Research Article

Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field

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Science  04 Jan 2019:
Vol. 363, Issue 6422, eaat9077
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 recycling of glycolate. RNAi suppresses the native glycolate/glycerate transporter PLGG1 to prevent glycolate from leaving the chloroplast and entering the native pathway (gray).

  • Fig. 1 Alternative photorespiratory pathways.

    (A) Model of three alternative photorespiration pathway designs. AP1 (red) converts glycolate to glycerate using five genes from the E. coli glycolate pathway encoding the enzymes glycolate dehydrogenase, glyoxylate carboligase, and tartronic semialdehyde reductase. AP2 (dark blue) requires three introduced genes encoding glycolate oxidase, malate synthase, and catalase (to remove hydrogen peroxide generated by glycolate oxidase). AP3 (blue) relies on two introduced genes: Chlamydomonas reinhardtii glycolate dehydrogenase and Cucurbita maxima malate synthase. (B) qRT-PCR analysis of the two transgenes in AP3 and the target gene PLGG1 of the RNAi construct. Results for three independent transformation events are shown with (1, 5, and 8) and without (8, 9, and 10) PLGG1 RNAi. Error bars indicate SEM. * indicates statistical difference at P < 0.05 compared 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 membrane fraction, using custom antibodies raised against the indicated target genes, cytosolic marker actin, and chloroplast-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 as numerous malate synthase enzymes, have been previously characterized (table S3) (17).

  • Fig. 2 AP plant lines are more photoprotective under photorespiration stress.

    (A) Representative photos of 9-day-old T2 transgenic tobacco lines during the chlorophyll fluorescence photoprotection screen for AP pathway function showing AP3 protecting photosystem II from photodamage under severe photorespiratory conditions. (B) Combined values of the three AP construct designs with and without RNAi targeting the glycolate-glycerate transporter PLGG1. Error bars indicate SEM. * indicates statistical difference compared to WT based on one-way ANOVA at P ≤ 0.05, ** P ≤ 0.001. Fvʹ/Fmʹ for individual lines is described in supplementary data set 1. Actual significant P values are shown in supplementary data set 15.

  • Fig. 3 Photorespiration AP lines increase biomass under greenhouse conditions.

    (A) Photos of 6-week-old AP3 and WT plants grown in the greenhouse. Individual plant lines are indicated in the labels below the plant. (B) Percent difference in total dry weight biomass of the indicated combined plant 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.

  • Fig. 4 Photorespiratory and AP3 metabolic intermediates.

    (A to F) Relative amount of the indicated metabolite 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 is the 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 fresh weight. Error bars indicate SEM, n = 4 leaf samples. Statistical differences between AP3 designs and WT based on one-way ANOVA, with P values indicated. All P values are listed in dataset 15.

  • Fig. 5 Photosynthetic efficiency of greenhouse-grown plants.

    Data are the combined result of three independent transformants (hereafter referred to as combined) with and without PLGG1 RNAi. (A) CO2 assimilation based on intercellular [CO2] (Ci). (B) Combined apparent CO2 compensation point: Ci* calculated using the common intercept method and slope regression (29). (C) Combined maximum rate of RuBisCO carboxylation (Vcmax). Vcmax values are presented at 25°C and modeled from photosynthetic response under changing CO2 concentration. Gray bars indicate constant Ci*; green bars indicate derived values based on measured Ci*. Error bars indicate SEM. P values for statistical comparison to WT based on one-way ANOVA are given.

  • Fig. 6 Plant productivity and photosynthetic performance in 2017 field trials.

    (A) Percent difference 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. * indicates significance compared to WT and ** indicates significance between WT and AP3-only lines compared to AP3 with RNAi lines. P values are shown in supplementary data set 15. (B) Total combined accumulated leaf starch for indicated lines extracted from 10 mg of fresh weight leaf material. (C) Combined apparent quantum efficiency of photosynthesis (Φa) determined by linear regression of assimilation based on available light-response curves. (D) Combined accumulated assimilation of CO2 (A′) based on diurnal analysis of photosynthesis. (E) Combined accumulated electrons 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.

Supplementary Materials

  • Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field

    Paul F. South, Amanda P. Cavanagh, Helen W. Liu, Donald R. Ort

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Figs. S1 to S15 
    • Tables S1 to S3 
    Data S1-11
    Data S15
    Data S12
    Data S13
    Data S14
     
    Correction (10 January 2019):The legend to fig. S13 has been corrected to describe that panels A to C are from the 2017 field trial and panels D and E are data from a greenhouse-grown experiment.
    The original version is accessible here.

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