes.

   The oceanic strain belongs to the widely distributed oligotrophic
   subcluster 5.1 clade II (YX04‐1), while the coastal strain belongs to
   subcluster 5.2 clade CB5 (XM24), which has been previously reported to
   be halotolerant in the coastal waters of the East China Sea and
   Chesapeake Bay (Choi and Noh [55]2009; Chen et al. [56]2006). We
   cultured these strains at two temperatures (24°C and 27°C,
   Figure [57]1, Table [58]S1) that lie within the annual range of the
   present‐day South China Sea (Jin et al. [59]2023). At each temperature,
   we examined the effects of two Fe concentrations (2 and 250 nM) by
   measuring physiological responses such as growth, carbon fixation, and
   cellular Fe quotas. We also sequenced their transcriptomes across all
   four treatments to compare potential acclimation strategies in response
   to Fe‐warming interactions in these two strains. This study provides
   insights into how marine Synechococcus from taxonomically divergent
   coastal and oceanic clades regulate their transcriptional networks and
   short‐term responses to iron limitation in combination with moderate
   ocean warming in these adjacent but contrasting environments.

FIGURE 1.

   FIGURE 1
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   Experimental design schematic for both Synechococcus strains utilised
   in the study. Schematic overview of the coastal strain (Synechococcus
   XM‐24, green) and the oceanic strain (Synechococcus YX04‐1, blue) from
   the South China Sea, each grown at two temperatures (24°C and 27°C) and
   under two Fe conditions (Fe‐limited or Fe‐replete). Six treatment
   comparisons were performed: (1) −Fe@24°C versus +Fe@24°C, (called
   “−Fe@24°C”), (2) −Fe@27°C versus +Fe@27°C (called “−Fe@27°C”), (3)
   −Fe@27°C versus −Fe@24°C (called “27°C@‐Fe”), (4) + Fe@27°C versus
   +Fe@24°C (called “27°C@+ Fe”), (5) −Fe@27°C versus +Fe@24°C (called
   “‐Fe + warming”), and (6) + Fe@27°C versus −Fe@24°C (called “+Fe +
   warming”). The gold circles represent Fe‐replete cultures, whereas
   flasks without circles represent Fe‐limited cultures. The dashed
   rectangles denote cultures in two temperatures (24°C and 27°C). The
   coastal strain, Synechococcus XM24, is denoted by green bottles, while
   the oceanic strain, Synechococcus YX04‐1, is represented by blue
   bottles. Taxonomic and isolation site information is included for the
   two strains. Further detailed description of experimental design can be
   found in the Experimental Procedure section and in Table [61]S1.

2. Results

2.1. Physiological Responses in Coastal and Oceanic Strains

   In this section, we explored how Fe‐warming interactions affect
   physiology in both Synechococcus isolates, including growth, carbon
   fixation, and cellular Fe quotas. In both the coastal and oceanic
   Synechococcus isolates, Fe limitation led to a significant decrease
   (p < 0.05, tested by two‐way ANOVA and a Tukey multiple comparison
   test) in growth rates (Figure [62]2A,B), carbon fixation rates
   (Figure [63]S1A,B), and Fe quotas (Fe/P, Figures [64]2C,D and Fe/C,
   [65]S1C,D) at both high and low temperatures. Notably, the coastal
   strain had a much higher Fe quota than the oceanic strain, as the mean
   replete and limited Fe:P ratios of the coastal strain were about 3–4
   fold higher than those of the oceanic strain (Table [66]S2). In both
   the coastal and oceanic strains, the Fe quota (Fe/P) was increased with
   warmer temperatures under Fe replete conditions (Table [67]S2,
   Figure [68]2).

FIGURE 2.

   FIGURE 2
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   Specific growth rates and Fe quotas in the oceanic and coastal
   Synechococcus strains. Specific growth rates (d^−1) for the oceanic
   strain (A) and the coastal strain (B) and Fe quotas as the iron (Fe) to
   phosphorus (P) ratio (mmol/mol) for the oceanic strain (C) and the
   coastal strain (D) in an experimental matrix of two temperatures (x
   axis) and two Fe conditions (pink replete and blue limiting). Error
   bars are standard deviations of triplicates. Mean values that do not
   share the same letter are significantly different from one another with
   p < 0.05 (tested by two‐way ANOVA).

