RESEARCH ARTICLE SUMMARY◥

PLANT SCIENCE

Synthetic glycolate metabolismpathways stimulate crop growth andproductivity in the fieldPaul F. South, Amanda P. Cavanagh, Helen W. Liu, Donald R. Ort*

INTRODUCTION:Meeting food demands forthe growing global human population requiresimproving crop productivity, and large gainsare possible through enhancing photosyntheticefficiency. Photosynthesis requires the carbox-ylation of ribulose-1,5-bisphosphate (RuBP) byribulose-1,5-bisphosphate carboxylase-oxygenase(RuBisCO), but photorespiration occurs inmostplants such as soybean, rice, andwheat (knownas C3 crops) when RuBisCO oxygenates RuBPinstead, requiring costly processing of toxicbyproducts such as glycolate. Photorespirationcan reduce C3 crop photosynthetic efficiencyby 20 to 50%. Although various strategies existfor lowering the costs of photorespiration,chamber- and greenhouse-grown plants with

altered photorespiratory pathways within thechloroplast have shown promising results, in-cluding increased photosynthetic rates andplant size.

RATIONALE:To determine if alternative pho-torespiratory pathways could effectively im-prove C3 field crop productivity, we tested theperformance of three alternative photorespi-ratory pathways in field-grown tobacco. Onepathway used five genes from the Escherichiacoli glycolate oxidation pathway; a second path-way used glycolate oxidase and malate syn-thase fromplants and catalase from E. coli; andthe third pathway used plant malate synthaseand a green algal glycolate dehydrogenase. All

enzymes in the alternative pathway designswere directed to the chloroplast. RNA inter-ference (RNAi)was also used to down-regulatea native chloroplast glycolate transporter inthe photorespiratory pathway, thereby limitingmetabolite flux through the native pathway.The three pathways were introduced with

and without the trans-porter RNAi constructinto tobacco, which is anideal model field crop be-cause it is easily trans-formed, has a short lifecycle, produces large quan-

tities of seed, and develops a robust canopysimilar to that of other field crops.

RESULTS: Using a synthetic biology approachto vary promoter gene combinations, we gen-erated a total of 17 construct designs of thethree pathways with and without the trans-porter RNAi construct. Initial screens for pho-toprotection by alternative pathway functionunder high–photorespiratory stress conditionsidentified three to five independent transfor-mants of each design for further analysis. Geneand protein expression analyses confirmed ex-pression of the introduced genes and suppres-sion of the native transporter in RNAi plants.In greenhouse screens, pathway 1 increasedbiomass by nearly 13%. Pathway 2 showed nobenefit compared to wild type. Introduction ofpathway 3 increased biomass by 18% withoutRNAi and 24% with RNAi, which were con-sistent with changes in photorespiratory me-tabolism and higher photosynthetic rates.Ultimately, field testing across two differentgrowing seasons showed significant increasesin biomass of pathway 3 plants with RNAicompared toWT of 20% in 2016 (P = 0.04) andby 24% in 2017 (P = 0.018). In addition, thispathway increased the light-use efficiency ofphotosynthesis by 17% in the field.

CONCLUSION: Engineering more efficientphotorespiratory pathways into tobacco whileinhibiting the native pathway markedly in-creased both photosynthetic efficiency andvegetative biomass. We are optimistic that sim-ilar gains may be achieved and translated intoincreased yield in C3 grain crops because pho-torespiration is common to all C3 plants andhigher photosynthetic rates under elevated CO2,which suppresses photorespiration and in-creases harvestable yield in C3 crops.▪

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South et al., Science 363, 45 (2019) 4 January 2019 1 of 1

The list of author affiliations is available in the full article online.*Corresponding author. Email: d-ort@illinois.eduThis is an open-access article distributed under theterms of the Creative Commons Attribution license(https://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.Cite this article as P. F. South et al., Science 363, eaat9077(2019). DOI: 10.1126/science.aat9077

Alternative photorespiratory pathways in tobacco. Three alternative pathways [1 (red),2 (dark blue), and 3(light blue)] introduced into tobacco chloroplasts for more efficient recyclingof glycolate. RNAi suppresses the native glycolate/glycerate transporter PLGG1 to prevent glycolatefrom leaving the chloroplast and entering the native pathway (gray).

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RESEARCH ARTICLE◥

PLANT SCIENCE

Synthetic glycolate metabolismpathways stimulate crop growth andproductivity in the fieldPaul F. South1,2, Amanda P. Cavanagh2, Helen W. Liu3*, Donald R. Ort1,2,3,4†

Photorespiration is required in C3 plants to metabolize toxic glycolate formed whenribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylatesribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration canreduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed intotobacco chloroplasts synthetic glycolate metabolic pathways that are thought to bemore efficient than the native pathway. Flux through the synthetic pathways wasmaximized by inhibiting glycolate export from the chloroplast. The synthetic pathwaystested improved photosynthetic quantum yield by 20%. Numerous homozygoustransgenic lines increased biomass productivity between 19 and 37% in replicated fieldtrials. These results show that engineering alternative glycolate metabolic pathways intocrop chloroplasts while inhibiting glycolate export into the native pathway can driveincreases in C3 crop yield under agricultural field conditions.

Population growth, increasing global afflu-ence, and an expanding bioeconomy areconspiring to increase mid-century agri-cultural demand by 60 to 120% over 2005levels, a challenge that current rates of crop

productivity improvement averaging <2% peryear cannot meet (1–3). In the 45 years after1960, global crop productivity increased 135%from 1.84 to 3.96 metric tons per hectare (4).The increased use of pesticides, fertilizers andirrigation, and mechanization, along with theadoption of higher-yielding crop varieties thatdrove this remarkable global increase in produc-tivity, are now largely optimized for major cropsand are unlikely to generate sufficient yield in-creases tomeetmid-century agricultural demand.However, photosynthetic efficiency remains stand-ing as a determinant of yield potential with theimprovement capacity to double crop producti-vity (1–3, 5, 6). In C3 crops such as wheat, rice,and soybeans, photorespiration reduces the pho-tosynthetic conversion efficiency of light intobiomass by 20 to 50%, with the largest lossesoccurring in hot dry climates where yield in-creases are sorely needed. Whereas ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO)carboxylates ribulose-1,5-bisphosphate (RuBP) dur-ing photosynthesis, the unproductive and energy-intensive process of photorespiration results from

oxygenation of RuBP byRuBisCO,which becomesmore prevalent at higher temperatures and underdrought conditions (6, 7). Toxic by-products of theRuBisCO oxygenation reaction (2-phosphoglycolateand glycolate) and of the glycine decarboxylationreaction (ammonia) are recycled by photorespi-ration into nontoxic products but at the expenseof energy and net loss of fixed carbon (6, 7). Somephotosynthetic algae, bacteria, and plants haveevolvedmechanisms to reduce the oxygenationreaction by RuBisCO via carbon-concentratingmechanisms (CCMs), including C4 photosynthesis(8, 9), inspiring efforts to introduce CCMs into C3plants (8–12). Here we have taken an alterna-tive approach of introducing non-native andsynthetic metabolic pathways to recycle the pro-ducts of RuBisCO oxygenation more efficiently(13). Previously, two alternative photorespiratorypathways implemented inArabidopsis improvedphotosynthesis and plant size in chamber andgreenhouse experiments (14, 15). These resultsinspired us to optimize these alternative photo-respiratory pathways in tobacco, a useful agri-cultural model crop, for field trials. Computermodeling of these alternative pathways revealedthe importance of optimized expression of non-native genes to achieve maximum flux throughthe alternative pathway and thus maximize thebenefits for crop plants under field conditions(16). Additionally, we sought to minimize fluxthrough the native photorespiratory pathway andmaximize flux through the introduced pathwaysby inhibiting glycolate export from the chloroplast.

