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An oxygen-sensing mechanism for angiosperm adaptation to altitude

Abstract

Flowering plants (angiosperms) can grow at extreme altitudes, and have been observed growing as high as 6,400 metres above sea level1,2; however, the molecular mechanisms that enable plant adaptation specifically to altitude are unknown. One distinguishing feature of increasing altitude is a reduction in the partial pressure of oxygen (pO2). Here we investigated the relationship between altitude and oxygen sensing in relation to chlorophyll biosynthesis—which requires molecular oxygen3—and hypoxia-related gene expression. We show that in etiolated seedlings of angiosperm species, steady-state levels of the phototoxic chlorophyll precursor protochlorophyllide are influenced by sensing of atmospheric oxygen concentration. In Arabidopsis thaliana, this is mediated by the PLANT CYSTEINE OXIDASE (PCO) N-degron pathway substrates GROUP VII ETHYLENE RESPONSE FACTOR transcription factors (ERFVIIs). ERFVIIs positively regulate expression of FLUORESCENT IN BLUE LIGHT (FLU), which represses the first committed step of chlorophyll biosynthesis, forming an inactivation complex with tetrapyrrole synthesis enzymes that are negatively regulated by ERFVIIs, thereby suppressing protochlorophyllide. In natural populations representing diverse angiosperm clades, we find oxygen-dependent altitudinal clines for steady-state levels of protochlorophyllide, expression of inactivation complex components and hypoxia-related genes. Finally, A. thaliana accessions from contrasting altitudes display altitude-dependent ERFVII activity and accumulation. We thus identify a mechanism for genetic adaptation to absolute altitude through alteration of the sensitivity of the oxygen-sensing system.

Main

Around 25% of the Earth’s land surface, containing at least 30% of plant species diversity4, is mountainous. Although the altitude at which an individual plant grows may never change, it is critical that individuals (and populations) are adapted to survive at that altitude, and this is an important component of plant ecology4. Altitude and latitude have been considered collectively to impart a syndrome of developmental and physiological characteristics linked mostly to climatic adaptation (including dwarfism, resistance to ultra-violet light, low temperature tolerance, flowering time and others4,5). However, no consistent trait has been associated with specific environmental components of altitude, such as pO2, which might reveal mechanisms underlying direct altitudinal adaptation. Here we investigate the idea that adaptation to altitude involves direct sensing of oxygen concentration across altitudinal ranges. Mammalian adaptation to very high altitude involved mutation of components of the hypoxia-inducible factor (HIF) oxygen-sensing system6,7,8 (which is mechanistically different to the plant oxygen-sensing pathway9), indicating the importance of matching metabolism with altitude. Oxygen sensing in plants through Met1–Cys2 ERFVII transcription factors is mediated by the PCO branch of the PROTEOLYSIS 6 (PRT6) N-degron pathway10,11. Following Met1 removal, the amino-terminal cysteine is oxidized by PCOs12 using molecular O2, arginylated by arginyl transferase13 and recognized by the E3 ligase PRT6 for ubiquitin-mediated destruction (Fig. 1a).

Fig. 1: Atmospheric oxygen sensing regulates tetrapyrrole synthesis via FLU.
figure 1

a, Schematic representation of the PCO branch of the PRT6 N-degron pathway28. MetAP, methionine amino-peptidase; ATE, arginyl transferase; oxCys, oxidized cysteine. The position of oxygen and possible positions of nitric oxide (NO) in the pathway are shown. Oxygen is used by PCOs to oxidize amino-terminal Cys of ERFVIIs. b, Steady-state Pchlide, measured by fluorescence at 636 nm, in etiolated seedlings of different species grown at different ambient O2 concentrations. c, d, Steady-state Pchlide in Col-0 and erfVII at different ambient O2 concentrations (c) with expression of individual stabilized Cys2Ala mutant ERFVIIs controlled by their native promoters (d)18 (p). e, Amount of FLU RNA transcript in Col-0 and erfVII grown at different ambient O2 concentrations. f, Regulation of FLU orthologue mRNA in P. somniferum (PSOM) and S. lycopersicum (Solyc) grown at various ambient O2 concentrations. g, Schematic of the A. thaliana FLU gene, showing potential ERFVII binding sites (left) and chromatin immunoprecipitation (ChIP) analysis of RAP2.3–HA and HRE2–HA occupancy of FLU gene regions (range indicated by colons); including known positive and negative regulatory sequences20,29 using anti-HA antibody. All experiments were carried out using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l.) unless otherwise stated. Data are mean ± s.d.; one-way ANOVA. Significantly different groups are indicated by letters in d. n = 3 biologically independent experiments. AU, arbitrary units; R2, coefficient of determination.