   Under Fe‐limited conditions, only the oceanic strain showed increased
   growth (~80%) with warming (Figure [70]2A, Table [71]S2). Additionally,
   the growth of the oceanic strain was less impaired by Fe limitation at
   warming conditions. However, warming intensified the negative effects
   of Fe limitation on carbon fixation (Figure [72]S1A). Carbon fixation
   rates showed minimal responses to warming (Figure [73]S1A, 27°C@‐Fe).
   Under Fe limitation, carbon fixation rates were less affected by
   warming (Figure [74]S1A) compared to the significant increase in
   growth. Warming alone, regardless of Fe availability, did not stimulate
   growth in the coastal strain.

2.2. Overview of Transcriptional Responses in Both Strains

   To investigate the overall profiles of transcriptomes in two
   Synechococcus isolates in response to Fe‐warming interactions, we
   identified the total differentially expressed genes (DEGs) and the most
   responsive DEGs under different conditions. The number of upregulated
   and downregulated genes that are differentially expressed in both
   strains is shown in Figure [75]3. The genome sizes of both strains are
   similar, at 2.4 MB for the oceanic strain and 2.5 MB for the coastal
   strain. The oceanic strain displays a greater number of DEGs (300–800)
   than the coastal strain (100–300) across all comparisons between
   treatments. The overall gene expression heatmap (Figure [76]S2) showed
   that gene expression changes from warming are more consistent in the
   oceanic strain, whereas changes due to Fe limitation are more uniform
   in the coastal strain.

FIGURE 3.

   FIGURE 3
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   The differentially expressed gene (DEG) numbers in treatments for the
   oceanic strain (A) and the coastal strain (B). Red denotes upregulated
   DEGs, and blue denotes downregulated DEGs, and numbers of each are
   marked on bars. The heatmap of genes involved in the ABC transporter
   and two‐component system for the oceanic strain (C). The treatment
   columns include –Fe@24°C, –Fe@27°C, 27°C@‐Fe, 27°C@+ Fe, −Fe + warming,
   +Fe + warming. Detailed descriptions of each treatment comparison are
   given in the Methods. Each row denotes one gene with ID and annotation
   listed. The asterisk denotes differential expression with a fold change
   > 2 and an adjusted p < 0.05.

   The top 20 most responsive DEGs for each treatment in both strains are
   summarised in Tables [78]S3 and [79]S4. They are involved in pathways
   of photosynthesis (psbA, petJ, ndhD, cpeAB), carbon and nutrient
   metabolism (cysK, glnB, nirA, urtA‐D), Fe transport (idiA, afuC), and
   heat shock response (hsp20, clpB). KEGG pathway enrichment analysis
   (Figures [80]S3 and [81]S4) reveals that DEGs in the oceanic strain are
   involved in ABC transporter and two‐component systems during Fe
   limitation under warming (Figure [82]S3A, −Fe@27°C), warming under Fe
   limitation (Figure [83]S3C, 27°C@‐Fe), and Fe/warming interactions
   (Figure [84]S3E, −Fe + warming; Figure [85]S3F, +Fe + warming).

2.3. Transcriptional Responses of Photosynthetic Pathways in Coastal and
Oceanic Strains

   To investigate the distinct photosynthetic apparatus responses in both
   strains to Fe‐warming interactions, we compared the transcriptional
   responses of Fe limitation under low temperature and warming conditions
   (−Fe@24°C, −Fe@27°C), warming under high Fe and low Fe conditions
   (27°C+ Fe, 27°C@‐Fe), and Fe‐limited warming and Fe‐replete warming
   conditions (−Fe + warming, +Fe + warming). In the oceanic strain, at
   low temperature under Fe limitation (−Fe@24°C) most downregulated DEGs
   were involved in light harvesting and photosynthetic electron transport
   pathways (Figure [86]4A), including photosystem I (PSI) (psaA‐I),
   photosystem II (PSII) (psbA‐F, psbL, psbT, psbUV, psbX), cytochrome b6f
   complex (petA‐D), ferredoxin (petF), and cytochrome c6 (petJ), and
   phycobilisome (apcABC, cpcAB, cpcG, cpeA‐D) genes. ATPase and oxidative
   phosphorylation were barely affected by Fe limitation. Similarly, under
   warming and Fe‐limited conditions, the oceanic strain also upregulated
   genes involved in photosynthesis, such as PSI (psaEF, psaK, psaM), PSII
   (psb28, psbBC, psbEF, psbUV), the cytochrome b6f complex (petC), and
   phycobiliproteins, as well as those for NADPH production
   (Figure [87]4A).

FIGURE 4.

