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).
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).
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).
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.