Source data

We reasoned that plant biochemical pathways that require oxygen may be subject to evolutionary pressure in relation to altitude. Tetrapyrrole synthesis, which leads to chlorophyll, is dependent on ambient O2 at several points (Extended Data Fig. 1a). Following germination, during etiolated growth in the dark, the chlorophyll biosynthesis intermediate protochlorophyllide (Pchlide) accumulates because angiosperms possess only a light-activated chloroplast enzyme for Pchlide reduction14 (light-dependent NADPH-protochlorophyllide oxidoreductase (L-POR, hereafter POR)). We set out to determine whether differences in pO2 with altitude could influence the flux through the tetrapyrrole pathway through oxygen sensing and be a target for evolutionary adaptation. We first investigated whether the ambient O2 concentration regulates steady-state Pchlide levels in plant species representing diverse angiosperm clades, A. thaliana (rosid), Solanum lycopersicum (asterid), Papaver somniferum (basal dicot) and Brachypodium distachyon (monocot) (Fig. 1b). This showed that decreasing ambient O2 levels from 27% (hyperoxia) to 5% (hypoxia) decrease Pchlide levels in etiolated seedlings. To show that this is not just the result of hypoxia-related flux restrained by O2-requiring enzymes of the pathway we also analysed steady-state Pchlide levels for the A. thaliana flu mutant. FLU directly inhibits the first committed enzyme of tetrapyrrole synthesis, glutamyl tRNA reductase (GluTR)—the major form of which is encoded by HEMA1—thereby inhibiting synthesis of 5-aminolevulinic acid (ALA), the precursor of all tetrapyrroles15, which results in prevention of accumulation of free Pchlide (Extended Data Fig. 1a). The proposed mechanism for FLU activity involves POR, Pchlide, GluTR and CHL27 (a component of the tetrapyrrole synthesis enzyme Mg-protoporphyrin monomethylester cyclase3); in the dark, free Pchlide (which cannot be converted to chlorophyllide without light) binds POR as part of a complex with CHL27 and FLU. This inactivation complex enables FLU to interact with and inhibit GluTR activity, reducing ALA synthesis and therefore Pchlide levels16,17. Under mild hypoxia, compared with normoxia and hyperoxia, steady-state Pchlide levels were unrestrained in the flu mutant, indicating that the observed oxygen-associated Pchlide levels in different species are regulated by oxygen sensing (Fig. 1b, Extended Data Fig. 1b–d). In A. thaliana accession Col-0 (originally collected less than 100 m above sea level (a.s.l.)) grown at 48 m a.s.l. (pO2 = 21.2 kPa), this decrease was largely abolished in the absence of all 5 ERFVII transcription factors (using the pentuple related to apetala (rap) and hypoxia responsive erf (hre) mutant18 rap2.12 rap2.2 rap2.3 hre1 hre2 (hereafter referred to as erfVII)) (Fig. 1c), demonstrating that oxygen sensing via ERFVIIs is required for this response. A Cys-to-Ala mutation (Cys2Ala) (which removes the Cys N-degron) in all ERFVIIs significantly reduced Pchlide levels in etiolated seedlings, similar to prt6 seedlings (in which all ERFVIIs are stable) (Fig. 1d). Mutation of either PRT6 or ERFVII genes led to opposite stable changed steady-state Pchlide levels (lower in prt6, higher in erfVIIs); mutation of RAP2.3 had the largest effect, indicating that all ERFVIIs contribute to this function and that RAP2.3 may have a predominant role (Extended Data Fig. 2a, b). A prt6 flu double mutant showed high levels of Pchlide equivalent to those in the flu mutant, indicating that stabilized ERFVIIs act upstream of FLU (Extended Data Fig. 2c). Because hypoxia suppressed Pchlide levels, we analysed the role of ERFVIIs in regulating expression of genes encoding components of the inactivating complex—including FLU, HEMA1, CHL27 and POR (which is encoded by three genes, two of which (PORA and PORB) are expressed in etiolated seedlings19)—and CHLM, which encodes a chlorophyll synthesis enzyme previously shown to be regulated by oxygen sensing18. Accumulation of FLU transcripts was positively regulated by ERFVIIs via PRT6, whereas CHL27, PORA, PORB and CHLM were negatively regulated, and HEMA1 was not regulated, by this pathway (Extended Data Fig. 2d). The amount of FLU RNA increased strongly with increasing hypoxia, a response that was abolished in the erfVII mutant, whereas expression of tetrapyrrole synthesis genes showed the opposite trend (Fig. 1e, Extended Data Fig. 2e). Reflecting the transcript data, FLU protein levels were enhanced by hypoxia via ERFVIIs, whereas POR protein accumulation was repressed by hypoxia through ERFVIIs (Extended Data Fig. 2f). Oxygen-controlled repression of FLU-orthologous RNA accumulation was conserved in S. lycopersicum and P. somniferum (Fig. 1f).

In A. thaliana, the C-terminally haemagglutinin (HA)-tagged ERFVIIs HRE2 and RAP2.3 associated with the FLU gene in a region containing an evolutionarily-conserved ERFVII binding site20, adjacent to the initiating ATG (similar binding was previously observed for HRE2 at the FLU locus during the response of light-grown seedlings to hypoxia20). HRE2–HA and RAP2.3–HA also showed differential binding to two genes (PCO1 and CRU1) previously shown to be regulated by ERFVIIs. HRE2 similarly associated with ATG-proximal regions of CHL27 and CHLM genes, and also with gene regions of PORA and PORB but not with HEMA1 (Fig. 1g, Extended Data Fig. 3). These data demonstrate that O2 regulation of Pchlide synthesis occurs via oxygen sensing of the PCO N-degron pathway through ERFVII-regulated control of expression of components of the GluTR inactivation complex, in particular the negative regulator FLU.