   FIGURE 4
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   Heatmap presenting gene expression patterns of the photosynthetic
   pathways under different treatments in the oceanic strain (A) and the
   coastal strain (B). The comparison treatment columns include –Fe@24°C,
   –Fe@27°C, 27°C@‐Fe, 27°C@+ Fe, −Fe + warming, +Fe + warming. Detailed
   descriptions of each treatment comparison are given in the Methods.
   Each row denotes one gene with ID and annotation listed. The asterisk
   denotes differential expression with a fold change > 2 and an adjusted
   p < 0.05. Upregulated genes are represented in red, while downregulated
   genes are depicted in blue.

   For the oceanic strain under Fe‐replete conditions, warming
   downregulated most DEGs related to ATP production (atpEF, atpHI) and
   photosynthesis (psbA, psbI, psbJ, psbL in PSII, psaB in PSI, petH,
   petN). However, one gene in PSI (psaM) was upregulated by warming under
   Fe‐replete conditions (Figure [89]4A, 27°C@+ Fe). In contrast, warming
   upregulated several genes related to oxidative phosphorylation (ndhA,
   ndhD‐G, ndhO, COX15) (Figure [90]4A, −Fe@27°C).

   In the coastal strain, photosynthetic pathways were much less sensitive
   to Fe limitation, with only 15 DEGs in total compared to 66 DEGs in the
   oceanic strain (Figure [91]4B). However, the gene petJ encoding Fe‐rich
   cytochrome c6 was notably downregulated at both temperatures
   (Figure [92]4B, −Fe@27°C, −Fe@24°C). Although most genes in the
   photosynthetic pathways were downregulated, multiple copies of psbA2
   were upregulated and psbA1 remained unaffected under Fe limitation at
   both temperatures (Figure [93]4B, −Fe@27°C, −Fe@24°C). The DEGs under
   Fe limitation were similar at high and low temperatures, where psbA2
   copies were all upregulated, and petJ and cpeC were downregulated
   (Figure [94]4B, −Fe@27°C, −Fe@24°C). Surprisingly, in the coastal
   strain, only two genes in PSII (psbK, psbM) were upregulated with
   warming regardless of Fe conditions (Figure [95]4B, 27°C@+ Fe,
   27°C@‐Fe), corresponding to a minimal increase in growth
   (Figure [96]4B).

   In contrast, under the warming condition, gene expression in the
   oceanic strain was largely unaffected by Fe limitation (Figure [97]4A,
   −Fe@27°C). Rubisco (ribulose bisphosphate carboxylase) genes rbcS and
   rbcL were downregulated (Figure [98]S5A, −Fe@27°C). The downregulation
   of the Rubisco small subunit rbcS at both Fe concentrations was
   observed (Figure [99]S5A). Under warmer and Fe‐limited conditions, the
   DEGs related to carbon fixation showed no notable difference to warming
   in the oceanic strain (Figure [100]S5A).

2.4. Comprehensive Nutrient Assimilation Network in the Oceanic Strain

   To understand how nutrients were differently assimilated and utilised
   in both strains under Fe‐warming interactions, we compared DEGs linked
   to nutrient transport, regulation, and metabolism systems. The DEGs in
   the oceanic strain related to the ABC transporter (Figure [101]3C) are
   involved in the transport of Fe (idiA, afuBC), phosphonate (phnCD),
   urea (urtABC, urtE), manganese (Mn) (mntAB), biotin (bioY), sugars
   (msmX), polysaccharides (kpsE, kpsM, kpsT), and vitamin B[12] (bacA).
   The DEGs in the two‐component system (Figure [102]3C) are involved in
   regulating P (phoB) and heme (ctaA).

   Fe limitation alone upregulated most DEGs involved in nutrient
   metabolism in the oceanic isolate (Figure [103]5A, −Fe@24°C, −Fe@27°C).
   More DEGs, particularly those related to N and P transport systems,
   were upregulated under warming in Fe limited cultures, including the
   nitrate/nitrite transporter narK, global N regulator glnA, nitrate
   reductase (narB, nirA), ammonium transporter amt, urea transporters
   (urtABC), and the phosphate transporter pstS (Figure [104]5A). However,
   warming alone negatively affected nutrient metabolism regardless of Fe,
   with a few exceptions, including genes coding for urease accessory
   proteins (ureE, ureG) and sulfite reductase sir (Figure [105]5A,
   27°C@‐Fe and 27°C@+ Fe). The PII signalling gene glnB was upregulated
   with warming alone (Figure [106]5A, 27°C@‐Fe, 27°C@+ Fe).