ResultsTransgene assembly

We transformed Nicotiana tabacum cv. PetiteHavana (tobacco) with three different photores-

piratory alternative pathway (AP) designs, ex-pressing as many as seven genes in single con-structs (Fig. 1A and table S1). Tobacco is anideal model crop for these studies because of itscompletely sequenced genome, short life cycle(3 months from seed to seed), well establishedhigh-efficiency transformation protocols, and theability to form a fully closed canopy like othercrops in the field. The AP1 construct targets thefive genes of the Escherichia coli glycolate oxi-dation pathway to the chloroplast (Fig. 1A) (14).AP2 includes Arabidopsis glycolate oxidase (GO)and Cucurbita maxima (pumpkin) malate syn-thase (MS), along with a catalase (CAT) fromE. coli (Fig. 1A) (15). AP3 also contains C.maximaMS sequence but replaces the plant GO used inAP2 with Chlamydomonas reinhardtii glycolatedehydrogenase (CrGDH) to avoid hydrogen per-oxide production when glycolate is convertedto glyoxylate (Fig. 1A). With this modification,expression of E. coli CAT in the chloroplast isunnecessary (17). Using multigene constructsassembled from modular parts by Golden Gatecloning, we generated multiple promoter genecombinations and within-construct position ef-fects to optimize AP performance. We generatedfive iterations of AP1, three iterations of AP2, anda single design of AP3 for testing (table S1). Inaddition to the expression of the AP genes, wedesigned a long hairpin RNA interference (RNAi)construct and added it to the library of multigeneconstructs to reduce the expression of the chloro-plast glycolate-glycerate transporter PLGG1 withthe goal of minimizing glycolate flux out of thechloroplast and into the native pathway (Fig. 1 andtable S1) (18, 19). In total, we successfully trans-formed 17 different constructs of the three APdesigns into tobacco with and without the in-clusion of an RNAi module targeting the PLGG1transporter.

Gene and protein analysisconfirm chloroplast-localizedtransgene expression

Transgene expression analysis conducted on threeindependent transformants of each AP designselected for further analysis confirmed strongexpression of the transgenes along with ~80%RNAi suppression of PLGG1 expression (Fig. 1Band fig. S1). Immunoblot analysis of whole-cellextract was normalized on the basis of total pro-tein content and verified using antibodies againstthe RuBisCO large subunit and actin (fig. S2).Immunoblot analysis of isolated intact chlo-roplasts from AP3 plants (Fig. 1C) verified thatthe construct design of AP3 directs CrGDH andMS protein to the chloroplast and that RNAisuppresses expression of the PLGG1 transporterprotein. The cytoplasmic marker protein actinwas undetectable in the isolated chloroplast frac-tion, ensuring that the AP3 proteins in the chloro-plast fraction was not a result of cytoplasmiccontamination (Fig. 1C). Moreover, the chloro-plast marker PGL35 was only faintly detectablein the whole-leaf extracts but was greatly en-riched in the isolated chloroplast fraction (Fig.1C). WhereasMSwas also greatly enriched in the

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1Global Change and Photosynthesis Research Unit, UnitedStates Department of Agriculture–Agricultural ResearchService, Urbana, IL 61801, USA. 2Carl R. Woese Institute forGenomic Biology, University of Illinois, Urbana, IL 61801, USA.3Department of Crop Sciences, University of Illinois, Urbana, IL61801, USA. 4Department of Plant Biology, University of Illinois,Urbana, IL 61801, USA.*Present address: Department of Plant and Microbial Biology,University of California, Berkeley, CA 94720, USA.†Corresponding author. Email: d-ort@illinois.edu

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chloroplast fraction, CrGDH appeared to bemuchless enriched in this fraction (Fig. 1C). Glycolatedehydrogenases have been shown to be stronglyassociated with membranes in both chlorophytesand bacteria (20, 21) and thus may have been inef-ficiently extracted from our chloroplast prepara-tion (17). Isolation of the insoluble membranefraction from the chloroplast extraction showedthat a large fraction CrGDH in tobacco chloro-plasts was associated with the membranes (Fig.

1C) and that CrGDH was enriched relative toPGL35 in the membrane fraction.

AP plants are resistant tophotorespiration stress

Following selection for construct expression byselectable marker screening [BASTA resistance(bar) gene added to all constructs] (table S1) andgenotyping selection for single-insert homozygoustransgenic plants, all independent constructs of

the three AP designs were assessed for resistanceto photorespiration stress in a high-throughputchlorophyll fluorescence assay. Photorespiratorymutants typically display impaired growth andphotosynthesis when transferred from elevatedCO2 concentrations ([CO2]) to ambient air, whichis accompanied by the onset of photoinhibitionthat can be diagnosed by monitoring chlorophyllfluorescence (19, 22–24). We hypothesized thatAP function would be photoprotective under highphotorespiratory stress, thus protecting photo-system II operating efficiency (i.e., Fv′/Fm′) fromphotodamage (19, 22). Previously, this methodof monitoring Fv′/Fm′ after illumination in low[CO2] enabled identification of photorespirationmutants that cause photoinhibition (19, 22, 24).Using this protocol to monitor AP function,we exposed thousands of single-insert homozy-gous T2 seedling plants to 24 hours of high lightintensity (1200 mmol m−2 s−1) and very low [CO2](1 to 38 mbar CO2) and then compared Fv′/Fm′ inthe transformants with azygous wild-type (WT)and empty vector (EV) controls (fig. S3). Manyindependent transformants (66% of AP1, 54%of AP2, and 84% of AP3 plants) were significantlymore photoprotective under this severe photo-respiratory stress. Versions of AP1 and AP3sustained 33 to 48% higher Fv′/Fm′ values com-pared toWT and EV controls (Fig. 2, A and B, anddata set S1). Under ambient [CO2], there were noobserved differences in Fv′/Fm′ between the APand control lines. However, PLGG1 RNAi inhibi-tion of glycolate efflux from the chloroplast reducedFv′/Fm′ when these plants were shifted from ele-vated [CO2] to ambient (fig. S4). This photo-inhibited phenotype of the PLGG1 RNAi plantswas not only rescued by transgenic complemen-tation with AP1 or AP3 constructs, but was alsosubstantially more resistant to photoinhibitionthanWTandEV controls (Fig. 2B and dataset S1).