Because pO2 decreases with altitude (Fig. 2a), and could thus affect steady-state Pchlide levels, we investigated the relationship between Pchlide levels and altitude. We analysed Pchlide in accessions of A. thaliana (collected from Eurasia and Africa), Solanum habrochaites (collected from South America), Solanum cheesmaniae (collected from the Galapagos Islands) and B. distachyon (collected from Turkey) from natural populations growing at different altitudes from sea level to more than 3,000 m a.s.l. with different latitudinal ranges (Fig. 2a, b, Extended Data Fig. 4a, b, Supplementary Table 1a). When analysed at 48 m a.s.l. (pO2 21.2 kPa), all species showed a positive correlation between altitude of accession collection and Pchlide steady state level. Furthermore, the amount of Pchlide in A. thaliana and S. habrochaites was dependent on the ambient O2 concentration, and hypoxia resulted in greater reduction of Pchlide levels in accessions from higher altitudes (Fig. 2b, c). In A. thaliana, the steady-state Pchlide level was not related to submergence tolerance (Extended Data Fig. 4c, Supplementary Table 1c). As a result of increased Pchlide (which produces singlet oxygen15 under light), following transfer to light, dark-grown seedlings of accessions collected at higher altitude accumulated substantially more reactive oxygen species (ROS) at 48 m a.s.l. than those collected at a lower altitude; this effect was dependent on the ambient O2 level and functional PRT6 (Extended Data Fig. 5). These results show that there is a relationship between Pchlide accumulation and the altitude of accession collection, suggesting adaptation of oxygen sensing and/or downstream signalling through the PCO N-degron pathway that fine-tunes steady-state Pchlide level to local atmospheric O2 levels, potentially avoiding damaging light-associated singlet-oxygen ROS production. Consistent with such a relationship, we found that accumulation of FLU transcript in A. thaliana grown at 48 m a.s.l. (pO2 = 21.2 kPa) was strongly influenced by the altitude at which the accession was collected (that is, there was less FLU transcript in high-altitude accessions) and by ambient O2 level (Fig. 2d). There was a similar relationship between altitude of collection and FLU expression for S. habrochaites (FLU expression was lower in high-altitude accessions) (Fig. 2e). Conversely, the amounts of PORA, PORB and CHLM (but not CHL27 or HEMA1) transcripts were higher in accessions from higher elevations, and lower at 15% oxygen compared with those grown in 21% oxygen (Extended Data Fig. 6a). POR protein accumulated to higher levels in the high-altitude A. thaliana accession Sha (originally collected in Tajikistan at 3,400 m a.s.l, pO2 = 13.9 kPa) than in the low-altitude Col-0, and by introgressing the prt6-1 transfer-DNA insertion mutation from Col-0 through eight back-crosses into Sha (prt6Sha), we showed that this increase was controlled through the oxygen-sensing pathway; conversely FLU protein accumulation was increased in prt6Sha compared with Sha (Extended Data Fig. 6b). In contrast to the observed relationship between the steady-state Pchlide level and elevation in wild species, an altitudinal cline of cultivated Chenopodium quinoa (quinoa, recently domesticated in the high Andes21) did not show altitude-dependent Pchlide accumulation (Fig. 2f).

Fig. 2: Steady-state levels of Pchlide and FLU expression are determined by altitude.
figure 2

a, Pchlide levels in A. thaliana accessions collected at different altitudes and geographic locations30, grown at a pO2 of 21.2 kPa. Grey dots show pO2 at the different altitudes from which the accessions were collected. Genomic groups are indicated in different colours. IP, Iberian Peninsula b, Pchlide levels in S. habrochaites grown at 48 m a.s.l. with 21% and 15% ambient O2. c, Effect of ambient O2 concentration on Pchlide levels in A. thaliana accessions grown at 48 m a.s.l. (R2 values from Supplementary Table 1b). d, e, Effect of ambient O2 concentration on amount of FLU mRNA in A. thaliana (d) and S. habrochaites (e) grown at 48 m a.s.l. f, Pchlide levels in cultivated domesticated C. quinoa accessions obtained from different altitudes grown at pO2 21.2 kPa. g, Comparison of Pchlide levels in A. thaliana accessions collected at different altitudes and grown at 48 m a.s.l. with pO2 = 21.2 kPa or 15% ambient O2, or at 2,479 m a.s.l.(pO2 = 15.7 kPa). h, FLU RNA accumulation in A. thaliana accessions collected at different altitudes and grown at 48 m a.s.l. (pO2 = 21.2 kPa) or at 2,479 m a.s.l. (pO2 = 15.7 kPa). All experiments were carried out using etiolated seedlings after 5 days growth. Data are mean ± s.d. Accessions used are listed in Supplementary Table 1a. n = 3 biologically independent experiments.

Source data

Reduced pO2 is just one altitude-dependent parameter: others include atmospheric pressure. We therefore assessed steady-state Pchlide levels and FLU expression in etiolated seedlings at two sites located at extreme altitudes (site SB: 52.829809° N −1.249732° E, 48 m a.s.l., pO2 = 21.2 kPa; and site ESPE: −0.312917° N −78.445157° E, 2,479 m a.s.l., pO2 = 15.7 kPa) (Extended Data Fig. 7). The Pchlide level was much lower in the high-altitude accession Sha when it was grown at the high-altitude site ESPE compared with the low-altitude site SB, whereas in the erfVII mutant Pchlide level remained similar at both sites (Extended Data Fig. 8a). Steady state levels of Pchlide in an A. thaliana altitudinal cline were reduced at ESPE, compared to SB at pO2 21.2 kPa, and were similar to accumulation at SB in 15% ambient O2 (Fig. 2g). The amount of FLU transcript was also increased in plants grown under ambient O2 concentration at ESPE compared with SB, particularly in high-altitude accessions, and was similar to the amount of FLU transcript in plants grown at SB under 15% O2 (Fig. 2d, h), whereas the amounts of PORA, PORB and CHLM transcripts were lower in the plants grown under atmospheric O2 concentration at ESPE compared with SB (Extended Data Fig. 8b). These data demonstrate that O2 is the major component sensed by altitudinal clines when controlling steady-state Pchlide levels. They also indicate that high-altitude populations have adapted to lower ambient O2 through increased O2 sensitivity, and that this has occurred in phylogenetically distant angiosperm species. Reduced accumulation of FLU transcripts in high-altitude accessions at pO2 21.2 kPa further suggests that they exhibit lower ERFVII activity compared with low-altitude accessions. In summary, the sensitivity to O2 determines downstream Pchlide level, which is tailored to the local ambient pO2 via regulation of FLU expression mediated by ERFVIIs.