FIGURE 5.

   FIGURE 5
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   Heatmap of the gene expression patterns involved in nutrient metabolism
   under different treatments in the oceanic strain (A) and the coastal
   strain (B). The treatment columns include –Fe@24°C, –Fe@27°C, 27°C@‐Fe,
   27°C@+ Fe, −Fe + warming, +Fe + warming. Red denotes upregulated DEGs,
   and blue denotes downregulated DEGs. Detailed descriptions of each
   treatment comparison are given in the Methods. Each row denotes one
   gene with ID and annotation listed. The asterisk denotes differential
   expression with a fold change > 2 and an adjusted p < 0.05.

   Fe limitation notably upregulated phosphonate transporters (phnC, phnD)
   at low temperatures. Warming under Fe limitation led to the
   downregulation of phnC, phnD, and the phosphate starvation‐inducible
   gene phoH, while phosphate regulators phoB were upregulated. In
   contrast, phosphate transporter genes were not significantly affected
   by either warming or Fe limitation. Instead, Fe limitation under
   warming conditions in the coastal strain upregulated ferredoxin‐nitrate
   reductase narB, glutamine synthetase glnA, and a urea transport gene
   urtA (Figure [108]5B, −Fe@27°C). More DEGs were observed when Fe
   limitation was induced under warming conditions (Figure [109]5B,
   −Fe@27°C) than at low temperatures (Figure [110]5B, −Fe@24°C). Warming
   under Fe‐replete conditions (27°C@+ Fe) upregulated nitrite reductase
   nirD while downregulating all the urea transport genes urtA‐D.

2.5. Transcriptional Responses for Fe Transport, Regulation, and Storage

   To explore how Fe‐related metabolism responded under different
   Fe‐warming conditions, we characterised the DEGs involved in Fe
   transport, regulatory, and storage systems in the two isolates. Both
   strains exhibited a significant upregulation of Fe stress biomarkers
   such as idiA, afuB, and afuC under Fe limitation regardless of
   temperature (Figure [111]6A,B). Fe porin genes were significantly
   expressed under various Fe‐warming conditions (Figure [112]6A,B).

FIGURE 6.

   FIGURE 6
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   The heatmap presents the gene expression patterns involved in Fe
   utilisation and heat shock response under different treatments in the
   oceanic strain (A) and the coastal strain (B). The treatment columns
   include –Fe@24°C, –Fe@27°C, 27°C@‐Fe, 27°C@+ Fe, −Fe + warming,
   +Fe + warming. Red denotes upregulated DEGs, and blue denotes
   downregulated DEGs. Detailed descriptions of each treatment comparison
   are given in the Methods. Each row denotes one gene with ID and
   annotation listed. The asterisk denotes differential expression with a
   fold change > 2 and an adjusted p < 0.05.

   Warming upregulated genes like afuC (27°C@‐Fe), fur (27°C@+ Fe), and
   Fe‐S cluster regulator sufR (27°C@‐Fe) and idpA (27°C@+ Fe) in the
   oceanic strain (Figure [114]6A). The suf operon transcriptional
   repressor sufR was downregulated with warming under Fe limitation. The
   dps gene (encoding the DNA binding protein in starved cells) was
   upregulated under +Fe + warming conditions (Figure [115]6A). Similarly,
   bacterioferritin in the coastal strain was downregulated under
   −Fe + warming conditions (Figure [116]6B). In the coastal strain, the
   Fe^3+ transporter idiA, Fe uptake porins, and the Fe uptake factor piuC
   were all responsive to warming (Figure [117]6B).

   Heat shock genes (groEL/groES (hsp60/hsp10), dnaK/dnaJ/grpE
   (hsp70/hsp40/nucleotide exchange factor), htpG/hsp90, and clpB/hsp100)
   were more responsive to Fe limitation than to warming in both strains,
   but were especially prominent under the −Fe+ warming condition,
   indicating an important role for Fe and warming interactive stress
   protection by these chaperone proteins (Figure [118]6, −Fe
   vs. + Fe@24°C, −Fe vs. + Fe@27°C). Warming alone led to the
   upregulation of antioxidant genes in the oceanic strain, such as those
   coding for methionine sulfoxide reductase and thioredoxins. The oceanic
   strain upregulated SOD1 (Cu‐Zn family superoxide dismutase) with
   warming when Fe was limiting (Figure [119]S6A).

