Multigene manipulation of photosynthetic carbon metabolism enhances the photosynthetic capacity and biomass yield of cucumber under low-CO2 environment

Abstract

Solar greenhouses are important in the vegetable production and widely used for the counter-season production in the world. However, the CO₂ consumed by crops for photosynthesis after sunrise is not supplemented and becomes chronically deficient due to the airtight structure of solar greenhouses. Vegetable crops cannot effectively utilize light resources under low-CO₂ environment, and this incapability results in reduced photosynthetic efficiency and crop yield. We used cucumber as a model plant and generated several sets of transgenic cucumber plants overexpressing individual genes, including β-carbonic anhydrase 1 (CsβCA1), β-carbonic anhydrase 4 (CsβCA4), and sedoheptulose-1,7-bisphosphatase (CsSBP); fructose-1,6-bisphosphate aldolase (CsFBA), and CsβCA1 co-expressing plants; CsβCA4, CsSBP, and CsFBA co-expressing plants (14SF). The results showed that the overexpression of CsβCA1, CsβCA4, and 14SF exhibited higher photosynthetic and biomass yield in transgenic cucumber plants under low-CO₂ environment. Further enhancements in photosynthesis and biomass yield were observed in 14SF transgenic plants under low-CO₂ environment. The net photosynthesis biomass yield and photosynthetic rate increased by 49% and 79% compared with those of the WT. However, the transgenic cucumbers of overexpressing CsFBA and CsSBP showed insignificant differences in photosynthesis and biomass yield compared with the WT under low-CO₂.environment. Photosynthesis, fluorescence parameters, and enzymatic measurements indicated that CsβCA1, CsβCA4, CsSBP, and CsFBA had cumulative effects in photosynthetic carbon assimilation under low-CO₂ environment. Co-expression of this four genes (CsβCA1, CsβCA4, CsSBP, and CsFBA) can increase the carboxylation activity of RuBisCO and promote the regeneration of RuBP. As a result, the 14SF transgenic plants showed a higher net photosynthetic rate and biomass yield even under low-CO₂environment.These findings demonstrate the possibility of cultivating crops with high photosynthetic efficiency by manipulating genes involved in the photosynthetic carbon assimilation metabolic pathway.

Keywords: carbon metabolism; cucumber; greenhouse; highphotosynthetic efficiency; low-CO2; multigene manipulation.

Figure 1

Phylogenetic tree of βCA (A), FBA (B) and SBP (C) in plant species. Unrooted phylogenetic tree was calculated with the Maximum-Likelihood method, using JTT modeling with gamma-distributed rates and 1000 bootstrap replications based on the multiple sequence alignments of βCA and SBP. The final tree was then rooted in the single clade consisted of members from C. reinhardtii. Phylogenetic tree was also constructed with JTT model for FBA proteins and the tree was rooted in the middle of two groups of members from C. reinhardtii. Bootstrap values are indicated at the base of each clade. All target protein in cucumber genome were colored with red in those trees.

Figure 2

Expression patterns of target genes in different tissues in cucumber. (A) Tissue-specific expression profiles of four genes in different tissues, including leaf, flower, and growing tip tissue. The relative expression level in flower and growing tip were determined with the expression level of leave as internal standards to normalize. (B) Expression profiles of four genes in five full expanded true leaves. Small letters represent significant differences (P< 0.05). Labels in the figures and tables below are the same.

Figure 3

Generation and identification of transgenic plants. (A) Schematic diagram of vectors for cucumber transformation. (i) Separate expression vectors. (ii) The multigene expression vector. 2A: 2A linker peptide. TP: target peptide. T: flag protein tag. (B) 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. (C) Characterization of cucumber transgenic lines, (i) CsβCA1, (ii) CsβCA4, (iii) 14SF (Four multigene overexpression transgenic cucumber plants), (iv) CsSBP, or (v) CsFBA, by PCR and Western blotting. The upper parts are the results of PCR detection, and the lower part are the results of Western blotting. M: DL2000 DNA marker. WT: the wild-type cucumber leaves. Different numbers represent plants from representative transgenic lines.

Figure 4

Expression analysis of transgenic lines. (A–D) qRT-PCR analysis of target gene transcript abundance in WT and transgenic cucumber lines. (E) qRT-PCR analysis of four genes in WT and 14SF lines. (F–I) ELISA analysis of target gene coding protein content of CsβCA1 (F), CsβCA4 (G), CsSBP (H), and CsFBA (I) in different transgenic lines. Small letters in each figure represent significant differences among samples by Student’s t-test (P < 0.05).

Figure 5

Overexpression of indicated genes increase the RuBisCO carboxylase activities in transgenic cucumber leaves. Overexpression of CsβCA1 (A) , CsFBA (B), CsβCA4 (D), or CsSBP (E) increased the RuBisCO carboxylase activities in transgenic cucumber leaves. (C) A multigene combination 14SF transgenic lines showed the increased RuBisCO carboxylase activities in transgenic cucumber leaves. Small letters in each figure represent significant differences among samples by Student’s t-test (P < 0.05).

Figure 6

Comparison of photosynthetic and rate of WT and transgenic cucumber leaves. The net photosynthesis rate (A), net photosynthesis rate dynamics in a time-course manner (B), Fv/Fm (C) and YII (D) were determined. Values represent the means ± SD (n = 3) of three plants per line. Small letters in each figure represent significant differences among samples by Student’s t-test (P < 0.05).

Figure 7

Morphological comparison of different transgenic cucumber lines after 42 days-growth in low-CO₂ concentrations.

Figure 8

Superposition of plant photosynthetic carbon assimilation pathway. The highlighted part represents the locations of βCA1, βCA4, SBP and FBA enzymes.