Since the analyses carried out at different altitudes showed that the sensitivity to atmospheric O2—as indicated by accumulation of Pchlide and its regulatory component FLU—increases with altitude, we examined the expression of classical hypoxia-associated genes. The expression of ADH1, PDC1, PGB1 and SUS4 transcripts (members of the core 49 conserved hypoxia-induced genes22 that are regulated by ERFVIIs through the PCO N-degron pathway23) was highly dependent on both the altitude of accession collection and ambient oxygen level in both A. thaliana and S. habrochaites (Fig. 3a, Extended Data Figs. 8c, d). This indicates that altitude adaptation is not restricted to Pchlide accumulation and is a central conserved feature of oxygen sensing in angiosperms. Furthermore, it suggests that—similar to FLU regulation—at high oxygen levels, ERFVIIs are more active in low-altitude accessions than in high-altitude accessions. We next tested whether ERFVIIs are active in etiolated seedlings of the low-altitude accession Col-0 at pO2 21.2 kPa. Pchlide level in Col-0 was intermediate between those in prt6 and erfVII mutants (Fig. 1d), indicating ERFVII repressive activity. Increasing PCO2 activity in Col-0 led to increased levels of Pchlide, suggesting that active ERFVIIs can be destabilized, but this over-accumulation was inhibited by removal of PRT6 activity (Fig. 3b).

Fig. 3: Genetic mechanisms linking oxygen sensing to altitude adaptation.
figure 3

a, Effect of ambient O2 on RNA accumulation of hypoxia-associated ADH1 in S. habrochaites and A. thaliana accessions. b, Pchlide levels in prt6-5, 35S:PCO2, prt6-5 35S:PCO2 and Col-0. c, d, Pchlide level and FLU, PORA and PORB transcript expression in prt6-1 mutants and transgenic plants expressing wild-type or Cys2Ala mutant Col-0 RAP2.3 or HRE2 (driven by their own promoters) in Sha and Col-0 genetic backgrounds. e, ChIP analysis of HRE2–HA occupancy at FLU −49:+84 and hypoxia-related genes in Col-0 and Sha seedlings grown with pO2 21.2 kPa or 15% ambient oxygen. f, Western blot analysis of HRE2–HA in Sha and Col-0 accessions grown at pO2 21.2 kPa. The experiment was repeated independently three times with similar results. BZ, bortezomib. g, A model for angiosperm adaptation to altitude through oxygen sensing. Wedges indicate decreasing pO2 with increasing altitude. Blocked arrows indicate repression. Arrow-crossed box is international standard symbol for a rheostat. The inactivation complex model is adapted from ref. 16. All experiments were carried out using etiolated seedlings after 5 days growth at 48 m a.s.l. Data are mean ± s.d.; one-way ANOVA. Significantly different groups are indicated by letters in c, d. n = 3 biologically independent experiments.

Source data

To understand the genetic mechanisms involved in altitude adaptation through oxygen sensing, we investigated the influence of components of the ERFVII–PCO N-degron pathway in the high-altitude A. thaliana accession Sha. We transformed wild-type and Cys2Ala stable versions of the Col-0 ERFVIIs RAP2.3 and HRE2 (expression driven by their own promoters) into Sha. In prt6Sha, RAP2.3(Cys2Ala)-expressing Sha and HRE2(Cys2Ala)-expressing Sha (but not in Sha expressing wild-type Col-0 RAP2.3), FLU, PORA and PORB expression were markedly affected, resulting in reduced Pchlide (Fig. 3c, d). In addition, expression of hypoxia-related genes was enhanced in prt6Sha and RAP2.3(Cys2Ala)-expressing Sha (Extended Data Fig. 9a, b). This indicates that constitutively stabilized ERFVIIs reduce Pchlide as effectively in Sha as in Col-0. Therefore, components downstream of stabilized ERFVIIs, including FLU function, are unaltered in Sha (as otherwise altering upstream components would not affect the high Pchlide level in Sha). Expression of components of the PCO branch of the PRT6 N-degron pathway were not significantly different between Col-0 and Sha, and the sequences of Col-0 and Sha FLU genes were identical (Extended Data Fig. 9c, d). Etiolated seedlings from Sha × erfVII reciprocal crosses showed low Pchlide steady-state levels, indicating that ERFVII activity (in repressing Pchlide levels) in Sha has the potential to be as strong as that in Col-0 (Extended Data Fig. 10). This result also suggests the presence of dominant repressor(s) of oxygen sensing in the low-altitude accession Col-0. We further analysed ERFVII activity in Col-0 and Sha by measuring HRE2–HA occupancy on FLU and hypoxia-related genes (which have been shown to bind HRE2 (ref. 20)). At a pO2 of 21.2 kPa, HRE2–HA occupancy of these genes was high in the prt6 mutant in both Sha and Col-0 backgrounds (in which HRE2 is stabilized), whereas in the wild type, HRE2–HA displayed lower occupancy of these genes in Sha than in Col-0 (Fig. 3e). Occupancy increased in Sha and Col-0 when grown in 15% ambient O2. Thus, at high ambient oxygen ERFVII occupancy is higher in the low-altitude genetic background, but HRE2 activity in both low- and high-altitude genetic backgrounds responds to hypoxia. Finally, Western blot analysis showed that at pO2 21.2 kPa, HRE2–HA abundance was higher in Col-0 than in Sha, but treatment with the proteasome inhibitor bortizomib resulted in equivalent markedly higher HRE2–HA abundance in both accessions (Fig. 3f).