2.6. Fe‐Warming Interactions in Both Strains

   In addition to examining the individual effects of Fe concentrations
   and temperatures, we explored their interactive effects (Fe‐warming
   interactions) to determine whether there are non‐linear effects of
   changes in both factors simultaneously. The interactions between Fe and
   warming have a more pronounced effect on gene expression than either
   factor alone in both strains (Figure [120]3A). In the oceanic strain,
   many gene expression patterns in photosynthesis were similar between
   −Fe@27°C and 27°C@‐Fe (Figure [121]4A). The DEGs involved in oxidative
   phosphorylation were upregulated under the −Fe + warming comparison
   treatment, consistent with warming at Fe limitation but opposite to Fe
   limitation at warming comparison treatments. These indicate a
   predominant impact of temperature. However, antenna proteins were
   generally unaffected by either Fe limitation or warming alone, yet were
   downregulated under −Fe + warming (‐Fe@27°C vs. + Fe@24°C;
   Figure [122]4A). Furthermore, the DEG patterns under −Fe + warming were
   consistent with 27°C@‐Fe (Figure [123]5), and the patterns under
   +Fe + warming conditions were similar to 27°C@+Fe, indicating warming
   plays an important role in nutrient metabolism in the oceanic strain.
   Most Fe utilisation and heat shock genes showed opposite trends under
   +Fe + warming and −Fe + warming conditions (Figure [124]6A), indicating
   that these genes are more affected by Fe concentrations.

   In comparison, the coastal strain showed gene expression patterns under
   −Fe + warming that were similar to those under Fe limitation at both
   temperatures, and patterns under +Fe + warming are opposite to those
   under‐Fe at 27°C (Figure [125]4B). This indicates that Fe plays a
   predominant role in photosynthetic activities in the coastal strain.
   Unlike in the oceanic strain, 27°C at‐Fe and −Fe + warming had minimal
   effect on nutrient metabolism in the coastal strain, indicating the
   nuanced effect of temperature at low Fe on the coastal strain.
   Furthermore, warming had no effect on Fe utilisation and only one heat
   shock gene, hsp20, was upregulated at 27°C at‐Fe (Figure [126]6B),
   indicating that these pathways were regulated by Fe concentrations. In
   summary, under −Fe + warming conditions, the oceanic strain regulated
   pathways including photosynthesis, nutrient metabolism, heat shock, and
   Fe transport (Figure [127]7A), and the coastal strain regulated similar
   pathways but with many fewer genes (Figure [128]7B).

FIGURE 7.

   FIGURE 7
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   Summary figure depicting gene expression under –Fe + warming
   interactive conditions in the oceanic strain (A) and the coastal strain
   (B). Red denotes gene upregulation and blue denotes gene
   downregulation. 2‐OG, 2‐oxoglutarate; 6PGL, 6‐Phosphogluconolactone;
   BGP, 1,3‐bisphosphoglycerate; Cyt b[6]f, cytochrome b[6]f complex; F6P,
   fructose 6‐phosphate; FBP, fructose‐1,6‐bisphosphate; Fd, ferredoxin;
   FNR, ferredoxin‐NADP(+) oxidoreductase; G1P, glucose 1‐phosphate; G6P,
   glucose‐6‐phosphate; GAP, glyceraldehyde‐3‐phosphate; NDH, NADH
   dehydrogenase; OM, outer membrane; PBS, phycobilisomes; PC/Cyt c[6],
   plastocyanin/cytochrome c[6]; PEP, phosphoenolpyruvate; PGA,
   3‐phosphoglycerate; PSI, photosystem I; PSII, photosystem II; Pyr,
   pyruvate; Ru5P‐ribulose‐5‐phosphate; RuBP, ribulose‐1,5‐bisphosphate;
   S7P, sedoheptulose‐7‐phosphate; SBP, Sedoheptulose 1,7 bisphosphate;
   TM, thylakoid membrane.

3. Discussion

3.1. Differential Sensitivity to Fe‐Warming Interactions

   We explored how the sensitivity of the two isolates can influence their
   acclimation to environmental changes. The oceanic strain is more
   responsive to Fe‐warming interactions and may experience greater
   selective pressure from both factors in oligotrophic regions. Both Fe
   and temperature changes significantly affect physiology and central
   metabolism, including photosynthesis and nutrient assimilation, which
   aligns with previous studies in marine Synechococcus (Liu and
   Qiu [130]2012; Mackey et al. [131]2015).