AP plants show enhanced biomassaccumulation in greenhousegrowth studies

Following the initial photoprotection screen andexpression analysis, we determined the impactof the three APs on plant growth in greenhousegrowth studies. Both the AP1 and AP3 designssignificantly increased dry-weight biomass rela-tive to the WT plants. Overall, AP1 plants in-creased dry weight biomass by 13%, but thebenefit was lost when the PLGG1 RNAi modulewas present (Fig. 3B). AP2 introduction did notsignificantly alter dry weight (Fig. 3B). ThreeAP3 lines that sustained much higher Fv′/Fm′values (200-8,9,10) compared to WT and EVwere taller (Fig. 3A) and showed the largestincreases in biomass in greenhouse studies,with a 24% increase with and 18% increasewithout the PLGG1 RNAi module compared toWT (Fig. 3B). We also tested an AP3 line thathad the same Fv′/Fm′ as WT and EV (200-4),which showed no increase in biomass, and oneline that had an intermediate Fv′/Fm′ (200-6)that showed a small but statistically significantbiomass increase in greenhouse studies (fig. S5,A and C). Transcript expression analysis of AP3

South et al., Science 363, eaat9077 (2019) 4 January 2019 2 of 9

Fig. 1. Alternative photorespiratory pathways. (A) Model of three alternative photorespirationpathway designs. AP1 (red) converts glycolate to glycerate using five genes from the E. coli glycolatepathway encoding the enzymes glycolate dehydrogenase, glyoxylate carboligase, and tartronic semi-aldehyde reductase. AP2 (dark blue) requires three introduced genes encoding glycolate oxidase, malatesynthase, and catalase (to remove hydrogen peroxide generated by glycolate oxidase). AP3 (blue) relieson two introduced genes: Chlamydomonas reinhardtii glycolate dehydrogenase and Cucurbita maximamalate synthase. (B) qRT-PCR analysis of the two transgenes in AP3 and the target gene PLGG1 of theRNAi construct. Results for three independent transformation events are shown with (1, 5, and 8) andwithout (8, 9, and 10) PLGG1 RNAi. Error bars indicate SEM. * indicates statistical difference at P < 0.05compared to WT based on one-way ANOVA. Actual P values are shown in supplementary data set 15.(C) Immunoblot analysis from whole leaves and isolated chloroplasts, including the insoluble membranefraction, using custom antibodies raised against the indicated target genes, cytosolic marker actin, andchloroplast-specific marker platoglobulin 35 (PGL35). Five micrograms of protein was loaded per lane.Arrows indicate detected protein based on molecular weight.The kinetic properties of CrGDH, as well asnumerous malate synthase enzymes, have been previously characterized (table S3) (17).

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events 200-4 and 200-6 revealed that CrGDH andMS expression was greatly reduced comparedto transgenic events 200-8,9,10 (fig. S5B).

AP3 plants have an alteredphotorespiratory metabolite profile

We further investigated theAP3plants that showedthe greatest growth stimulation and gene expres-sion to determine the effect of AP3 enzymes onthe leaf photorespiratory metabolite profile. Weperformed gas chromatography followed by massspectrometry on leaf samples from greenhouse-grown WT and AP3 plants to analyze the pho-torespiratory intermediates glycolate, glyoxylate,glycine, and serine and the AP3-specific inter-mediate pyruvate (Fig. 4). AP3 introductionwith and without the RNAi module increasedglyoxylate and pyruvate concentration comparedto WT, suggesting altered native photorespira-tion and possibly flux through the alternativepathway (Fig. 4, B and F). AP3 plants with andwithout the RNAi module also had decreasedconcentrations of the photorespiratory inter-mediates serine, for which photorespirationis amajor source (25), and glycerate, possibly dueto a diversion of carbon away from the nativephotorespiratory pathway (Fig. 4, D and E).Glycine concentrations were similar in AP3 andWT plants (Fig. 4C). AP3 with the RNAi moduletargeting the glycolate-glycerate transporter PLGG1had increased glycolate accumulation compared toWT in a manner similar to the Arabidopsis T-DNAinsertion mutant plgg1-1 (Fig. 4A) (18, 19).

AP3 plants exhibit increasedphotosynthetic rate andchloroplast [CO2]

To test if altered photorespiration due to intro-duction of the AP3 design in plants affects ratesof photosynthesis, we compared CO2 assimila-tion rates (A) as a function of intercellular CO2

concentrations (Ci) under saturating light in AP3andWT plants. AP3 plant lines with andwithoutthe PLGG1 RNAi module had increased rates ofphotosynthesis compared toWT (Fig. 5A). Model-ing of the A/Ci curves showed increases in themaximum rate of RuBisCO carboxylation (Vcmax)visualized in the initial slope of the A/Ci curve inAP3 lines (Fig. 5, A and C). We observed no sta-tistical differences in the maximum rate of elec-

tron transport (Jmax) in any AP design (fig. S6).Increases inVcmax, which is a property of RuBisCOenzymatic activity, could be due to increasedRuBisCO protein content or increased availabil-ity of CO2 as a substrate for RuBisCO. Immuno-blot analysis shows no difference in RuBisCOcontent on a permicrogramprotein basis (fig. S2),suggesting that the observed difference is basedon increased availability of CO2 at the site of car-boxylation in the chloroplast. Increases in CO2

availability for RuBisCO carboxylation could arise

from increasedmesophyll conductance (gm; i.e., thediffusion of CO2 intomesophyll cell chloroplasts)or from the direct release of photorespiratoryCO2 in the chloroplast by the decarboxylation ofmalate and pyruvate in the plastid (Fig. 1A), bothof which would result in observed increase inVcmax determined from A/Ci curves (26). Thereis no apparent reason to expect that the intro-duction of these alternative pathways would de-crease the resistance for the movement of CO2

from the mesophyll intercellular air space to the

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Fig. 2. AP plant lines are more photoprotective under photo-respiration stress. (A) Representative photos of 9-day-old T2transgenic tobacco lines during the chlorophyll fluorescencephotoprotection screen for AP pathway function showing AP3protecting photosystem II from photodamage under severephotorespiratory conditions. (B) Combined values of the threeAP construct designs with and without RNAi targeting theglycolate-glycerate transporter PLGG1. Error bars indicate SEM.* indicates statistical difference compared to WT based onone-way ANOVA at P ≤ 0.05, ** P ≤ 0.001. Fvʹ/Fmʹ forindividual lines is described in supplementary data set 1.Actual significant P values are shown in supplementarydata set 15.

Fig. 3. Photorespiration AP lines increase biomass under greenhouse conditions. (A) Photos of6-week-old AP3 and WT plants grown in the greenhouse. Individual plant lines are indicated in thelabels below the plant. (B) Percent difference in total dry weight biomass of the indicated combinedplant lines. * indicate statistical difference based on one-way ANOVA. Error bars are SEM, n = 7(plants measured), P < 0.05 values listed in data set 15.