Here we demonstrate that altitude adaptation involves genetic modifications of the sensitivity to atmospheric O2 through oxygen sensing, mediated by ERFVII accumulation and activity. We show that this adaptation, through ERFVII regulation, influences two distinct features: hypoxia-related gene expression and steady-state levels of Pchlide (mainly via oxygen-regulated expression of FLU and POR). Prevention of free Pchlide accumulation is a result of the regulated rate of ALA synthesis, mediated by the POR–Pchlide–CHL27 complex that triggers FLU inactivation of GluTR24. Coupling of POR expression to the ambient oxygen concentration may enable matching of POR protein to Pchlide levels, allowing POR to bind to free Pchlide. It may be ecologically important to match tetrapyrrole flux (which requires molecular O2) to the ambient O2 concentration to provide the most effective Pchlide level once seedlings arrive at the soil surface and chlorophyll synthesis commences in the light. Altitude adaptation involves fine-tuning the activity of the oxygen-sensing system, which acts like a rheostat measuring altitude (Fig. 3g), perhaps through negative regulation of the PCO N-degron pathway. Oxygen sensing is transduced by ERFVIIs to influence outputs including expression of hypoxia-related genes, steady-state Pchlide levels (through regulation of ALA synthesis by inactivation complex components) and potentially other biochemical pathways that require molecular oxygen. Altitude adaptation enables decoding of the ambient oxygen level (determined by the local pO2) to provide equivalent outputs at different altitudes, resulting in, for example, equivalent Pchlide levels in a low-altitude (high pO2)-adapted accession grown at low altitude to those in in a high-altitude (low pO2)-adapted accession grown at high altitude.

Although this study only investigated etiolated seedlings, other stages of development (including analogous stages such as subsurface rhizome-derived etiolated shoots) may also be subject to a similar adaptive mechanism, as the steady-state Pchlide level is an important regulator of chlorophyll synthesis and FLU has been shown to influence the chlorophyll supply in light conditions24. Notably, regulation of tetrapyrrole synthesis by oxygen sensing also occurs in cyanobacteria, which share a common ancestor with chloroplasts25. Previous studies have shown that oxygen sensing is an important feature of skotomorphogenesis18. Here we demonstrate that oxygen sensing during this important developmental stage is associated with genetic adaptation to altitude. We show that local ambient absolute O2 concentration regulates expression of hypoxia-related genes and steady-state levels of Pchlide—a biochemical intermediate of chlorophyll biosynthesis—in altitudinal clines of diverse species through the oxygen-sensing system. This provides a general mechanism for adaptation to absolute elevation that is likely to be conserved throughout angiosperms. In addition, as this mechanism appears to not have been selected in breeding of quinoa, it may represent an untapped trait for crop improvement at unadapted altitude26. It also represents a component that deserves investigation in relation to plant ecological adaptation. The relevance of this mechanism will be of increasing importance as global warming leads to displacement of wild and crop plants to higher altitudes27.

Methods

No statistical methods were used to predetermine sample size. Experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Plant materials

Information on A. thaliana, S. habrochaites, S. cheesmaniae, B. distachyon P. somniferum and C. quinoa accessions (Supplementary Table 1): A. thaliana accessions were obtained from NASC, UK. The A. thaliana prt6-1, −5, erfVII and individual mutant ERFVII lines (all in Col-0 accession background) were described previously18,31. The flu-1 (Col-0 background) mutant32 was obtained from M. Terry. The 35S:PCO2-FLAG12 transgenic (Col-0 background) was obtained from F. Licausi. Solanum accessions were obtained from TGRC, UC Davis, USA. C. quinoa cultivated accessions and B. distachyon accessions were obtained from the US National Plant Germplasm Collection Germplasm Resources Information Network (GRIN), Beltsville, US Department of Agriculture, Agricultural Research Service (http://www.ars-grin.gov/), and the B. distachyon accession Bd21 was provided by L. Mur. P. somniferum (‘Lauren’s grape’) seeds were obtained from Mr. Fothergill’s seeds, UK, and tomato Solanum lycopersicum (Marmande) were obtained from Sutton Seeds, UK. The Col-0 accession prt6-1 T-DNA insertion was introgressed through eight back-crosses into the Sha accession following BASTA resistance conferred by the transgene. The 35S:HRE2-3×HA transgene10 from Col-0 was introgressed into Sha through five back-crosses following BASTA resistance conferred by the transgene, genotyping across all five chromosomes was used to confirm absence of detectable Col-0 genomic DNA away from both transgenes.

Growth conditions for etiolated seedlings

As previously described18, surface-sterilized seeds were plated on 0.5× MS media (including 1% w/v sucrose) and chilled for 4 days at 4 °C before being exposed to constant white light at 20 °C for 8 h to activate germination. Subsequently, unless indicated otherwise, plates were incubated in darkness at 20 °C for 5 days. For experiments using different oxygen levels, open plates were placed in a methacrylate chamber (1,120 × 180 × 270 mm) (Epica) and flushed until equilibrium with water-saturated premixed gas combinations (BOC) and left for 5 days in the dark. Oxygen levels in the chamber were measured at the beginning and end of the experiment using an oxygen meter attached to the outlet pipe (KANE 250 Compact Flue Gas Analyzer-Kane International).

