Rubisco from two ecotypes ofPlantago lanceolataL. that are native to sites differing in atmospheric CO2levels

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Hannon , View ORCID Profile Arnold J. Bloom doi: https://doi.org/10.1101/2024.07.13.603406 Xiaoxiao Shi 1 Department of Plant Sciences, University of California at Davis , Davis, CA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xiaoxiao Shi Nathan M. Hannon 1 Department of Plant Sciences, University of California at Davis , Davis, CA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathan M. Hannon Arnold J. Bloom 1 Department of Plant Sciences, University of California at Davis , Davis, CA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnold J. Bloom For correspondence: ajbloom{at}ucdavis.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Rubisco, the most prevalent protein on the planet, initiates the conversion of CO 2 into carbohydrates during photosynthesis. Responses of this process to atmospheric CO 2 fluctuations daily, seasonally, and over millennia is still poorly understood. We have hypothesized that higher plants maintain carbon-to-nitrogen homeostasis by adjusting the balance of magnesium and manganese in chloroplasts to alter their relative carbon fixation and nitrogen assimilation rates. The following study examined the influence of magnesium and manganese on rubisco carboxylation and oxygenation in protein purified from two ecotypes of Plantago lanceolata : one adapted to the high atmospheric CO 2 that occurs near a natural CO 2 spring and the other adapted to more typical CO 2 atmospheres that occur nearby. The plastid DNA coding for the large unit of rubisco were similar in both ecotypes. The kinetics of rubiscos from the two ecotypes differed more when they were associated with manganese than magnesium. Specificity for CO 2 over O 2 ( S c/o ) for rubiscos from both ecotypes were higher when the enzymes were bound to magnesium than manganese. This disparity may account for the adaptation of this species to different CO 2 environments. Introduction Global atmospheric CO 2 will increase from its current concentration of 421 ppm to somewhere between 450 and 1130 ppm by the end of the century ( IPCC, 2021 ). Experiments on the responses of higher plants to rising CO 2 subject them to atmospheres containing higher than 550 ppm CO 2 ( Broberg et al ., 2019 ; Tcherkez et al ., 2020 ), a concentration that is 40% above the current ambient. Such treatments accelerate carbon fixation, inhibit photorespiration ( Cousins and Bloom, 2004 ), decrease protein concentrations by over 6% ( Medek et al ., 2017 ; Myers et al ., 2014 ; Taub et al ., 2008 ), and increase plant carbon-to-nitrogen ratio (C/N) by 20% ( Butterly et al ., 2015 ; Krämer et al ., 2022 ; Sardans et al ., 2012 ; Wang et al ., 2019 ; Ziska et al ., 2016 ). Central to these responses is rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), the enzyme that constitutes 50% of the protein in a leaf, 20% of the protein on Earth, and 3% of the global biomass ( Bar-On and Milo, 2019 ; Ellis, 1979 ). Rubisco catalyzes several competing reactions ( Bloom and Lancaster, 2018 ): One reaction is the carboxylation of ribulose 1,5-bisphosphate (RuBP) to two molecules of 3-phosphoglyceric acid (3PGA) that initiates C 3 carbon fixation, the foundation of primary productivity. A second reaction is the oxygenation of RuBP to one molecule of 3PGA and one molecule of 2PG that initiates photorespiration, a process generally considered to dissipate more than 25% of the energy in C 3 plants as waste heat ( Smith et al ., 2024 ; South et al ., 2019 ). Photorespiration, however, enhances assimilation of nitrate (NO 3 − ) and sulfate (SO 4 − ) into amino acids in plant shoots ( Bloom, 2015a , b , 2019 ; Bloom et al ., 2010 ; Bloom et al ., 2014 ; Bloom and Kameritsch, 2017 ; Bloom et al ., 2020 ; Bloom and Lancaster, 2018 ; Bloom and Plant, 2021 ; Bloom et al ., 2012 ; Bloom et al ., 2002 ; Carlisle et al ., 2012 ; Dietterich et al ., 2015 ; Easlon and Bloom, 2013 ; Foyer et al ., 2009 ; Myers et al ., 2014 ; Rachmilevitch et al ., 2004 ; Rubio-Asensio and Bloom, 2017 ; Rubio-Asensio et al ., 2015 ; Smart and Bloom, 2001 ; Smart et al ., 1998 ). Therefore, plant protein concentrations, growth, and yields decrease when elevated CO 2 atmospheres inhibit photorespiration for long periods ( Bloom, 2015a ; Bloom et al ., 2014 ; Bloom and Plant, 2021 ; Myers et al ., 2014 ). A handful of studies conducted more than four decades ago indicated that binding of rubisco to Mg 2+ instead of Mn 2+ accelerates RuBP carboxylation and inhibits RuBP oxygenation, but the data were highly variable ( Christeller, 1981 ; Christeller and Laing, 1979 ; Jordan and Ogren, 1983 ; Martin and Tabita, 1981 ; Wildner and Henkel, 1979 ). These studies purified rubisco via ammonium sulfate precipitation followed by centrifugation, a process that can adversely influence enzyme structure and activation ( Iñiguez et al ., 2021 ; Wingfield, 1998 ). In a recent study ( Shi et al ., 2024 ), we used fast protein liquid chromatography to purify rubisco from five model C 3 species while preserving the metal-binding characteristics of the native protein ( Barnett et al ., 2012 ; Hagège et al ., 2015 ; Montes-Bayón et al ., 2016 ) and developed new methods to assess rubisco carboxylation and oxygenation that are indifferent to the presence of Mg 2+ or Mn 2+ ( Shi et al ., 2024 ). The maximum velocity of carboxylation ( V cmax ) was faster and the Michaelis constants of rubisco for CO 2 ( K c ) was greater when rubisco was bound to Mg 2+ rather than Mn 2+ . Both S c/o (rubisco specificity for CO 2 over O 2 ) and V cmax / V omax were greater when the enzyme was bound to Mg 2 rather than Mn 2+ ( Shi et al ., 2024 ). Plantago lanceolata L. may not be a model plant species, but its leaves have been widely used in herbal medicines (Drava et al. 2019, Gonçalves and Romano 2016, Wichtl 2004) and