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chloroplast stroma or from the mitochondria tothe chloroplast stroma. However, an increase ingm contributing to increased CO2 availabilitywithin the AP3 plant chloroplast is difficult torule out, largely because modeling of gm requiresknowledge of, or assumptions about, the con-ductance of CO2 released from the mitochondriaduring the conversion of glycine to serine to thechloroplast, which is directly affected by the in-troduction of the alternative pathway.Theory predicts that the release of photores-

piratory CO2 in the chloroplast by the AP path-ways, instead of in themitochondria through thenative photorespiratory pathway, would lowerCi*, the intercellular [CO2] at which the chloro-plast [CO2] reaches G*, the [CO2] in the chloro-plast at which the rates of RuBisCO oxygenationand carboxylation are equal (27–29). To deter-mine Ci*, we measured the internal [CO2] atwhich CO2 response curves measured at differ-ent subsaturating illumination intensities intersect(29). AP3 plants with the RNAi module targetingPLGG1 showed a significant reduction of 10% inCi* and AP3 plants without the RNAi moduleshowed a significant reduction of 6.4% in Ci*compared toWT (Fig. 5B). The observeddecreasesin Ci*, coupled with unaltered respiration (fig. S7),are consistent with elevated chloroplastic [CO2]due to decarboxylation of malate and pyruvatewithin the introduced pathway (Figs. 1A and 5Band fig. S6), which would also explain the signifi-cantly higher values of Vcmax in AP3 plants com-pared to WT plants (Fig. 5, A and C, and fig. S6).Accounting for the observed Ci* in A/Ci curveanalysis reduces the apparent change in Vcmax

further indicating that the difference in Vcmax

was not due to changes in RuBisCO content oractivity but rather by increased chloroplastic[CO2] (Fig. 5C).

AP plants show increasedphotosynthetic rates, quantumefficiency, and biomass accumulation inreplicated field trialsIn the 2016 growing season, we tested four in-dependent events of AP1, two independent eventsof AP2, and five independent transformationevents of AP3, along with two WT and two EVcontrols in the field, using a randomized singleblock design experiment (fig. S8). Biomass in-creased by 16% in AP1 lines and 10% in one ofthe AP2 lines tested (fig. S9). The three AP3 linesthat showed the largest biomass increases inthe greenhouse consistently showed the largestincreases in dry-weight biomass, with total bio-mass increasing by as much as 23% relative toWT (fig. S9). Independent AP3 events 200-4and 200-6, in which CrGDH and MS expressionwas significantly lower compared to other trans-genic events (fig. S5B) and showed less or noimprovement in greenhouse biomass (fig. S5C),also showed no increases in total biomass in the2016 field season (fig. S9). We anticipated, owingto their lower energetic requirements, that the APpathways would improve themaximumquantumefficiency of net CO2 assimilation (Fa) relative tothe native pathway.Fa increased in lines of all APpathways, in many cases by >20%, includingthose containing the RNAi module targeting thePLGG1 transporter (fig. S10) but not in AP3 events200-4 and 200-6 (fig. S11A). The high–biomass-producing AP3 plant lines exhibited an increasedlight-saturated rate of assimilation compared toWT, to several AP1 lines, and to all AP2 plantlines (fig. S10C) and to AP3 events 200-4 and200-6 (fig. S11).To validate the 2016 field results and improve

the statistical power of comparisons with AP3plants under agricultural conditions, we tested

five randomized replicate blocks of three AP3 in-dependent transformed lines with and withouttheRNAimodule targetingPLGG1 in comparisonto WT during the 2017 growing season (fig. S12).The AP3 plant lines showed a 17% difference in2016, but in 2017 the 10% increase in dry-weightbiomass (4% leaf, 24% stem) was not signifi-cant. The inclusion of the PLGG1 RNAi modulein AP3 designs further increased leaf dry biomassto 22%, stem dry biomass to 28%, and total drybiomass to 24% compared to WT (Fig. 6A). ThatAP3 plant lines with the RNAi module showeda significant leaf and total dry weight biomassincrease (17% and 13%, respectively) over theAP3-only plants supports our hypothesis thatforcing greater glycolate flux through the syn-thetic pathway by inhibiting flux through thenative photorespiratory pathway drove the in-creased productivity. Total mid-day starch con-tent in AP3 plants increased by ~70% and in AP3with PLGG1 RNAi by ~40% compared to the

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Fig. 4. Photorespiratory and AP3metabolic intermediates. (A to F) Relative amount of the indicatedmetabolite detected from ~40 mg of leaf tissue (fresh weight; FW) sampled in the late morning.Metabolite concentrations were reported as concentrations relative to the internal standard, which isthe target compound peak area divided by peak area of hentriacontanoic acid: Ni (relative concentration) =Xi (target compound peak area) * X−1IS (peak area of hentriacontanoic acid) per gram freshweight. Error bars indicate SEM, n = 4 leaf samples. Statistical differences between AP3 designsand WT based on one-way ANOVA, with P values indicated. All P values are listed in dataset 15.

Fig. 5. Photosynthetic efficiency ofgreenhouse-grown plants. Data are thecombined result of three independent trans-formants (hereafter referred to as combined)with and without PLGG1 RNAi. (A) CO2 assim-ilation based on intercellular [CO2] (Ci).(B) Combined apparent CO2 compensationpoint: Ci* calculated using the common inter-cept method and slope regression (29).(C) Combined maximum rate of RuBisCOcarboxylation (Vcmax). Vcmax values arepresented at 25°C and modeled from photo-synthetic response under changing CO2 con-centration. Gray bars indicate constant Ci*;green bars indicate derived values based onmeasured Ci*. Error bars indicate SEM. P valuesfor statistical comparison to WT based onone-way ANOVA are given.

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WT control (Fig. 6B). The apparent quantumefficiency of photosynthesis increased in bothAP3plant pathways; by 7% with and 17% withoutPLGG1 RNAi for the 2017 field season (Fig. 6Cand fig. S13). Because plants with both AP3 de-signs exhibited increases in the quantum efficien-cy of photosynthesis and decreases in Ci*, wehypothesized that total daily net carbon gainthrough photosynthesis would be higher com-pared to WT, resulting in the observed increasesin biomass over the growing season (Figs. 5B and6, A and C). Indeed, modeled daily net carbongain from measurements of photosynthesis overa diurnal time course in plants containing AP3showed an increase of 5 to 8% in CO2 assimila-tion (A′ ) and increases in electron use in photo-synthesis (J ′) compared to WT (Fig. 6, D and E).