Generation of transgenic plants

Individual C2A and WT ERFVII transgenes, including 2 kbp of sequence upstream of the initiating ATG and introns in accession Col-0 were described previously18. These were transformed into A. thaliana accession Sha as previously described33.

Biochemical analyses

Measurements of Pchlide and ROS

Pchlide was assayed from cotyledons isolated from etiolated seedling as described34. Cotyledons (or whole coleoptiles of B. distachyon) of etiolated seedlings were homogenized in 1 ml ice-cold 80% (v/v) acetone overnight at 4 °C. Extractions were vortexed and centrifuged, 800 μl was aliquoted into a fresh tube and 200 μl was used to measure the relative fluorescence at room temperature (excitation: 440 nm; emission: 550–750 nm) using either a Varioskan Flash (Thermo Fisher Scientific) for measurements at SB, or BioTek Cytation 5 Multi-Mode Reader (for measurements at ESPE). To account for differences in cotyledon size between accessions, images of 20 representative cotyledons for each accession in each experiment were taken using a Leica MZ75 and the area was measured using Fiji (https://imagej.net/). Reported Pchlide values represent the florescence (either from 550 to 700 nm or at 636 mn (arbitrary units)) per cotyledon in a 1 mm2 area.

ROS were detected in 5 days old etiolated seedlings 1 day after transfer to light, 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was incubated with seedlings for 30 min and then washed in 10 mM MES, 0.1 mM CaCl2, pH 6, for 1 h at 22 °C. Dye excitation was at 480 nm and emitted light was detected at 535–550 nm with a Leica DM5000 B. ROS and chlorophyll quantification was carried out using Fiji.

ChIP, gene expression and protein analyses

ChIP was performed as described35. Chromatin was extracted from ~3.5 g of etiolated seedlings. Ten microgrammes of anti-HA (Sigma, H3663-200UL) was used for immunoprecipitation. EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (EIF4A1, AT3G13920) and ACTIN 2 (ACT2, AT3G18780) were used as negative targets. CRUCIFERINA (CRU1, AT5G44120) and PLANT CYSTEINE OXIDASE 1 (PCO1, AT5G15120) served as a known positive targets for RAP2.3 and HRE2 (refs. 20,29). Chromatin was purified using QIAquick PCR Purification Kit (Qiagen). Quantitative PCR values of the immunoprecipitation product were normalized against the no-antibody samples. Data presented shows the average of three independent biological repeats. Oligonucleotide primers for ChIP are listed in Supplementary Table 2. For quantitative real time (QrtPCR), RNA was extracted from etiolated seedlings using a RNeasy mini kit (Qiagen) and DNase I on-column digestion (Sigma). First strand cDNA was synthesized using a qScript cDNA Synthesis Kit (Quantabio) from 0.8 µg RNA. rtPCR was performed using PerfeCTa SYBR Green FastMix (Quantabio) and the BioRad CFX96 qPCR system. Relative values were normalized over housekeeping genes in the appropriate species. Data presented shows the average of three independent biological repeats. Oligonucleotide primers used for rtPCR are shown in Supplementary Table 2.

Western blots were carried out as previously described36. The mouse anti-HA antibody (Sigma-Aldrich) was used at a concentration of 1:1,000 dilution, anti-POR (Agrisera AS05 067-10) was used at 1:4,000 dilution, and anti-FLU (obtained from B. Grimm) was used at 1:2,000 and the secondary antibody, goat anti-mouse IgG1 horseradish peroxidase conjugate (Thermo Fisher Scientific) was used at 1:20,000 dilution. Proteins were extracted from 5-day-old etiolated seedlings under green light as previously described37. Bortizomib treatment was carried out in 6-well tissue culture plates (Fisher Scientific); a total reaction volume of 3 ml of 50 μM Bortezomib (Santa Cruz Biotechnology) was added to each well. Negative controls contained an equivalent volume of DMSO (Sigma-Aldrich) (DMSO was used to reconstitute the Bortezomib powder). Five-day-old etiolated seedlings were transferred to each well by laying the seedlings on top of the solution in each well gently ensuring that the roots of each seedlings were immersed in the reaction solution. The plate was covered with aluminium foil and placed on a flatbed shaker (60 rpm) for 2 h, after which material was dried with paper towel to remove excess water and frozen in liquid nitrogen immediately.

Phylogenetic analyses

Genomic DNA and CDS sequences for FLU, CHLM and hypoxia-related gene orthologues were obtained from KEGG: Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/kegg/). FLU, CHLM genomic sequences were searched manually for potential ERFVII binding sites and HRPE-like sequences20.

Statistics

For experimental analysis of Pchlide and quantitative reverse transcription PCR, three independent replicates with different biological material are reported for each experiment. For ChIP, three independent replicates with different biological material were used per experiment. Each experiment was repeated at least twice. For analysis of ROS via staining and microscopy 8–15 seedlings were analysed and images taken for representative samples, experiment was repeated at least three times. In all cases measurements were taken from distinct samples. Differences in ROS content were tested by general linear model (GLM) with two factors (genotype and oxygen) with fixed effects. GLM tests were carried out with SPSS v.27. All graphs were produced using Graphpad software (Version 8), also used to calculate standard deviation of the mean for all samples tested. Relationships between dependent and independent variables were assessed by linear regression analysis using standard parameters for coefficiency of determination (R2) in Graphpad.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Full versions of all blots are provided in Supplementary Fig 1. Unique identifiers for genes from all species analysed are listed in the text. Where appropriate, seeds of accessions and transgenic lines are available from the corresponding author. Source data are provided with this paper.