food preparations (Guarrera and Savo 2016). Research has shown that the root fractions of P. lanceolata L. possess antibacterial properties as demonstrated through Phytochemical Analysis by Gas Chromatography-Mass Spectrometry in vitro (Rahamouz-Haghighi et al. 2022). Furthermore, P. lanceolata leaf extract has been found to selectively inhibit the proliferation of CAL51 triple-negative breast cancer cell proliferation (Alsaraf et al. 2019) and have anti-obesity effects on mice in vivo (Yoshida et al., 2013). Several flavonoids present in the inflorescences and leaves of P. lanceolata have been discovered to have anti-allergic and anti-inflammatory effects (Budzianowska et al. 2022, Murai et al., 1995, Antunes-Ricardo et al. 2015). The following study compared the kinetics of rubisco purified from two ecotypes of Plantago lanceolata L.: one collected near a CO 2 spring has experienced an average daytime concentration of 791 ppm CO 2 for many generations, and the other collected 200 m from the spring has experienced ambient CO 2 concentrations of about 421 ppm ( Saban et al ., 2019 ; Saban et al ., 2020 ; Watson-Lazowski et al ., 2016 ). We also extracted plastid DNA from the two P. lanceolata ecotypes and compared the sequences for the large subunit of rubisco ( rbcL ) to determine if multi-generational exposures to different atmospheric CO 2 levels might result in genetic adaptations in rubisco. Materials and methods Plant growth conditions Prof. G. Taylor provided us with seeds from of Plantago lanceolata L. collected in May 2008 from nine plants growing in naturally elevated CO 2 atmospheres near a CO 2 spring at Bossoleto, Italy (Lat. 43°17’, Long. 11°35’) and at a nearby (ca. 200 m apart) ambient CO 2 control site ( Saban et al ., 2020 ). Seeds obtained from the two sites were grown for one generation in the glasshouse at the University of Southampton and then crossed within maternal families to standardize parental effects. We planted the two ecotypes into 5 × 5 cm containers containing Sunshine Mix 4 (Sungro, Agawam, MA). The plants grew in a controlled environmental chamber for 21 d under 450 ppm or 750 ppm CO 2 , 60% relative humidity (light/dark), air temperature 25°/18°C (light/dark), and 16 h of 500 μmol m −2 s −1 PPFD at canopy height. Rubisco extraction and purification We collected about 40 g of leaves and froze them in liquid N 2 . Manual grinding with a mortar and pestle lysed the plant cells to a fine powder. We extracted the fine powder in 100 ml of a buffer (50 mM Tris-HCl pH 7.4, 20 mM MgCl 2 , 20 mM NaHCO 3 , 0.1 mM Na 2 EDTA, 10% glycerol, 50 mM mercaptoethanol, and 1 mM PMSF), filtered the extract through four layers of Miracloth, centrifuged it at 12100 × g at 4°C for 15 min to clarify it, and passed the supernatant through a 0.22 μm syringe filter before loading it onto an FPLC column. We purified rubisco using an ENrich™ SEC (Size Exclusion Column) 650 10 × 300 Column (BioRad, Hercules, CA) followed by a HiScale 16/20 6 mL SOURCE 30Q column (GE Healthcare Life Sciences, Pittsburgh, PA) on a NGC™ FPLC system (BioRad, Hercules, CA). All buffers included 2 mM dithiothreitol to prevent intermolecular disulfide bond formation. Pre-equilibration of the size exclusion column involved eluting five column volumes (CV) of 85% buffer A (50 mM Tris-HCl, 1 mM EDTA, 0.1 mM PMSF, pH 7.4) and 15% buffer B (50 mM Tris-HCl, 1 M NaCl 1 mM EDTA, 0.1 mM PMSF, pH 7.4). We loaded the protein sample onto the column and eluted it with 2 CV of 15% buffer B. UV absorption at 280 nm confirmed the protein peak. We ( a ) concentrated the protein solution to around 1 mM, ( b ) exchanged the buffer into buffer A, ( c ) filtered the sample, ( d ) loaded it onto a SOURCE 30Q column pre-equilibrated with 5 CV of buffer A, ( e ) eluted the protein with 50% buffer B with a linear gradient over 10 CV, ( f ) pooled the protein peak at 280 nm, ( g ) exchanged the buffer into buffer A with the addition of 20% glycerol, ( h ) and stored the protein solution at -80°C. We checked the purity of the protein on SDS-PAGE gels, Western Blots, and an Evolution 201 UV/Vis spectrometer coupled with an Evolution 1-cell Peltier temperature control system (Thermo Fisher Scientific, Waltham, MA). Rubisco carboxylation colorimetric reaction We developed a colorimetric assay for rubisco carboxylation that is accurate in the presence of Mg 2+ or Mn 2+ ( Shi et al ., 2024 ): To estimate v c and V cmax , we assessed the production of 3PGA in the presence of high levels of CO 2 by iPGM (cofactor-Independent PhosphoGlycerate Mutase) conversion of 3PGA to 2PGA ( Raverdy et al ., 2007 ; Zhang et al ., 2004 ), followed by a commercial assay kit (2-phosphoglycerate Assay kit, Abcam ab174097) that converts 2PGA into PEP, then pyruvate, H 2 O 2 , and resorufin. Absorption at 570 nm monitored resorufin production. This assay can detect 2PGA levels below 20 pmol and works in the presence of either metal cofactor. In more detail, we activated rubisco before the assays as follows. We added an assay buffer containing 20 mM Tris-HCl, 1 mM EDTA, 100 mM Triethanolamine at pH 7.8 to one 5 mL tube; added 1 or 2 μL of rubisco (0.5 – 1 μM), 10 μL of 250 mM NaHCO 3 , 0.5 μL carbonic anhydrase (∼2.5 units), 5 μL of iPGM (10 μM), 4 μL of MgCl 2 (final concentration 20 mM) or MnCl 2 (5 mM); mixed thoroughly; and allowed the mixture to sit for 5 minutes. The final assay volume in the tube was 1750 μL. We started the reaction by adding 45 μL of 10 mM RuBP to the tube, split the mixture into five tubes, and stopped the reaction after 1, 2, 3, 4, or 5 mins by adding 0.5 N HCl to each tube. We added KOH to each reaction tube to adjust the pH to about pH 7.8 and added to the tubes equal amounts of freshly mixed 2PGA colorimetric cocktail that we prepared from a 2-phosphoglycerate assay kit (ab174097, Abcam, Cambridge, MA), mixed well, and moved the tubes to an opaque box. We measured OD 570nm after 40 min for each of the tubes and