Discussion

We showed that installing synthetic glycolatemetabolic pathways into tobacco chloroplastsdrove large increases in biomass accumulation inboth greenhouse conditions and in the field un-der agricultural conditions. Because AP3 plantsexhibited the greatest growth stimulation, weselected this pathway for more in-depth char-acterization. In summary, the AP3 transgene pro-ducts CrGDH and MS localized to the chloroplast(Fig. 1C). Evidence that these transgene productsfunction in the chloroplast to catalyze the reac-tions depicted in Fig. 1 include the stimulation ofthe rate of photosynthesis (Fig. 5) and improve-

ment of photosynthetic quantum yield (Fig. 6 andfig. S10), the lowering of Ci* (Fig. 5) and increasein the initial slope of an A/Ci relationship (Fig. 5)that both indicate increased [CO2] in the chloro-plast, and the altered photorespiratory metab-olite profile (Fig. 4). In addition, the direct roleof this pathway in chloroplast glycolate metabo-lism is supported by its ability to prevent photo-inhibitionandrescue the reducedgrowthphenotypeof PLGG1 RNAi tobacco lines (fig. S4). Moreover,AP3 lines that contain the full transgene constructbut with reduced transgene expression showedless, or no improvement in greenhouse (fig. S5C)or field biomass (fig. S9), and Fa values similartoWT (fig. S11) provide evidence that the amountof expression of the introduced alternative photo-respiratory pathway drove the extent of im-proved growth and increased photosyntheticefficiency.Of the two alternative pathways to photores-

piration that inspired our designs (13, 14), AP2showed limited improvements in plant produc-tivity, and 24% of the independent transgenicAP2 lines resulted in stunted growth and yellowleaves (fig. S14C). The AP1 design improved pro-ductivity in tobacco, but the enhancement asso-ciatedwithAP1was eliminated in both greenhouseand field settings when the PLGG1 RNAi modulewas added (Fig. 3 and fig. S9). Modeling (16)predicted that directing the complete flux of gly-colate through the AP1 pathway by inhibitingglycolate export from the chloroplast would re-

sult in the largest increase in energy savings,photosynthetic efficiency, and growth among alldesigns. Elimination of the AP1 enhancementsby the PLGG1 RNAi module implies that thisintroduced pathway may not have had sufficientkinetic capacity to handle the full glycolate fluxunder high rates of RuBisCO oxygenation. Fur-ther optimization of expression of AP1 genesand/or use of AP1 genes of different origins andkinetic properties may lead to achieving the fullbenefits that modeling predicts for this design.The AP3 design containing C. maxima MS andCrGDH reliably increased plant biomass and im-proved photosynthetic efficiency (Figs. 3, 5, and 6),and the phenotype is dependent on the level ofexpression of the transgenes in the independenttransformation events (fig. S5). The inclusion ofan RNAi module to reduce expression of thePLGG1 chloroplast glycolate-glycerate transporterin numerous independent transformant plantlines increased postharvest dry-weight biomasscompared to AP3 introduction alone by 13% (P =0.0805) (Figs. 3 and 6A and fig. S13). Without analternative photorespiration pathway in place,inhibition of PLGG1 expression by RNAi decreasedplant growth and led to photoinhibition (i.e., re-duced Fv′/Fm′) when these plants were transfer-red from elevated [CO2] to ambient air (fig. S4),as was reported previously for the plgg1-1 T-DNAknockout line inArabidopsis (23). Thus, the genet-ic complementation of the low-growth photo-inhibited phenotype and the significant increasein biomass in AP3 lines with RNAi over AP3 aloneare consistentwith the expected benefit of direct-ing a greater proportion of the glycolate fluxthrough the synthetic pathway in the chloro-plast and away from the native photorespiratorypathway outside of the chloroplast (18, 19). In-deed, forcing greater glycolate flux through thesynthetic pathway by inhibiting glycolate trans-port out of the chloroplast through PLGG1 intothe native photorespiratory pathway resulted ingrowth stimulation in field experiments between19 and 37% (with an average effect size of 24%,P = 0.028) for the AP3 plants with RNAi. Theglycolate-glycerate exchange transporter PLGG1works in tandemwith a second glycolate exporterBASS6 to stoichiometrically balance the exportof two glycolate molecules with the import ofone glycerate molecule during photorespiration(18). Thus, targeting the expression of both trans-portersmay further test AP3 kinetic capacity andmay drive even greater growth stimulation. Rec-ognizing that these alternative pathways areintervening in the central metabolism of photo-synthetic cells, it will be important to validatethe biochemistry that is occurring as AP pathwayintermediates may well have destinations thatare different from those depicted in Fig. 1A.Although inhibiting photorespiration under

normal oxygen-containing atmospheres invaria-bly results in inhibited photosynthesis and growth(7), some evidence indicates that stimulatingphotorespiratory flux can enhance photosyn-thetic rate and plant growth. Overexpression ofthe H-protein in the glycine decarboxylase com-plex or overexpression of plant glycolate oxidase

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Fig. 6. Plant productivity and photosynthetic performance in 2017 field trials. (A) Percentdifference from WT for stem, leaf, and total biomass of AP3 with and without the PLGG1 RNAi module.Data are the combined result of three independent transformants with and without PLGG1 RNAi.(B) Total combined accumulated leaf starch for indicated lines extracted from 10 mg of fresh weightleaf material. (C) Combined apparent quantum efficiency of photosynthesis (Fa) determined bylinear regression of assimilation based on available light-response curves. (D) Combined accumulatedassimilation of CO2 (A′) based on diurnal analysis of photosynthesis. (E) Combined accumulatedelectrons used in electron transport determined from assimilation based on diurnal photosynthesis.Error bars indicate SD, and P values are indicated based on two-way ANOVA.

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(GO) can lead to increased photosynthesis andbiomass production (30, 31). In both of these re-ports, the overexpression of these photorespira-tion genes was accompanied by an increase instomatal conductance that itself would be ex-pected to increase photosynthesis and growthunder water-replete conditions. Conversely, fourdifferent photorespiration mutants (pglp1, shm1,hpr1, and glyk1) partially lost stomatal respon-siveness to altered CO2 availability, possibly in-dicating that alternative pathways could influenceplant adaptation through stomatal signaling (32).We saw no statistical differences in stomatal con-ductance (fig. S15, A and B) or the expression ofGO (fig. S15C) in AP3 tobacco plants, indicatingthat neither of these contributed to the stimula-tions observed in AP3 plant lines. Whether theinstallation of these alternative pathways mayaffect global changes in the transcriptome andthe proteome and if that may have secondaryimpacts on plant growth outside of changes toprimary metabolism remain to be determined.Energy demand calculations suggest that AP3would consumemore adenosine 5´-triphosphatethan native photorespiration, similar to AP2 (33).It is likely that CrGDH uses the electron trans-port chain as an electron acceptor (17), and thedecarboxylation of malate and pyruvate generatereducing equivalents (Fig. 1). However, the globaleffect of AP3 and PLGG1 repression on energybalance, as well as the possible fate of inter-mediates from AP3 in sucrose synthesis or thetricarboxylic acid cycle, will need to be assessed(17, 33).Tobacco was selected for these proof-of-concept

