Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield

Abstract

In photosynthetic organisms, the photosystem II (PSII) complex is the primary target of thermal damage. Plants have evolved a repair process to prevent the accumulation of damaged PSII. The repair of PSII largely involves de novo synthesis of proteins, particularly the D1 subunit protein encoded by the chloroplast gene psbA. Here we report that the allotropic expression of the psbA complementary DNA driven by a heat-responsive promoter in the nuclear genome sufficiently protects PSII from severe loss of D1 protein and dramatically enhances survival rates of the transgenic plants of Arabidopsis, tobacco and rice under heat stress. Unexpectedly, we found that the nuclear origin supplementation of the D1 protein significantly stimulates transgenic plant growth by enhancing net CO2 assimilation rates with increases in biomass and grain yield. These findings represent a breakthrough in bioengineering plants to achieve efficient photosynthesis and increase crop productivity under normal and heat-stress conditions.

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Fig. 1: Expressing the chloroplast gene psbA in the nucleus enhances D1 abundance.
Fig. 2: Identification of the nuclear-encoded D1 proteins localized in thylakoid membranes.
Fig. 3: Nuclear expression of psbA enhances plant heat tolerance.
Fig. 4: Expressing psbA in the nucleus stimulates plant growth.
Fig. 5: Expressing psbA in the nucleus enhances lincomycin resistance and increases CO2 assimilation rate.
Fig. 6: Expressing psbA in the nucleus increases biomass and grain yield in rice.

Data availability

All data generated or analysed during this study are included in the published article and Supplementary Information. Source data for Figs. 1 and 3–6, and Extended Data Figs. 3 and 7–10 are included with the paper.

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Acknowledgements

This study was supported by the Chinese Academy of Sciences (the Strategic Priority Research Programme, grant no. XDB27040105), the Ministry of Science and Technology of China (National Key R&D Programme of China, grant no. 2016YFD0100405) and the National Natural Science Foundation of China (grant nos. U1812401, 31770314, 31570260 and 31600225). We thank H.-L. Zhao and X.-G. Zhu (Institute of Plant Physiology & Ecology, Chinese Academy of Sciences) for suggestions and technical assistance and X.-Y. Gao, J.-Q. Li and Z.-P. Zhang (Institute of Plant Physiology & Ecology, Chinese Academy of Sciences) for assistance with electron microscopy.

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F.-Q.G. conceived the project and provided supervision. F.-Q.G., J.-H.C. and S.-T.C designed the experiments. J.-H.C., S.-T.C. and N.-Y. H. analysed the data. J.-H.C. and S.-T. C. carried out most of the experiments partially with the contributions of N.-Y. H., Q.-L.W., Y.Z. and W.G. F.-Q.G., J.-H.C. and N.-Y. H. wrote the manuscript.

Corresponding author

Correspondence to Fang-Qing Guo.

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Extended data

Extended Data Fig. 1 The integrated transgene RbcSPTP-psbA cDNA under control of the AtHsfA2 promoter in the transgenic lines of Arabidopsis, tobacco and rice.

a, Schematic diagram of the pHsfA2::RbcSPTP-psbA cDNA construct with 2kb of HsfA2 promoter sequence and the plastid-transit peptide sequence of RbcS (RbcSPTP) fused in frame with the cDNA of the Arabidopsis plastid gene psbA at N-terminal. The specific primers (RbcSPTP-F and psbA-R), derived from the plastid-transit peptide sequence RbcSPTP and coding region of the psbA cDNA respectively, were used to amplify the indicated fragment (283 bp) of the integrated transgene RbcSPTP-psbA cDNA. b, The indicated fragments (283 bp) were amplified from the nuclear genome samples isolated from the indicated transgenic lines of Arabidopsis, tobacco and rice by PCR. No corresponding fragments could be amplified from the wild-type samples. Arrows indicate the amplified 283-bp fragments. The amplified fragments were confirmed by sequencing. Three times these experiments were repeated independently with similar results.

Extended Data Fig. 2 RT-PCR analysis of expression of the integrated transgene pHsfA2::RbcSPTP-psbA cDNA in the transgenic lines of Arabidopsis, tobacco and rice under control or subjected to heat stress.

a, Schematic diagram of the pHsfA2::RbcSPTP-psbA cDNA construct with 2kb of HsfA2 promoter sequence and the plastid-transit peptide sequence of RbcS (RbcSPTP) fused in frame with the cDNA of the Arabidopsis plastid gene psbA at N-terminal. b-d, The expression levels of the integrated transgene pHsfA2::RbcSPTP-psbA cDNA in detached leaves of the transgenic lines of Arabidopsis (b, A1, A2 and A3), tobacco (c, T6, T13 and T58) and rice (d, R3, R13 and R23) under control or subjected to heat treatments (for Arabidopsis, 41 °C, 1 h; Tobacco, 42oC, 1 h; Rice, 44oC, 1 h) using the specific primers (RbcSPTP-F and psbA-R), derived from the plastid-transit peptide sequence RbcSPTP and coding region of the psbA cDNA respectively by RT-PCR analysis. No corresponding fragments could be amplified from the wild-type samples. Three times these experiments were repeated independently with similar results.