calculated the 2PGA concentration in each reaction tube based on a calibration curve determined by adding specified quantities of a 1 mM 2PGA standard solution that the assay kit provided. The carboxylation turnover rate v c = ½ (resorufin production rate – v o ), where O 2 optode measurements of O 2 depletion estimated v o as detailed below. We estimated V cmax from the Michaelis-Menten equation: and the relative specificity S c/o from: We conducted separate calibration runs in the presence of the buffer and Mg 2+ or Mn 2+ in the same concentrations used in the rubisco reactions ( Fig. S1 ) and calculated the ratio between the slopes of the runs using only the 2PGA buffer and the slopes of the runs containing rubisco. Linear regressions for each combination of plant and metal ion, which included a constant term for each run, expressed the change in 2PGA as a function of time. IPGM expression and purification We transformed a 6xHis-tagged C. elegans nematode iPGM plasmid into BL21(DE3) competent cells and incubated them at 37°C overnight. We selected one colony for culturing at 37°C overnight in 50 mL LB media (peptone 10 g, yeast extract 5 g, NaCl 10 g L −1 ) and 50 μL 100 g mL −1 kanamycin stock solution. We added 10 mL of the small culture to 1 L of the same media and grew it until OD 600 = 0.7. We added isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture flask to bring it to a final concentration of 0.2 mM and reduced the temperature and stirring speed to 30°C and 100 rpm, respectively. We grew the culture for 24 h, centrifuged it for 20 min at 4700 × g , and suspended the pellet in 30 mL buffer A (20 mM Tris-HCl, 2 mM DTT, pH 6.8). We stored the cell suspension at −80°C before purification. We thawed 30 mL of the cell suspension, lysed the cells by adding 30 mg of lysozyme and 30 μg of DNAse, and passed the cells twice through a French press. We centrifuged the solution at 12100 × g for 60 min, concentrated the supernatant using Amicon Ultra Centrifuge Filters (EMD Millipore, Burlington, MA), and filtered the resulting solution through a 0.22 μm filter before loading it onto a column. We pre-equilibrated a Bio-Scale Mini Profinity IMAC cartridge (Bio-Rad, Hercules, CA) with five column volumes (CV) of buffer A (50 mM Tris-HCl, 25 mM imidazole, 1 mM EDTA, 0.1 mM PMSF, pH 7.4), loaded the protein sample onto the column, washed the column with 5 CV of buffer A, and eluted the protein with a linear gradient over 10 CV of buffer B (50 mM Tris-HCl, 500 mM imidazole, 1 mM EDTA, 0.1 mM PMSF, pH 7.4), confirmed the protein peak by UV absorption at 280 nm, and loaded the fractions onto an SDS-PAGE gel. We stored the fractions with iPGM protein at –80°C. Rubisco oxygenation A needle-type micro-optode OXF50-OI (PyroScience GmbH, Breman, Germany) on a FireSting O 2 optical oxygen and temperature meter (FSO2-4) monitored changes in O 2 concentration. We conducted oxygenation experiments under three sets of conditions at 25°C: ( a ) ambient (79% N 2 , 20.96% O 2 , and 0.04% CO 2 ), ( b ) elevated CO 2 (78.96% N 2 , 20.96% O 2 , and 0.08% CO 2 ), and ( c ) reduced O 2 (89% N 2 , 10.96% O 2 , and 0.04% CO 2 ). Precision mass flow controllers (Apex Vacuum, Canton, GA) calibrated against soap bubble flowmeters mixed pure N 2 , O 2 , and CO 2 to the desired concentrations. A non-dispersive Infrared Gas Analyser (Li-cor, Lincoln, NE) checked the CO 2 concentration. We conducted a two-point calibration of the oxygen optode in both air and air-saturated water. The final assay volume in each tube was 3000 μL. Before the oxygenation assay, we activated rubisco as follows. We added assay buffer (20 mM Tris-HCl, 1 mM EDTA, 10 mM NaHCO 3 , 100 mM triethanolamine, pH 7.8) to a covered, temperature-controlled, 4 mL quartz cuvette; added 10 μL of rubisco (0.5 – 1 μM), 50 μL of iPGM (10 μM), 5 μL carbonic anhydrase (∼25 units), and 40 μL of MgCl 2 (final concentration 20 mM) or MnCl 2 (5 mM); and mixed the contents of the cuvette thoroughly. We allowed the mixture to sit for 5 minutes before inserting the oxygen sensor and sparging for 1 minute with the gas mixture corresponding to the experiment’s conditions. After about 30 s, once the oxygen sensor reading stabilized, we started the reaction by adding 100 μL of 10 mM RuBP (pre-equilibrated with the same gas mixture) and began collecting data. We did not observe any apparent protein deactivation and degradation during more than 300 s; therefore, we estimated the oxygenation turnover rate v o from the linear trend in oxygen consumption for at least 200 s divided by rubisco content. Michaelis-Menten kinetics predicts the oxygenation turnover rate v o to be: where V omax , K o , and K c depend on species and the associated metal, Mn 2+ or Mg 2+ , but are independent of [O 2 ] and [CO 2 ]. Values of v o under multiple sets of conditions for [O 2 ] and [CO 2 ], therefore, provided estimates of the three parameters V omax , K o , and K c . For the reduced O 2 experiments, we performed blank runs with RuBP and all other components but without rubisco. We calculated O 2 consumption rates before and after adding RuBP for both the blank and actual runs and determined the O 2 consumption rate as where R denotes the rate of change in oxygen levels in the presence or absence of rubisco and RuBP. Statistics In general, we used R for Windows version v.4.3.3 and will provide the R code upon request. From the measured rates of rubisco reactions, kinetic parameters were calculated using a Bayesian method. Briefly, random sets of kinetic parameters were generated from a lognormal prior distribution based on the approximate range in which kinetic parameters for C 3 plants have been calculated, and the relative likelihood of observing the values in our experiments was computed for each set of kinetic parameters. The result of each experiment was assumed to be normally distributed with a fixed variance, combining the regression standard error in the measurements