experiments not only for its ease of genetic trans-formation but also because it is an ideal modelcrop that is robust in the field, forms a fully closedcanopy, and produces large quantities of seed,circumventing the need for numerous seed am-plification generations, further accelerating thetimeline to field testing. The photorespiratorymechanism is common to all C3 plants, althoughenergetic costs and yield reductions depend onspecies-specific kinetic properties of RuBisCO, aswell as the temperature and [CO2] under whichthe crop is growing. Previous work has demon-strated that alternative photorespiration pathwaysshow a benefit to crop plants Camelina sativa (34)and potato (35) in greenhouse and chamber ex-periments, but it remains to be seen whether theincrease in vegetative biomass that we observedin tobaccowith AP3 in the field can be translatedinto increased seed or tuber production in cropssuch as soybean, cowpea, and potato. In green-house studies, only one AP3 line containing thePLGG1 RNAi module showed a significant in-crease in total seed weight (fig. S13E), but seedis not a major sink in tobacco as it is in graincrops. However, because increased photosynthe-tic efficiency due to the suppression of photo-respiration in C3 crops grown in elevated [CO2]results in increased seed yield (5, 36), we are op-timistic that use of alternative metabolic path-ways to photorespirationwill also lead to increasesin seed yield. Indeed, in this work, the observedstimulation of whole-plant biomass production

was much larger than the stimulation of photo-synthesis on a leaf area basis (5 to 8% increase inCO2 assimilation resulting in 10 to 24% increasein dry-weight biomass; compare Fig. 6Awith Fig.6, D and E), showing the benefit of compoundinterest from creating greater leaf area earlier inthe growth cycle.

Materials and MethodsPlant genetic transformation

Nicotiana tabacum cv. Petite Havana was genet-ically transformed using Agrobacterium tume-faciens strain C58C1-mediated transformation(37). The 17 binary plasmids used in this studywere assembled as described and listed in tableS1 (19). AP1 genes originated from E. coli (14).AP2 genes originated from Arabidopsis thaliana(glycolate oxidase) andCucurbitamaxima (malatesynthase) andE. coli (catalase) sources as described(15, 38). AP3 genes originated from Chlamydomo-nas reinhardtii for glycolate dehydrogenase and asdescribed for AP2 for C. maxima malate synthase(15, 17). Targeting to the chloroplast was designedby the addition of either theArabidopsisRuBisCOsmall subunit (RbcS) or phosphoglucomutasetransit peptide sequence added to the N terminusof the gene constructs. The RNAi module thattargets the plastidic glycolate-glycerate trans-porter PLGG1 was designed using 300 base pairsof exon sequence derived from the Sol genomicsnetwork (https://solgenomics.net). All binaryplasmids contained the BASTA resistance (bar)gene as a selectable marker for plant transfor-mation. A minimum of 10 independent T0 trans-formationswere generated to produce T1 progeny.T-DNA copy number was determined on T1 plantsthrough quantitative reverse transcription–quantitative polymerase chain reaction (qRT-PCR)analysis (iDNA Genetics, Norwich UK) (dataset17) (39). From these results, a minimum of fiveindependent transformation events were selec-ted to self and produce T2 progeny. Copy-numberanalysiswas repeated to verify single insert homo-zygous lines for each transformation event. Non-single insert lines were not further characterized(for a representative timeline of characterizationof AP3 lines see dataset 20). All WT controls usedin this study were azygous plants, which havebeen through the transformation protocol butlost the construct including the selectable markerresistance during segregation.

Chlorophyll fluorescence measurements

Tobacco T2 seeds were germinated under am-bient air conditions on Murashige and Skoog(MS) plates with essential vitamins in a con-trolled environment chamber (EnvironmentalGrowth Chambers, Chagrin Falls, Ohio, USA)with 14 hours day (25°C)/10 hours night (22°C)and light intensity of 500 mmol m−2 s −1. Eightdays after germination, seedling plates weretransferred to a custom assembled low-[CO2]chamber inside the controlled environment growthchamber (fig. S1). The light levels were increasedto 1200 mmolm−2 s −1 for 24 hours and [CO2] wasmaintained below 38 mbar (fig. S1). For PLGG1RNAi-only plants, which have strongly depressed

photorespiratory capacity, T1 lines were germi-nated on soil under elevated [CO2] conditionsfor 9 days and transferred to ambient air for 3 daysprior to screening. Fv′/Fm′ was determined oneach plate using the CF Imager Technologica(http://www.technologica.co.uk/). Maximum flashintensitywas 6800 mmolm−2 s−1 for 800ms. Imagevalues were obtained for each individual plant bydetecting colonies within the fluorimager soft-ware programdefining each position as described(19, 22, 40).

Gene expression and protein detection

Plants were grown under greenhouse or fieldconditions as described below. Five leaf discswere harvested from three plants per line (2.9 cm2,~100 mg). RNA and protein were extracted fromthe same leaf samples using theNucleoSpinRNA/Protein kit (Macherey-Nagel GmbH & Co.KG,Düren, Germany). cDNA was generated fromextracted RNAusing theQuantinova reverse tran-scriptase kit (QIAGEN,USA). Aminimumof threebiological replicates, including three technicalreplicates each, were performed for all samples.Gene expression was analyzed using a Bio-RadCFX connect real-time PCR system (Bio-Rad Lab-oratories, USA). Relative changes in transcriptlevels were determined using the DDCt methodwith primers directed toward the transgenetranscripts and the L25 gene as a standard con-trol gene (41). cDNA was amplified using a SSOadvanced SYBR greenmaster mix (Bio-Rad), andprimer sequences are described in table S2.Total protein fromAP3was extracted using the

Nucleospin protein/RNA kit described above orfrom frozen leaf material ground in liquid nitro-gen, resuspended in lysis buffer [50 mM HEPES(pH 7.6), 300 mM sucrose, 2 mM MgCl2] plusplant protease inhibitor cocktail (Sigma-Aldrich).Protein was quantified using the protein quan-tification assay (Macherey-Nagel GmbH & Co.KG, Düren, Germany). Unless indicated other-wise, 5 mg of protein was loaded per lane andseparated by 10% SDS–polyacrylamide electro-phoresis (SDS-PAGE). PAGE gels were transferredto polyvinylidene difluoride (PVDF) membranes(Immobilon-P, Millipore, USA) using a Bio-Radsemi-dry transfer system or the Bio-Rad Trans-Blot turbo system. After blocking in a 6% milkTBS solution, membranes were incubated withcustom antibodies raised against the malate syn-thase (MS) and PLGG1 (Agrisera, Vännäs, Sweden)and glycolate dehydrogenase (GDH) (Genscript,USA). As a protein loading control, antibodiesraised against the large subunit of RuBisCO (RbcL)and actin were used (Agrisera, Vännäs, Sweden).After subsequent washing and incubation withanti-rabbit secondary antibody (Bio-Rad, USA),chemiluminescence was detected with the Image-Quant LAS4010 scanner (GE Healthcare LifeSciences, Pittsburgh, USA).Chloroplasts were isolated in a manner sim-

ilar to that described (19), with tobacco-specificmodifications following (42). Leaf tissue was col-lected from 6-week-old WT and AP3 plants,briefly homogenized in extraction buffer [50mMMES-NaOH (pH6.1), 0.33Msorbitol, 2mMEDTA,