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Acknowledgements

Solanum sp. accessions were obtained from the UC Davis/C.M. Rick Tomato Genetics Resource Center maintained by the Department of Plant Sciences, University of California, Davis, USA; A. thaliana accessions were obtained from NASC, Nottingham, UK; and quinoa and B. distachyon altitudinal cline accessions were provided by the US Department of Agriculture’s National Plant Germplasm System, Plant Introduction Research Station, Ames, Iowa, USA. We thank M. Baldeon and O. Barreiro for generous use of equipment; B. Grimm for FLU antibody; G. Miño for technical support; and N. Crout and M. Blazquez for many helpful discussions. The work was funded by a Marie Curie CASCADE fellowship (600181) to M.A. and a Leverhulme Trust Research Project Grant (RPG-2018-172) to M.J.H. and was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/R002428/1) to M.J.H. C.A.-B. was funded by grant PID2019-104249GB-I00 from the Agencia Estatal de Investigación of Spain (AEI)/10.13039/501100011033.

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Authors and Affiliations

Authors

Contributions

M.J.H., M.A., C.A.-B. and K.P. designed the experiments. M.J.H., M.A., G.S., C.D. and J.M. carried out the experiments. M.A., C.A.-B. and M.J.H. conducted statistical analyses. M.J.H. and C.A.-B. wrote the manuscript with inputs from all co-authors. All co-authors read and approved the submitted manuscript.

Corresponding author

Correspondence to Michael J. Holdsworth.

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The authors declare no competing interests.

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Nature thanks Nico Dissmeyer, Bernhard Grimm, Margret Sauter and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Tetrapyrrole synthesis and Pchlide steady state levels in etiolated seedlings.

a, Schematic of tetrapyrrole synthesis showing points of oxygen requirement3. Blue, enzymes, black metabolites. Blocked arrows indicate repression. ALA, 5-aminolevulinic acid; Mg-Proto, Mg-protoporphyrin; GluTR, glutamyl-tRNA reductase; CHLM, MAGNESIUM-PROTOPORPHYRIN IX METHYLTRANSFERASE; HY1, HEME OXYGENASE1; POR, Light requiring PCHLIDE OXIDOREDUCTASE; FLU, FLOURESCENT IN BLUE LIGHT. Constituents of the GluTR inactivation complex are shown, diagram after16. b, Linearity of the measurement of Pchlide peak fluorescence at 636 nm, measured as arbitrary units (a.u.) per cotyledon per mm2 (to account for differing cotyledon sizes between accessions of the same species), using the Pchlide over-accumulating Col-0 mutant flu. c, Pchlide levels in Col-0 (wild type) and flu. d, Levels of Pchide in A. thaliana during 7 days of etiolated growth of Col-0 (wild-type) and flu. Experiments carried out using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l) unless otherwise stated. Means are plotted, error bars report SD. For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 2 The influence of the PRT6 N-degron pathway and ERFVIIs on Pchlide levels.

a, Pchide steady state levels in A. thaliana during 7 days of etiolated growth of Col-0 (wild-type) and N-degron pathway mutant prt6-1 and erfVII combinations. b, Pchlide levels at pO2 21.2 kPa (48 m a.s.l) in A. thaliana N-degron pathway mutant prt6-1 and erfVII combinations and individual ERFVII mutants. c, Pchlide levels in prt6, flu and prt6 flu. d, Analysis of RNA expression of FLU and genes of tetrapyrrole synthesis in Col-0 (wild type) and erfVII and prt6 mutants. e, Transcript levels in Col-0 and erfVII at different ambient oxygen levels. f, Accumulation of proteins for POR and FLU in Col-0 (wild type) and PRT6 N-degron pathway mutants at pO2 21.2 kPa (48 m a.s.l), and in Col-0 and erfVII at different ambient oxygen levels, repeated independently three times with similar results. All experiments carried out using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l) unless otherwise stated. Means are plotted, error bars report SD. Significant differences denoted with letters for one-way ANOVA (p < 0.05). Coefficient of determination (R2) is given. For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 3 ChIP analysis of HRE2 interaction with tetrapyrrole synthesis-related genes.

a, Conservation of ERFVII binding site (also known as EBP, GC box) and related HRPE (Hypoxia-Responsive Promoter Element38)-like element in the first coding exon of A. thaliana FLU and orthologues from selected angiosperms (initiating ATG highlighted). b, Schematic of the CHLM gene (repressed by ERFVIIs) showing ERFVII-binding sites and ChIP analysis of RAP2.3-3xHA and HRE2-3xHA occupancy of CHLM gene regions (including known positive and negative sequences29). c,d Conservation of ERFVII binding site in the first coding exons of CHLM and CHL27 respectively. e, ChIP analysis of HRE2-3xHA occupancy of gene regions of CHL27, PORA, PORB and HEMA1. f, Schematics of genes showing positions of ERFVII and ERFVII-like binding sites. White boxes untranslated and black boxes translated regions. All experiments carried out using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l). Means are plotted, error bars report SD. For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 4 Pchlide steady state levels and submergence tolerance for species accessions used in this study.