with the variance between the results of similar experiments. These likelihoods were used as weights to approximate a posterior distribution on the kinetic parameters from which we calculated posterior means and standard deviations of each parameter. To determine the overall effects of Mg 2+ and Mn 2+ across the two ecotypes, we aggregated each kinetic parameter, adjusted by their respective standard errors, and used it as input to a two-way ANOVA, with metal ion and ecotype as variables. rbcL sequences We isolated chloroplasts ( Bloom and Kameritsch, 2017 ) from the two P. lanceolata ecotypes and extracted the plastid DNAs using the modified high salt method (Shi et al. 2012). A 260 /A 280 ratios were verified by UV-Vis spectrophotometry, and samples with a ratio over 2 were sent for chloroplast genomic DNA sequencing (CD Genomics, Shirley, NY). After conducting the initial sample quality test, high-quality DNA samples were used to construct the library. The purified DNA samples were fragmented into 200-500 bp using Covaris S/E210 or Bioruptor. The overhangs resulting from the fragmentation were converted into blunt ends using T4 DNA polymerase, Klenow Fragment, and T4 Polynucleotide Kinase. Subsequently, an ‘A’ base was added to the 3’ end of the blunt phosphorylated DNA fragments, followed by the ligation of adapters to the ends of the DNA fragments. The desired fragments were then purified through gel-electrophoresis, selectively enriched, and amplified by PCR. Index tags were introduced into the adapter during the PCR stage, and a library quality test was performed. Finally, the qualified library was used for sequencing using Illumina PE150 Sequencing Platform, and the generated data were utilized for downstream bioinformatics analysis. GetOrganelle (v1.7.7.1) was used to construct the final cpDNA sequences. Custom R code (v4.3.3) was used to search for fragments containing variants of the cpDNA sequences and determine their frequencies. For each identified variant, a sequence of five codons centered on the changed nucleotide(s) was chosen, and the number of fragments containing the original and variant sequences was compared to calculate its frequency in both ecotypes. Results Rubisco Kinetics Rubisco purified from the ambient ecotype conducted both RuBP carboxylation and oxygenation at higher rates under all conditions than rubisco purified from the CO 2 spring ecotype ( Figs. 1 & 2 ). Rubisco from the ambient ecotype conducted significantly more carboxylation under 0.10% CO 2 than 0.04% CO 2 , whereas rubisco from the spring ecotype was much less affected by CO 2 enrichment ( Figs. 1 & 2 ). Rubiscos from both ecotypes conducted significantly more RuBP carboxylation and oxygenation when bound to Mn 2+ compared to Mg 2+ . ( Fig. 2 ). Download figure Open in new tab Fig. 1. Rates of RuBP carboxylation and oxygenation in turnovers per second by rubisco purified from two ecotypes of Plantago lanceolata L. One ecotype (Spring) collected near a CO 2 spring had experienced high CO 2 for many generations, and the other ecotype collected nearby (Ambient) had experienced ambient CO 2 atmospheres. The enzyme was bound to Mg 2+ or Mn 2+ and exposed to 0.04% or 0.10% CO 2 atmospheres. Shown are the mean ± SE, n= 4 – 10. Download figure Open in new tab Fig. 2. Tukey test comparing RuBP carboxylation and oxygenation rates under various combinations of metals (Mg vs. Mn), Plantago l anceolata ecotypes (Spr vs. Amb), CO 2 concentrations (0.04% vs. 0.1%), and O 2 concentrations (21% vs. 30%). Shown are mean difference ± SE, n = 4 –10. The kinetics of the rubiscos from the two ecotypes differed more when they were associated with Mn 2+ than Mg 2+ ( Fig. 3 & Table 1 ). The rubisco from the ambient ecotype exhibited a slightly higher V cmax and V omax than the rubisco from the spring ecotype when bound to magnesium and a much higher V cmax and V omax when bound to Mn 2+ ( Fig. 3 ). Specificity for CO 2 over O 2 ( S c/o ) for rubiscos from both ecotypes were higher when the enzymes were bound to Mg 2+ than Mn 2+ ( Fig. 3 ). Download figure Open in new tab Fig. 3. Influence of Mg 2+ or Mn 2+ (mean ± credible interval, n = 4 to 20) on the kinetics of rubisco purified from two ecotypes of Plantago lanceolata L.: one (Spring) collected near a CO 2 spring has experienced high CO 2 for many generations; the other collected nearby (Ambient) has experienced ambient CO 2 . V cmax is the maximum velocity of carboxylation, V omax is the maximum velocity of oxygenation, K c is the Michaelis constant of rubisco for CO 2 , K o is the Michaelis constant of rubisco for O 2 , S c/o is the specificity of rubisco for CO 2 over O 2 , and V cmax / V omax is the ratio of the maximum velocities. Table 1 provides statistical analysis of these data. View this table: View inline View popup Download powerpoint Table 1. 2-way ANOVA for the influence of Ecotype, Metal, or their interaction on the kinetic parameters shown in Fig. 3 for rubiscos purified from the two ecotypes in the presence of Mg 2+ or Mn 2+ . “Signif.” denotes significance where “.” P < 0.1, “*” P < 0.05, “**” P < 0.01, and “***” P < 0.001. cpDNA sequences Both ecotypes had similar plastid DNA coding for the large unit of rubisco (rbcL). The rbcL gene is located at positions 53249 to 54682 in the ambient ecotype and 53018 to 54451 in the elevated ecotype of the chloroplast DNA sequences (cpDNAs). Gene maps showing the rbcL encoding regions and the translation directions are displayed in Fig. 4 . The rbcL gene in the reconstructed sequences was identical to those in previously published sequences (Zhao et al. 2022, Saban et al. 2020 ). A small percentage of the fragments, however, differed from this sequence in one or, in one case, two nucleotides ( Table 2 ). The most common of these, by a significant margin, was a change in nucleotide 890 from A to C, which occurred in approximately 5.6% of fragments in the spring ecotype and 4.1% in the