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2 mM MgCl2, 1 mM MnCl2, 20 mM NaCl, 2 mMisoascorbic acid, and 1% polyvinypyrrolidone-40], filtered through three layers of Miracloth(Calbiochem), and centrifuged at 4°C for 4 minat 2500g to pellet chloroplasts. Pelleted chloro-plasts were resuspended in 5ml of buffer [50mMHEPES-NaOH (pH 6.8), 0.33 M sorbitol, 2 mMEDTA, 2 mM MgCl2, 1 mM MnCl2, 5 mM iso-ascorbic acid, 1 mM sodiumpyrophosphate, 5mMglutathione] using a fine paintbrush, applied to a20-ml Percoll density gradient [top to bottom:40% (v/v) and 90% (v/v) Percoll in resuspensionbuffer], and centrifuged at 4°C for 30 min at2500g. Intact chloroplasts accumulated at the40 to 90% interface andwere removed by aspira-tion, washed twice in 10 volumes of resuspensionbuffer, and collected by centrifugation for 10minat 2500 g.Plastid proteins were extracted by lysing the

chloroplasts in a hypotonic buffer [10 mM Tricine-NaOH (pH 8.0), 1% (v/v) plant protease inhibitorcocktail (Sigma-Aldrich), and 5 mM dithiothreitol(DTT)], followed by two freeze-thaw cycles. In-solublemembrane fractions from the chloroplastisolationwere isolated by centrifugation at 10,000 gfor 5min. The pellet was resuspended in 2× SDSsample buffer plus 10% DTT, then briefly soni-cated. The membrane fraction proteins werethen precipitated using ice-cold acetone. Aftercentrifugation (10,000 g for 5 min), the acetonewas removed, and the pellet was air dried. Theprotein pellet was then resuspended in SDS sam-ple buffer plus 10% DTT. Protein concentrationwas then determined using a total protein quan-tification kit (Macherey-Nagel GmbH & Co.KG,Düren, Germany).

Photorespiratory metabolite analysis

Metabolite analysis was performed as described(19). Briefly, ~40 mg of fresh leaf tissue was har-vested from 6-week-old greenhouse-grown plantstaken late morning (~10:00 to 11:00 a.m.) andflash frozen in liquid nitrogen. Leaf material wascrushed using a genogrinder (Biospec products)and extracted in 100% ice-coldmethanol. Sampleswere then submitted to the Metabolomics Center,Roy J. Carver Biotechnology Center, University ofIllinois at Urbana-Champaign and processed asdescribed (19). All known artificial peaks wereidentified and removed. To allow comparisonamong samples, all data were normalized to theinternal standard in each chromatogram and thesample freshweight. The spectra of all chromato-gram peaks were evaluated using the AMDIS2.71 program (NIST). Metabolite concentrationswere reported as concentrations relative to theinternal standard, which was the target com-pound peak area divided by peak area of hentria-contanoic acid: Ni (relative concentration) = Xi

(target compound peak area) * X−1IS (peak areaof hentriacontanoic acid) per gram fresh weight.The instrument variability was within the stan-dard acceptance limit of 5%.

Growth analysis (greenhouse)

Homozygous single-insert T2 seeds were germi-nated on LC1 Sunshine mix (Sun Gro 202 Horti-

culture, Agawam, MA, USA). Ten days aftergermination, seedlings were transferred to 4Lpots (400C, Hummert International, Earth City,MO, USA) with LC1 Sunshine mix supplementedwith slow-release fertilizer (Osmocote Plus 15/9/12, The Scotts Company LLC, Marysville, OH,USA). Pots were randomized within the green-house and positions were changed before eachwatering approximately every 4 to 5 days. Green-house growth conditions are tabulated in sup-plementary dataset 12. Aboveground biomass washarvested and dried for 2weeks to attain constantweight, and dry weights determined for stem andleaf fractions. Stem fractions included reproduc-tive material developed at time of final harvest.

Field experiments

In 2016, five independent transformation eventsof AP3, four events of AP1, and two independenttransformations of AP2, with two wild type (WT)and two empty vector (EV) controls, were plantedin a randomized single block design.Homozygoussingle-insert T2 seeds were germinated in potscontaining soil mix (Sun Gro 202 Horticulture,Agawam, MA, USA) on 14 May 2016 and grownfor 7 days before transferal to floating trays asdescribed (43). Plants were transplanted at theUniversity of Illinois Energy Farm field station(40.11°N, 88.21°W, Urbana, IL, USA) on 6 June2016 after the field was prepared as described(43). Each plot consisted of 6 × 6 plants spaced30 cm apart (fig. S8). The internal 16 plants perplot were the indicated transgenic plant linessurrounded by a border of 20 WT plants. An ad-ditional two-row border ofWT plants surroundedthe full experiment that consisted of 26 plots.Watering was provided as needed from six watertowers placed within the experiment. Weatherdata, including light intensity, air temperature,and precipitation, were measured for the 2016field season as described (43) (supplementarydata set 13).Apparent quantum efficiency of photosynthesis

(Fa) and the light-saturated rate of photosyntheticCO2 assimilation at ambient (400 mbar) and low(100 mbar) [CO2] were measured on the youngestfully expanded leaf 14 to 20 days after trans-planting in the field. Fa was determined fromassimilation measurements in response to lightlevels at the indicated [CO2]. Gas exchange mea-surements were performed using Li-Cor 6400XTinstrumentswith a 2-cm2 fluorescencemeasuringcuvette for which chamber leaks were correctedas outlined in the manual (LI-COR Biosciences,Lincoln, NE, USA). Measurements of CO2 as-similation were conducted at incidental lightintensities of 1200, 380, 120, 65, 40, 30, 25, 18,and 10 mmol m−2 s−1, and absorbed light wascalculated using an integrating sphere (Ocean-Optics, Largo, FL, USA) (23). Assimilation wasrecorded after a minimum of 120 s at each lightlevel. Fa was calculated from the slope of theinitial linear response of CO2 assimilation at lowlight levels. The saturating rate of assimilation(Asat) was determined at 1200 mmolm−2 s−1 lightintensity at the indicated [CO2]. Leaf and stembiomass were determined for 16 plants per plot

at 7 weeks post planting. Aboveground biomasswas harvested and separated into leaf and stemfractions. Plant material was dried at 65°C toconstant weight for aminimum of 2 weeks priorto biomass measurements.To increase the statistical power of the field

