Introduction of Exogenous Glycolate Catabolic Pathway Can Strongly Enhances Photosynthesis and Biomass Yield of Cucumber Grown in a Low-CO2 Environment

Abstract

Carbon dioxide (CO₂) is very important for photosynthesis of green plants. CO₂ concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO₂ by vegetable crops. Vegetables in greenhouses are chronically CO₂ starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO₂ environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO₂ environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO₂ environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO₂ was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO₂ environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.

Keywords: Cucumber; glycolate catabolic pathway; high-photosynthetic-efficiency; low-CO2 treatment; multigene co-overexpression.

FIGURE 1

Superposition of E. coli glycolate catabolic pathway in plant photosynthetic carbon assimilation pathway.

FIGURE 2

Generation and identification of transgenic plants. (A) Schematic diagram of vectors for cucumber transformation. (i) DEF expression vector. (ii) GT expression vector. 2A: 2A linker peptide. TP, Target peptide; T, The flag protein tag. (B) Enzyme cleavage site position and enzyme digestion verification result of expression vectors. (i) Enzyme cleavage site position of. DEF vector. (ii) Enzyme cleavage site position of. GT vector. (iii) Enzyme digestion verification result of DEF and GT vectors. M: 10 Kb marker ladder. (C) The construction of cucumber transformation. (i) Cucumber seeds, (ii) Seed germination, (iii) Agrobacterium infection, (iv) Elimination of Agrobacterium, (v) Bud regeneration, (vi) Root induction, (vii) Roots, (viii) Regeneration of seedling, (ix) Seedling. (D) Characterization of cucumber transgenic lines by PCR and Western blotting. The upper parts are the results of PCR detection, and the lower part are the results of western blotting. WT, The wild-type cucumber leaves. TUA, The reference gene. Different numbers represent plants from representative transgenic lines.

FIGURE 3

Daily variation curve of CO₂ concentration in an enclosed solar greenhouse for cucumber cultivation.

FIGURE 4

ELISA analysis of target gene coding protein content in different transgenic lines.

FIGURE 5

Typical Photographs of cucumber growth in low CO₂ concentrations. (a) Morphological comparison of different transgenic cucumber lines after 6 weeks-growth. (b) Comparison of single melon morphology of different transgenic cucumber lines.

FIGURE 6

Comparison of the growth parameters between overexpression lines and the control plants after 6 weeks-growth in a low CO₂ environment. Values represent the means ± SD (n = 3) of three plants per line. Capital letters in each figure represent extremely significant differences among samples by Student’s t-test (P < 0.01) and small letters represent significant differences (P < 0.05). Labels in the figures and tables below are the same.

FIGURE 7

Photosynthetic diurnal variation curve of transgenic plants and wild type control plants after 5 weeks-growth in a low CO₂ environment.

FIGURE 8

Comparison of photosynthetic enzyme activities between transgenic plants and wild type control plants after 5 weeks-growth in a low CO₂ environment.