ambient. This change replaces a methionine at residue 297 with a leucine. Download figure Open in new tab Fig. 4. cpDNA map of rbcL from the (A) CO 2 spring and (B) ambient ecotypes. The orange and green arrows indicate open reading frames (ORFs). The restriction enzymes shown are unique and dual cutters. View this table: View inline View popup Download powerpoint Table 2. Mutations that appeared in a significant portion of the fragments matching the rbcL gene in two ecotypes of Plantago lanceolata . “Positions” refer to the positions of the changed nucleotide(s) and are specified relative to the start of the rbcL gene. “Frequency” refers to the number of fragments containing the changed sequence as a percentage of the total number of fragments containing the original sequence and the changed sequence. Discussion Kinetics The ambient ecotype had overall higher rates of both carboxylation and oxygenation than the CO 2 spring ecotype. One possible explanation is differences in genomic DNA. We noticed larger average seed size and lower seed yield per flower and per plant for the CO 2 spring ecotype than for the ambient ecotype. Plantago lanceolata is able to change growth habit and phenology in different growth conditions; however, it is known that genetic variations exist based on significantly reduced seed yields compared to original and alien sites (Van Tienderen et al. 1991). Under limited water supply and high light exposure, Plantago lanceolata displayed higher water use efficiency (WUE), better nitrogen absorption, and higher bacterial frequency in roots (Miszalski et al. 2023). The photochemical activity of PSII stayed at the same level under the stressed conditions as under the no-stress conditions (Miszalski et al. 2023). Taken together, we think the extensive plasticity of Plantago lanceolata might be shared among many species from various geographic locations, and the variations in photosynthetic rates are genotype-specific. Exposure to elevated CO 2 atmospheres stimulated carboxylation rates in the ambient ecotype more than in the CO 2 spring ecotype. This would not be expected if faster carboxylation rates were always beneficial. Were balancing carboxylation and oxygenation advantageous, then elevated and, especially, fluctuating levels of CO 2 might select for a mechanism where carboxylation became less sensitive to atmospheric CO 2 . The nature of such a mechanism is unknown. The CO 2 spring ecotype has adapted to elevated CO 2 levels for many generations and might be expected to balance carboxylation and oxygenation more quickly. It is worth noting that the 0.1% CO 2 treatment used in this study is generally not sufficient to saturate rubisco carboxylation, so the lack of significant change in the CO 2 spring ecotype between ambient and 0.1% CO 2 conditions may indicate an additional regulatory mechanism beyond the usual kinetics. The change from methionine at residue 297 with a leucine is too rare to have a significant effect on the overall kinetics. Possibly, the small subunit of rubisco, whose gene is located in the nucleus and was not sequenced in this study, has some regulatory effect. In other C 3 species that have been studied, carboxylation rates are higher when rubisco is bound to Mg 2+ , whereas oxygenation rates are higher when bound to Mn 2+ ( Shi et al. 2024 ). By contrast, manganese Mn 2+ appears to promote both carboxylation and oxygenation in rubisco from Plantago lanceolata . Previous studies ( Bloom and Kameritsch 2017 ) indicate that plants respond to higher levels of CO 2 by increasing manganese levels. If rubisco from Plantago lanceolata thrives in the presence of Mn 2+ , this may explain why this species was able to adapt to the high CO 2 environment of the spring. When bound to Mg 2+ , kinetic parameters for rubisco from both ecotypes were roughly within the range of those for other C 3 plants ( Shi et al. 2024 ). Nonetheless, if the CO 2 spring ecotype has a mechanism that preserves the balance of carboxylation and oxygenation under elevated CO 2, as introduced above, then Michaelis-Menten kinetics might not fully characterize it because Michaelis-Menten kinetics predict that the ratio of carboxylation to oxygenation should be proportional to the ratio of the two gases with S c/o as the proportionality constant. Chloroplast DNA sequences The reconstructed sequences for rbcL from P. lanceolata were nearly identical to the previously published sequences for the rbcL gene (Zhao et al. 2022, Saban et al. 2020 ). This supports the generality that the rbcL gene is highly conserved among species ( Saban et al. 2020 ) and the P. lanceolata- specific findings of low overall genetic differentiation between the CO 2 elevated and ambient ecotypes and weak selection in exons ( Saban et al. 2020 ). Although DNA methylation may play a role in P. lanceolata ’s evolutionary divergence under exposure to elevated and ambient CO 2 for multiple generations ( Saban et al. 2020 ), we did not conduct cpDNA bisulfite sequencing because chloroplast DNA is relatively insensitive to methylation (Fojitova et al. 2001, Ahlert et al. 2009) and because in particular rbcL genes in tobacco, Arabidopsis , peas, and tomatoes are not affected by DNA methylation (Fojitova et al. 2001). Moreover, the BstNI/EcoRII assay, which had been used in previous studies, might not be accurate for analyzing low levels of methylation in chloroplast DNA sequences. (Marano et al. 1991, Ngernprasirtsiri et al. 1988, Ohta et al. 1991, Langdale et al. 1991). Increasing adenine or cytosine methylation by introducing cyanobacterial genes for adenine and cytosine DNA methyltransferases into the tobacco plastid genome through chloroplast transformation resulted in no phenotypic differences from wild-type plants and no alterations in plastid gene expression (Ahlert et al. 2009). The variant of the rbcL gene with a C, instead of an A, at position 890 occurred in both P. lanceolata ecotypes at a much