a, b, Pchlide levels at pO2 21.2 kPa in S. cheesmaniae and B. distachyon accessions collected at different altitudes and geographic locations. c, Relationship between A. thaliana accession and submergence tolerance, LT50 is defined as the number of days after which 50% of the plant population (for a particular accession) dies and was calculated from survival curves for each accession (data replotted from39), original data in Supplementary Table 1c. Coefficient of determination (R2) is given. Means are plotted, error bars report SD. For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 5 ROS and chlorophyll accumulation in 5 day old etiolated seedlings after one day of light in A. thaliana.

a. Relative fluorescence of ROS and chlorophyll. b. Example images of fluorescence of ROS and chlorophyll in different accessions at 21% and 15% ambient oxygen. Differences in ROS content were tested by GLM (General Linear Model) with two factors (Genotype and Oxygen) with fixed effects, both factors as well as the interaction where highly significant (Supplementary Table 1d). At least 8 seedlings were measured per accession, representative cotyledons are shown. For box and whisker plots whiskers go down to the minimum and up to the maximum values, boxes represent from 25th to 75th percentile and bars equal the median values.

Source data

Extended Data Fig. 6 Expression of RNA and protein for FLU, CHLM, HEMA1 and PORs in A. thaliana accessions.

a, Analysis of RNA expression in accessions collected at increasing altitude at pO2 21.2 kPa and 15% ambient oxygen. b, Accumulation of proteins for POR and FLU in Col-0 (wild type) and Sha, prt6 (Col-0 background) and Sha prt6, repeated independently three times with similar results. All experiments were carried out using etiolated seedlings after 5 days growth at 48 m a.s.l unless otherwise stated. Means are plotted, error bars report SD. Coefficient of determination (R2) is given. For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 7 Locations of sites used for reciprocal transplantation experiments.

Relationship between altitude and partial pressure of oxygen (pO2 kPa), showing positions of sites Sutton Bonington (SB): Latitude 52.829809° N longitude −1.249732° E pO2 21.2 kPa: 48 m a.s.l, and Sangolquí (ESPE): Latitude −0.312917° N longitude −78.445157° E pO2 15.7 kPa: 2479 m a.s.l. Maps obtained from http://www.ginkgomaps.com/.

Extended Data Fig. 8 Influence of ambient O2 on expression of hypoxia-induced RNAs in A. thaliana and S. habrochaites accessions.

a, Pchlide in A. thaliana high-altitude accession Sha, and erfVII (pentuple mutant obtained from the low altitude accession Col-0), measured at 48 m a.s.l. (SB, pO2 21.2 kPa) and 2479 m a.s.l. (ESPE, pO2 15.7 kPa). b, PORA/B, CHLM gene expression in A. thaliana accessions collected at different altitudes measured at 48 m a.s.l. (pO2 21.2 kPa, SB) or at 2479 m a.s.l. (pO2 15.7 kPa, ESPE). c, Hypoxia-related gene expression in A. thaliana accessions Col-0 (low altitude) and Sha (high altitude), at 48 m a.s.l. in different ambient levels of oxygen. d, Expression of S. habrochaites hypoxia-related genes at 15% and 21% ambient oxygen in accessions from different altitudes measured at 48 m a.s.l. Carried out using etiolated seedlings after 5 days growth (48 m a.s.l). Means are plotted, error bars report SD, coefficient of determination (R2) is given. For each n = 3 biologically independent experiments. For a, Sha (SB) was measured once.

Source data

Extended Data Fig. 9 Comparisons of genomic DNA and RNA expression patterns in A. thaliana accessions Col-0 and Sha.

a,b Introduction of the prt6-1 mutation (by introgression from low altitude accession Col-0) or C2A-RAP2.3 (by transformation) into A. thaliana high altitude accession Sha enhances the expression of hypoxia-related genes. c, Expression of RNAs for PCO PRT6 N-degron pathway components in accessions Sha and Col-0. RNA was extracted using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l). d, Comparison of DNA sequence of the first coding exon of FLU in accessions Col-0 and Sha. DNA sequence information was obtained from Arabidopsis 1001 web site: http://signal.salk.edu/atg1001/2.0/gebrowser.php. Shown are ERFVII binding site (also known as EBP, GCC box) and related HRPE (Hypoxia-Responsive Promoter Element2)-like element in the first coding exon of A. thaliana FLU (initiating ATG highlighted). All experiments carried out using etiolated seedlings after 5 days growth. Means are plotted, error bars report SD. Significant differences denoted with letters for one-way ANOVA (p < 0.05). For each n = 3 biologically independent experiments.

Source data

Extended Data Fig. 10 Pchlide steady state levels in reciprocal genetic crosses between A. thaliana high altitude accession Sha and erfVII (Col-0 accession).

Experiments carried out using etiolated seedlings after 5 days growth at pO2 21.2 kPa (48 m a.s.l). Means are plotted, error bars report SD. Significant differences denoted with letters for one-way ANOVA (p < 0.05). For each n = 3 biologically independent experiments.

Source data

Supplementary information

Supplementary Figure 1

All uncropped western blots

Reporting Summary

Supplementary Table 1

Lists of accessions for species used, R2 values for Fig. 2c, relationship between altitude of collection of A. thaliana accessions and submergence survival and comparison of ROS content among all A. thaliana genotypes

Supplementary Table 2

List of oligonucleotide primers used in this study.

Source data

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Abbas, M., Sharma, G., Dambire, C. et al. An oxygen-sensing mechanism for angiosperm adaptation to altitude. Nature 606, 565–569 (2022). https://doi.org/10.1038/s41586-022-04740-y

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