experiment, the 2017 growing season focused onsix independent transgenic AP3 lines. The fielddesign consisted of five replicate blocks withseven randomized 6 × 6 plants plots per block(fig. S11). The central 16 plants were the AP3transgenic line or WT surrounded by a WTborder. The entire 35 plot-area was surroundedby an additional row of WT as a border. Single-insert homozygous T2 lines generated from thesame harvest time were sown on LC1 Sunshinemix and germinated for 7 days. After 7 days,seedlings were transplanted to floating trays asdescribed above. Fourteen days after transplantto floating trays, plants were transplanted at theEnergy Farm field station at the University ofIllinois, Urbana, IL, USA, on 21 June 2017.Water-ing was provided as needed using parallel dripirrigation. Photosynthesismeasurements to deter-mineFawere performed 2 to 5 July, 2017, andFawas measured as described above. Measurementsof CO2 assimilation in response to light began pre-dawn and were conducted at light intensitiesof 0, 10, 18, 25, 30, 40, 65, 120, 380, 1200, and2000 mmol mol−1. Diurnal measurements of pho-tosynthesis were performed starting pre-dawnon 14 July 2017 and measured every 2 hours ontwo plants per plot per block. Light levels andchamber temperature was set to ambient valuesbased on incoming light levels using a PAR sensoron the Li-Cor 6400XT and a built-in temperaturesensor. Reference [CO2] was maintained at 400mbar. Diurnal measurements were continueduntil after dusk. At 49 days post-germination,eight plants per plot were harvested from allfive replicate blocks. Aboveground biomass wasseparated into leaf and stem fractions and driedin a drying oven for 2 weeks to constant weightbefore biomass measurements. For starch analy-sis, 10 mg of leaf material was collected on 14July, frozen in liquid nitrogen, and stored at−80°C. Starch was assayed using the Enzychromstarch assay kit (Bioassay Systems, Hayward, CA,USA). Colorimetric measurements were per-formed on a Biotek Synergy HT plate reader(Biotek Winooski, VT, USA).

Photosynthetic CO2 response

Photosynthetic compensation point (Ci*)measure-mentswere performedusing aLi-Cor 6800 (Li-CorBiosciences) equipped with a fluorescence cham-ber. Ci* was determined using the common in-tersection method by measuring the CO2 responseof photosynthesis under various subsaturatingirradiances (29, 44, 45). The common intersec-tionwas determined using slope–intercept regres-sion to produce more accurate and consistentvalues of Ci* (29). Plants were acclimated under250 mmol m−2 s−1 light at 150 mbar CO2 untilphotosynthesis reached steady state and thenmeasured at 150, 120, 90, 70, 50, and 30 mbarCO2 under light intensities of 250, 165, 120, 80,

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and 50 mmol m−2 s−1. The x-intersection pointwas converted to Ci* according to (29).To determine the net photosynthetic assimila-

tion rate from a CO2 dose response, the fifth leaffrom the base of 7-week-old N. tabacum plantswas measured using a Li-Cor 6800 infrared gasanalyzer (Li-Cor Biosciences, Lincoln, NE, USA)with leaf temperature controlled at 25°C andlight intensity set at 1500 mmol m−2 s −1. Leaveswere acclimated at a [CO2] of 400 mbar to achievea steady-state rate of assimilation. The [CO2] ofthe response curve was set at 400, 200, 100, 50,30, 400, 600, 800, 1000, 1500, 2000 mbar, andmeasurements were taken when assimilationreached a steady state rate. To determine themaximum rate of carboxylation (Vcmax) andmax-imum electron transport rate (Jmax), a model forleaf photosynthesis with temperature correctionswas used assuming a mesophyll conductance of0.57 mol−2 s−1 bar−1 (46), then adjusted using thedetermined value of Ci*.

Statistical analysis

Statistical analysis was performed using OriginPro 2016 (version 9.3.226, Origin Lab CorporationNorthampton, MA, USA) or R (version 3.4.2https://www.R-project.org/). For Fv′/Fm′ mea-surements, each plate contained a minimum of10 seedlings and the data shown reflect the aver-aged values. Significance was evaluated by one-way analysis of variance (ANOVA). Relative changesin gene expression were analyzed by one-wayANOVA with three technical replicates per bio-logical replicate from either greenhouse- or field-grown samples. Greenhouse biomass experimentswere analyzed by a one-way ANOVA with a min-imum of five biological replicates. Biomass datafrom the 2016 field season were analyzed by aone-way ANVOA with 16 biological replicates.Biomass data from the 2017 field season wereanalyzed by an ANOVA model that accountedfor fixed effects of transgenic pathway, indepen-dent line, and block using eight plants/genotypefor n = 5 blocks. Greenhouse photosyntheticmea-surements were analyzed by a one-way ANOVA,and three biological replicates per measurementand field photosynthetic measurements wereanalyzed by a two-way ANOVA with two plantreplicates per plot and five randomized replicateblocks. All ANOVA testing was followed with aTukey’s post-hoc test for means comparison.ANOVA tables for each analysis are included insupplementary data set 15.

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ACKNOWLEDGMENTS

We thank D. Drag and B. Harbaugh for plant care and managementin the greenhouse and field studies; and N. Ferrari, R. Field,G. Lambruschini, J. Ayers, M. Oraweic, R. Devries, R. Gossens,K. Brown, R. Edquilang, and C. Keller for assistance during laboratoryand field work. We thank M. Balasubmaranian for tobaccotransformation. We thank C. Benjamin for graphic design. We alsothank E. Ainsworth, S. Long, B. Walker, and R. Slattery for critical reviewof the manuscript. Funding: This work is supported by the researchproject Realizing Increased Photosynthetic Efficiency (RIPE) that isfunded by the Bill & Melinda Gates Foundation, Foundation for Foodand Agriculture Research, and the Department for InternationalDevelopment under grant no. OPP1172157. This work is licensed undera Creative Commons Attribution 4.0 International (CC BY 4.0) license,which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited. To view a copy ofthis license, visit https://creativecommons.org/licenses/by/4.0/. Thislicense does not apply to figures/photos/artwork or other content

included in the article that is credited to a third party; obtainauthorization from the rights holder before using such material. Authorcontributions: P.F.S, D.R.O, and A.P.C. designed experiments; P.F.S,A.P.C., and H.W.L performed experiments; and P.F.S, D.R.O, A.P.C.,and H.W.L analyzed data and wrote the manuscript. Competinginterests: The authors declare no competing interests Data materialsavailability: The data reported in this paper have been tabulated inthe supplementary materials. Plants and constructs reported areavailable from the University of Illinois for research purposes, subject tothe conditions of the Uniform Biological Material Transfer Agreement.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/363/6422/eaat9077/suppl/DC1Figs. S1 to S15Table S1 to S3Data S1 to S20

17 April 2018; accepted 20 November 201810.1126/science.aat9077

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Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the fieldPaul F. South, Amanda P. Cavanagh, Helen W. Liu and Donald R. Ort

DOI: 10.1126/science.aat9077 (6422), eaat9077.363Science 

, this issue p. eaat9077; see also p. 32Science40% more productive than wild-type tobacco plants.∼Weber). In field trials, these transgenic tobacco plants were

recaptures the unproductive by-products of photosynthesis with less energy lost (see the Perspective by Eisenhut and constructed a metabolic pathway in transgenic tobacco plants that more efficientlyet al.the cost of energy lost. South

atits efficiency. Photorespiration deals with these by-products, converting them into metabolically useful components, but In some of our most useful crops (such as rice and wheat), photosynthesis produces toxic by-products that reduce

Fixing photosynthetic inefficiencies

ARTICLE TOOLS http://science.sciencemag.org/content/363/6422/eaat9077

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REFERENCES

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