higher frequency than other mutations ( Table 2 ). This DNA change corresponds to a change from Met 297 to Leu 297 in the amino acid sequence, which we might understand by examining the related – and well-studied – spinach rubisco sequence. Rubisco crystal structures from spinach show residue 297 in close proximity to the substrate access channel ( Fig. 5 ). In an activated transition state analog that was achieved by binding to the tight-binding inhibitor CAP, magnesium coordination is in a distorted octahedron with longer than usual metal-R bond distances ( Fig.5A , Andersson 1996). This is the “closed” active site conformation. In another activated structure with the substrate RuBP and Ca 2+ , residue 297 moves to hover over the active site channel, whereas Lys334 moves away from the reaction center ( Fig. 5B ). Note that Ca 2+ in Fig. 5B can activate rubisco but does not turn it over (Taylor and Andersson 1997). The active site appears to be “open”. Download figure Open in new tab Fig. 5. The vicinities of the active sites in two published crystal structures of spinach rubisco. The red “+” symbols indicate water molecules. (A) Rubisco bound to Mg 2+ and CAP (PDB: 8RUC, Andersson 1996). (B) Rubisco bound to Ca 2+ and RuBP (PDB: 1RXO, Taylor and Andersson 1997). KCX denotes lysine peptide linkage and CAP denotes 2-carboxyarabinitol-1,5-diphosphate. Although interconversions of hydrophobic residues such as from methionine to leucine are common and enzyme function before and after such changes often remain the same, the significant location of residue 297 should not be ignored. It is located right at the entrance of the active site and at the end of the flexible part of a loop. Loop movements involving residue 297 might also interact with neighboring loops such as the loop containing the carbamylated Lys201 that is required for CO 2 activation or the loop on the distal side of the substrate containing Lys334, whose interactions with substrate-CO 2 complex are required for turning rubisco over (Cleland et al. 1998). Lys334 is also believed to stabilize the transition state and the product (Cleland et al. 1998) during the reactions when the active site is in the “closed” conformation ( Fig. 5A ). The distances of the C-alpha in Lys 334 to Met 297 changed from 20.6Å to 15.8Å from the open ( Fig. 5B ) to the closed state ( Fig. 5A ). The M297L variant encoded by this version of the gene might have properties that are advantageous under CO 2 enrichment such as regulating the opening and closing of the active site access tunnel, which might result in chloroplasts with this variant being favored in the CO 2 spring ecotype. Perhaps it occurred in the past and persists in a small percentage of chloroplasts in all ecotypes, but a high CO 2 environment selects for an increase in that percentage. As the most abundant protein on Earth, type I rubisco large subunit gene ( rbcL ) evolves slower than 98% of all enzymes (Bouvier et al. 2024). A phylogenetic study found that one nucleotide substitution and one amino acid change happen every 0.9 million years and 7.2 million years, respectively (Bouvier et al. 2024). This is consistent with the 4 to 5% occurrences of the mutation we observed at position 890 on the rbcL gene. Molecular dynamics simulations on extant and ancestral rubisco sequences showed that the small subunit ( rbcS ) consistently confined the structural dynamics of the large subunit throughout the enzyme’s evolutionary history (Kaustubh et al. 2024). The data were inconclusive, however, on the small subunit serving as a CO 2 substrate reservoir (Kaustubh et al. 2024), as some have proposed (Van Lun M et al. 2014). This may be because each species contains only one copy of the rbcL gene in the chloroplast, whereas multiple rbcS sequences are found encoded in genomic DNAs, and rbcS evolves faster (Bouvier et al. 2024). This might also explain the kinetic differences between spring and ambient genotypes, but clearly, additional studies on the function of rubisco small subunit are required. Conclusions The kinetic properties of rubisco from P. lanceolata generally align with those of other C 3 plant species. Nonetheless, notable differences exist. In particular, association of rubisco with Mn 2+ , promotes faster carboxylation and oxygenation in P. lanceolata than association with Mg 2+ . This disparity may account for the species’ ability to acclimate to high CO 2 environments. The rbcL gene, which is identical in both ecotypes except for a higher frequency of a rare variant in the spring ecotype, cannot explain differences between the two ecotypes. Data on rubisco kinetics from non-model plant species remain limited, underscoring the need to examine the factors contributing to the differences between P. lanceolata ecotypes and among P. lanceolata and model species. Elucidating the role of metals Mn 2+ vs. Mg 2+ within the photosynthetic and photorespiratory pathways should enhance our understanding of plant adaptations to climate change. Author contributions A.J.B. planned the research, obtained the funding, wrote the manuscript, and drew the figures and tables; X.S. developed the biochemical methods, edited the manuscript, and conducted experiments; N.M.H. also conducted experiments, performed the statistical analyses, and edited the manuscript. Data availability statement Data used in this work have been uploaded to Dryad and will be made public upon publication. Acknowledgments NSF grant CHE-19-04535 funded a portion of this research. References Cited ↵ Bar-On YM , Milo R. 2019 . The global mass and average rate of rubisco . Proceedings of the National Academy of Sciences 116 , 4738 – 4743 . OpenUrl Abstract / FREE Full Text ↵ Barnett JP , Scanlan DJ , Blindauer CA . 2012 . Protein fractionation and detection for metalloproteomics: challenges and approaches . Analyt. Bioanalyt. Chem . 402 , 3311 – 3322 . OpenUrl ↵ Bloom AJ . 