   In the oceanic strain, ABC transporters and two‐component regulatory
   systems were significantly enriched under Fe‐warming interactions,
   including those for Fe, nutrients, Mn, and polysaccharides. This
   indicates that under future warming in oligotrophic areas, the oceanic
   strain may have more ability to utilise various resources and sense and
   respond to environmental changes in the ocean.

3.2. Distinct Photosynthetic Strategies in Response to Fe‐Warming
Interactions

   We found that the two isolates employ distinct photosynthetic
   strategies to cope with Fe‐warming interactions. Physiological and
   transcriptomic data highlighted how each isolate adjusts phycobilisome
   organisation, electron flow, and carbon fixation under different
   Fe‐warming scenarios. Under Fe‐limited conditions at low temperature,
   the oceanic strain reduces the synthesis of Fe‐rich photosynthetic
   components, particularly PSII (2 Fe atoms), PSI (12 Fe atoms),
   cytochrome b6f (6 Fe atoms), and ferredoxin, to conserve Fe and
   minimise oxidative stress from impaired electron transport
   (Blanco‐Ameijeiras et al. [132]2017). Fe limitation also significantly
   reduced the expression of phycobiliprotein genes in the oceanic
   Synechococcus. Adjusting phycobilisome composition (including
   allophycocyanin, phycocyanin, and phycoerythrin) to better harvest
   green and blue wavelengths may be beneficial during deep heat waves,
   when warming conditions can extend into the lower euphotic zone (Chen
   et al. [133]2022; Santana‐Falcón and Séférian [134]2022; Fragkopoulou
   et al. [135]2023).

   Under Fe‐replete conditions, warming had little effect on the growth
   rates and photosynthesis in the oceanic strain. The oceanic strain may
   employ state transitions to modulate the distribution of energy
   absorbed by phycobilisomes. Under Fe‐limited conditions, warming may
   induce state 1, where the phycobilisome associates with PSII (Mackey
   et al. [136]2013). Under Fe‐replete conditions, warming may cause the
   oceanic strain to shift to state 2, where the PBS associates with PSI.
   The oceanic Synechococcus may utilise both linear electron flow and
   cyclic electron flow around PSI to generate ATP (Behrenfeld
   et al. [137]2008). These responses may indicate a strategy to balance
   the allocation of photosynthetic resources (ATP and NADPH) among
   cellular processes to maximise growth and metabolism. However, these
   proposed state transitions were based on the interpretations of the
   transcriptomic responses of photosynthetic pathways, and direct
   experimental validation will be needed in future studies.

   RuBisCO, the key enzyme for carbon fixation, was downregulated with
   warming. This may be compensated by increasing carboxysome numbers
   while reducing the amount of RuBisCO per carboxysome (Dedman
   et al. [138]2023). Cytochrome c oxidase (respiratory terminal oxidase)
   was upregulated, indicating a greater reliance on pathways by which
   electrons flow from PSII to a terminal oxidase, utilising O[2] as the
   electron acceptor (Blanco‐Ameijeiras et al. [139]2018). This may
   explain the higher carbon fixation rates observed with warming, despite
   no significant difference in growth rates.

   The coastal strain has a higher Fe quota, likely due to requirements in
   photosynthesis, particularly PSI. D1 genes in PSII were highly
   expressed, which require less Fe than PSI. The coastal strain has a
   higher turnover rate of D1 protein isoforms between D1:1 (coding by
   psbA) and D1:2 (coded by psbAII). D1:2 appears to be more
   stress‐resistant than D1:1 when excitation pressure on PSII increases
   due to light or temperature stress (Sane et al. [140]2002).
   Downregulation of cytochrome c oxidase (respiratory terminal oxidase)
   by warming leads to transient reduction in photosynthetic oxygen
   evolution during the period of D1:1/D1:2 exchange. Therefore, the
   coastal strain may upregulate PSII genes without greatly increasing Fe
   demand under Fe‐limited conditions to optimise energy use.

   Hence, the oceanic and the coastal Synechococcus use distinct
   strategies to adapt photosynthesis to Fe‐warming stress. These include
   potential state transitions of phycobilisomes between PSI and PSII to
   facilitate phycobilisome energy excitation (Bhatti et al. [141]2021),
   reducing oxidative stress, adjusting ATP and NADPH production to
   maximise metabolism in the oceanic strain, and the fast turnover of D1
   isoforms in PSII (Ahlgren and Rocap [142]2006) in the coastal strain.

3.3. Selective Nutrient Acquisition Preferences in the Two Strains