2015a . The increasing importance of distinguishing among plant nitrogen sources . Current Opinion in Plant Biology 25 , 10 – 16 . OpenUrl CrossRef PubMed Bloom AJ . 2015b . Photorespiration and nitrate assimilation: a major intersection between plant carbon and nitrogen . Photosynthesis Research 123 , 117 – 128 . OpenUrl CrossRef PubMed Web of Science ↵ Bloom AJ . 2019 . Metal regulation of metabolism . Current Opinion in Chemical Biology 49 , 33 – 38 . OpenUrl ↵ Bloom AJ , Burger M , Asensio JSR , Cousins AB . 2010 . Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis . Science 328 , 899 – 903 . OpenUrl Abstract / FREE Full Text ↵ Bloom AJ , Burger M , Kimball BA , Pinter PJ . 2014 . Nitrate assimilation is inhibited by elevated CO 2 in field-grown wheat . Nature Climate Change 4 , 477 – 480 . OpenUrl ↵ Bloom AJ , Kameritsch P. 2017 . Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants . Physiologia Plantarum 161 , 545 – 559 . OpenUrl ↵ Bloom AJ , Kasemsap P , Rubio-Asensio JS . 2020 . Rising atmospheric CO 2 concentration inhibits nitrate assimilation in shoots but enhances it in roots of C 3 plants . Physiologia Plantarum 168 , 963 – 972 . OpenUrl CrossRef ↵ Bloom AJ , Lancaster KM . 2018 . Manganese binding to Rubisco could drive a photorespiratory pathway that increases the energy effciency of photosynthesis . Nature Plants 4 , 414 – 422 . OpenUrl ↵ Bloom AJ , Plant RE . 2021 . Wheat grain yield decreased over the past 35 years, but protein content did not change . Journal of Experimental Botany 72 , 6811 – 6821 . OpenUrl CrossRef ↵ Bloom AJ , Rubio-Asensio JS , Randall L , Rachmilevitch S , Cousins AB , Carlisle EA . 2012 . CO 2 enrichment inhibits shoot nitrate assimilation in C 3 but not C 4 plants and slows growth under nitrate in C 3 plants . Ecology 93 , 355 – 367 . OpenUrl CrossRef PubMed Web of Science ↵ Bloom AJ , Smart DR , Nguyen DT , Searles PS . 2002 . Nitrogen assimilation and growth of wheat under elevated carbon dioxide . Proceedings of the National Academy of Sciences of the United States of America 99 , 1730 – 1735 . OpenUrl Abstract / FREE Full Text ↵ Broberg MC , Högy P , Feng Z , Pleijel H. 2019 . Effects of elevated CO 2 on wheat yield: Non-linear response and relation to site productivity . Agronomy 9 , 243 . OpenUrl ↵ Butterly CR , Armstrong R , Chen D , Tang C. 2015 . Carbon and nitrogen partitioning of wheat and field pea grown with two nitrogen levels under elevated CO 2 . Plant and Soil 391 , 367 – 382 . OpenUrl CrossRef ↵ Carlisle E , Myers SS , Raboy V , Bloom AJ . 2012 . The effects of inorganic nitrogen form and CO 2 concentration on wheat yield and nutrient accumulation and distribution . Frontiers in Plant Science 3 , 195 . OpenUrl ↵ Christeller JT . 1981 . The effects of bivalent cations on ribulose bisphosphate carboxylase/oxygenase . Biochemical Journal 193 , 839 – 844 . OpenUrl Abstract / FREE Full Text ↵ Christeller JT , Laing WA . 1979 . Effects of manganese ions and magnesium ions on the activity of soyabean ribulose bisphosphate carboxylase/oxygenase . Biochemical Journal 183 , 747 – 750 . OpenUrl Abstract / FREE Full Text ↵ Cousins AB , Bloom AJ . 2004 . Oxygen consumption during leaf nitrate assimilation in a C 3 and C 4 plant: the role of mitochondrial respiration . Plant, Cell and Environment 27 , 1537 – 1545 . OpenUrl CrossRef Web of Science ↵ Dietterich LH , Zanobetti A , Kloog I , Huybers P , Leakey ADB , Bloom AJ , Carlisle E , Fitzgerald G , Hasegawa T , Holbrook NM , Nelson RL , Ottman MJ , Raboy V , Sakai H , Sartor KA , Schwartz J , Seneweera S , Norton R , Fernando N , Usui Y , Yoshinaga S , Myers SS . 2015 . Impacts of elevated atmospheric CO 2 on nutrient content of important food crops . Scientific Data 2 , 150036 . OpenUrl ↵ Easlon HM , Bloom AJ . 2013 . The effects of rising atmospheric carbon dioxide on shoot-root nitrogen and water signaling . Frontiers in Plant Science 4 , 304 . OpenUrl ↵ Ellis RJ . 1979 . The most abundant protein in the world . Trends in Biochemical Sciences 4 , 241 – 244 . OpenUrl CrossRef Web of Science ↵ Foyer CH , Bloom AJ , Queval G , Noctor G. 2009 . Photorespiratory metabolism: Genes, mutants, energetics, and redox signaling . Annual Review of Plant Biology 60 , 455 – 484 . OpenUrl CrossRef PubMed Web of Science ↵ Hagège A , Huynh TNS , Hébrant M. 2015 . Separative techniques for metalloproteomics require balance between separation and perturbation . TrAC Trends in Analytical Chemistry 64 , 64 – 74 . OpenUrl ↵ Iñiguez C , Niinemets Ü , Mark K , Galmés J. 2021 . Analyzing the causes of method-to-method variability among Rubisco kinetic traits: from the first to the current measurements . Journal of Experimental Botany 72 , 7846 – 7862 . OpenUrl CrossRef ↵ Masson-Delmotte V , Zhai P , Pirani A , Connors SL , Péan C , Berger S , Caud N , Chen Y , Goldfarb L , Gomis MI , Huang M , Leitzell K , Lonnoy E , Matthews JBR , Maycock TK , Waterfield T , Yelekçi o , Yu R , Zhou B IPCC . 2021 . Summary for Policymakers . In: Masson-Delmotte V , Zhai P , Pirani A , Connors SL , Péan C , Berger S , Caud N , Chen Y , Goldfarb L , Gomis MI , Huang M , Leitzell K , Lonnoy E , Matthews JBR , Maycock TK , Waterfield T , Yelekçi o , Yu R , Zhou B , eds. Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change . Cambridge, In Press : Cambridge University Press . ↵ Jordan DB , Ogren WL . 1983 . Species variation in kinetic properties of ribulose 1, 5-bisphosphate carboxylase/oxygenase . Archives of Biochemistry and Biophysics 227 , 425 – 433 . OpenUrl CrossRef PubMed Web of Science ↵ Krämer K , Kepp G , Brock J , Stutz S , Heyer AG . 2022 . Acclimation to elevated CO 2 affects the C/N balance by reducing de novo N-assimilation . Physiologia Plantarum 174 , e13615 . OpenUrl ↵ Martin MN , Tabita FR . 1981 . Differences in the kinetic properties of the carboxylase and oxygenase activities of ribulose bisphosphate carboxylase/oxygenase . Febs Letters 129 , 39 – 43 . OpenUrl ↵ Medek DE , Schwartz J , Myers SS . 2017 . Estimated effects of future atmospheric CO 2 concentrations on protein intake and the risk of protein deffciency by country and region . Environmental Health Perspectives 125 , 0870021 – 0870028 . OpenUrl ↵ Michalke B Montes-Bayón M , Blanco-González E , Michalke B. 2016 . Metalloproteins . In: Michalke B , ed. Metallomics . Weinheim, Germany : Wiley-VCH Verlag GmbH & Co. KGaA , 339 – 357 . ↵ Myers SS , Zanobetti A , Kloog I , Huybers P , Leakey ADB , Bloom AJ , Carlisle E , Dietterich LH , Fitzgerald G , Hasegawa T , Holbrook NM , Nelson RL , Ottman MJ , Raboy V , Sakai H , Sartor KA , Schwartz J , Seneweera S , Tausz M , Usui Y. 2014 . Increasing CO 2 threatens human nutrition . Nature 510 , 139 – 142 . OpenUrl CrossRef PubMed Web of Science ↵ Rachmilevitch S , Cousins AB , Bloom AJ . 2004 . Nitrate assimilation in plant shoots depends on photorespiration . Proceedings of the National Academy of Sciences of the United States of America 101 , 11506 – 11510 . OpenUrl Abstract / FREE Full Text ↵ Raverdy S , Zhang Y , Foster J , Carlow CKS . 2007 . Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases . Molecular and Biochemical Parasitology 156 , 210 – 216 . OpenUrl CrossRef PubMed ↵ Rubio-Asensio JS , Bloom AJ . 2017 . Inorganic nitrogen form: a major player in wheat and Arabidopsis responses to elevated CO 2 . Journal of Experimental Botany 68 , 2611 – 2625 . OpenUrl CrossRef ↵ Rubio-Asensio JS , Rachmilevitch S , Bloom AJ . 2015 . Responses of Arabidopsis and wheat to rising CO 2 depend on nitrogen source and nighttime CO 2 levels . Plant Physiology 168 , 156 – 163 . OpenUrl Abstract / FREE Full Text ↵ Saban JM , Chapman MA , Taylor G. 2019 . FACE facts hold for multiple generations; Evidence from natural CO 2 springs . Global Change Biology 25 , 1 – 11 . OpenUrl ↵ Saban JM , Watson-Lazowski A , Chapman MA , Taylor G. 2020 . The methylome is altered for plants in a high CO 2 world: Insights into the response of a wild plant population to multigenerational exposure to elevated atmospheric [CO 2 ] . Global Change Biology 26 , 6474 – 6492 . OpenUrl ↵ Sardans J , Rivas-Ubach A , Peñuelas J. 2012 . The C: N: P stoichiometry of organisms and ecosystems in a changing world: a review and perspectives . Perspectives in Plant Ecology, Evolution and Systematics 14 , 33 – 47 . OpenUrl ↵ Shi X , Hannon NM , Bloom AJ . 2024 . Metals and other ligands balance carbon fixation and photorespiration in chloroplasts . Physiologia Plantarum In review . ↵ Smart DR , Bloom AJ . 2001 . Wheat leaves emit nitrous oxide during nitrate assimilation . Proceedings of the National Academy of Sciences of the United States of America 98 , 7875 – 7878 . OpenUrl Abstract / FREE Full Text ↵ Smart DR , Ritchie K , Bloom AJ , Bugbee BB . 1998 . Nitrogen balances for wheat canopies (Triticum aestivum cv. Veery 10) grown under elevated and ambient CO 2 concentrations . Plant, Cell and Environment 21 , 753 – 764 . OpenUrl CrossRef PubMed ↵ Smith K , Strand DD , Kramer DM , Walker BJ . 2024 . The role of photorespiration in preventing feedback regulation via ATP synthase in Nicotiana tabacum . Plant, Cell & Environment 47 , 416 – 428 . OpenUrl ↵ South PF , Cavanagh AP , Liu HW , Ort DR . 2019 . Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field . Science 363 , eaat9077 . OpenUrl Abstract / FREE Full Text ↵ Taub DR , Miller B , Allen H. 2008 . Effects of elevated CO 2 on the protein concentration of food crops: a meta-analysis . Global Change Biology 14 , 565 – 575 . OpenUrl CrossRef Web of Science ↵ Tcherkez G , Ben Mariem S , Larraya L , García-Mina JM , Zamarreño AM , Paradela A , Cui J , Badeck F-W , Meza D , Rizza F , Bunce J , Han X , Tausz-Posch S , Cattivelli L , Fangmeier A , Aranjuelo I. 2020 . Elevated CO 2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat . Journal of Experimental Botany 71 , 5990 – 5600 . OpenUrl ↵ Wang J , Liu X , Zhang X , Li L , Lam SK , Pan G. 2019 . Changes in plant C, N and P ratios under elevated [CO 2 ] and canopy warming in a rice-winter wheat rotation system . Scientific Reports 9 , 1 – 9 . OpenUrl ↵ Watson-Lazowski A , Lin Y , Miglietta F , Edwards RJ , Chapman MA , Taylor G. 2016 . Plant adaptation or acclimation to rising CO 2 ? Insight from first multigenerational RNA-Seq transcriptome . Global Change Biology 22 , 3760 – 3773 . OpenUrl ↵ Wildner GF , Henkel J. 1979 . The effect of divalent metal ions on the activity of Mg ++ depleted ribulose-1, 5-bisphosphate oxygenase . Planta 146 , 223 – 228 . OpenUrl CrossRef Web of Science ↵ Wingfield P. 1998 . Protein precipitation using ammonium sulfate . Current protocols in protein science 13 , A. 3F. 1-A. 3F. 8. ↵ Zhang Y , Foster JM , Kumar S , Fougere M , Carlow CK . 2004 . Cofactor-independent phosphoglycerate mutase has an essential role in Caenorhabditis elegans and is conserved in parasitic nematodes . Journal of Biological Chemistry 279 , 37185 – 37190 . OpenUrl Abstract / FREE Full Text ↵ Ziska LH , Pettis JS , Edwards J , Hancock JE , Tomecek MB , Clark A , Dukes JS , Loladze I , Polley HW . 2016 . Rising atmospheric CO 2 is reducing the protein concentration of a floral pollen source essential for North American bees . Proceedings of the Royal Society B: Biological Sciences 283 , 20160414 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 17, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Rubisco from two ecotypes of Plantago lanceolata L. that are native to sites differing in atmospheric CO2 levels Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Rubisco from two ecotypes of Plantago lanceolata L. that are native to sites differing in atmospheric CO 2 levels Xiaoxiao Shi , Nathan M. Hannon , Arnold J. Bloom bioRxiv 2024.07.13.603406; doi: https://doi.org/10.1101/2024.07.13.603406 Share This Article: Copy Citation Tools Rubisco from two ecotypes of Plantago lanceolata L. that are native to sites differing in atmospheric CO 2 levels Xiaoxiao Shi , Nathan M. 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