Extended Data Fig. 3 Immunodetection of the core subunits abundance of PSII in thylakoid membranes in wild-type and the transgenic lines of Arabidopsis under control or heat stress conditions (Related to Fig. 1e, the immunodetection of Arabidopsis D1 abundance).

Immunodetection of the core subunits (D2, CP43 and CP47) abundance of PSII in thylakoid membranes in wild-type and the transgenic lines of Arabidopsis (A1, A2 and A3) harboring the pHsfA2::RbcSPTP-psbA cDNA construct under control or heat stress conditions. Three times these experiments were repeated independently with similar results. Samples of thylakoid membranes were isolated from equal fresh weight of detached leaves for each genotype according to (Chen et al., 2017). Equal protein loading was confirmed with antiserum against CF1β. Source data

Extended Data Fig. 4 Alignments of derived amino acid sequences of the D1 protein homologs from Arabidopsis (AtD1), tobacco (NtD1) and rice (OsD1).

Amino acid sequences of AtD1 (NP_051039), NtD1 (NP_054477) and OsD1(AJC09319) were aligned as the identical amino acid residues and conservative changes were depicted in black and grey background, respectively. Red arrows indicate the AA differences between the Arabidopsis D1 and the tobacco and rice D1 protein sequences.

Extended Data Fig. 5 Expressing psbA in the nucleus protects thylakoids against heat stress.

a-c, Blue native-PAGE analysis of thylakoid membrane proteins from detached leaves of wild-type and the indicated transgenic lines of Arabidopsis (a, A1, A2 and A3), tobacco (b, T6, T13 and T58) and rice (c, R3, R13 and R23) harboring the pHsfA2::RbcSPTP-psbA cDNA construct under normal (control) or heat stress conditions (41oC, 4 h for Arabidopsis, 42oC, 3 h for tobacco and 44oC, 8 h for rice). Three times these experiments were repeated independently with similar results. d-f, Transmission electron micrographs of the chloroplast ultrastructure from detached, fully expanded leaves of wild-type and the transgenic lines of Arabidopsis (d), tobacco (e) and rice (f) challenged with heat treatments (41oC, 4 h for Arabidopsis, 42oC, 3 h for tobacco and 44oC, 8 h for rice). Two times these experiments were repeated independently with similar results. Scale bars for TEM panels = 0.5 µm.

Extended Data Fig. 6 Nuclear heat-responsive expression of the chloroplast gene psbA stabilizes thylakoid membranes against heat stress.

Transmission electron micrographs of the chloroplast ultrastructure from detached, fully expanded leaves of wild-type and the transgenic lines of Arabidopsis (a), tobacco (b) and rice (c) challenged with heat treatments (41oC, 4 h for Arabidopsis, 42oC, 3 h for tobacco and 44oC, 8 h for rice). Scale bars for TEM panels = 2 µm. Two times these experiments were repeated independently with similar results.

Extended Data Fig. 7 Expressing the chloroplast gene psbA in the nucleus stimulates growth in rice.

a, Seedling phenotypes of wild-type and transgenic rice lines (R3, R13 and R23) harboring the pHsfA2::RbcSPTP-psbA cDNA construct under field growth conditions. Scale bar=10 cm. Three times these experiments were repeated independently with similar results. b, The representative top second leaves detached from the plants of wild-type and transgenic rice lines (R3, R13 and R23) at the heading stage when grown under field growth conditions. Scale bar = 10 cm. c, Width of the top second leaves detached form the indicated genotypes as shown in (b) (n=20). Individual values (black-coded dots) and means are shown. Statistical analyses were performed (***P value < 0.001, two-sided Student’s t test). Error bars indicate SD. Source data

Extended Data Fig. 8 Phenotypes and SEM analysis of the top second leaves detached from wild-type and transgenic rice lines at flowering stage.

a, Phenotypes of the representative top second leaves detached from wild-type and transgenic rice lines (R3, R13 and R23) harboring the pHsfA2::RbcSPTP-psbA cDNA construct under field growth conditions. Three times these experiments were repeated independently with similar results. Scale bar=5 cm. b, Scanning electron microscopy (SEM) analyses showing adaxial epidermal cells at maximum width of leaves detached from wild-type and transgenic rice lines (R3, R13 and R23) as shown in (a). Scale bars = 20 μm. c, Average widths of the silica-phellem blocks (SPB) were measured based on the SEM images taken from 15 top second leaves each genotype. Widths of 100 silica-phellem blocks for each genotype were measured (n=100). Bars indicate standard deviation. Individual values (black-coded dots) and means are shown. (**P value<0.01, ***P value<0.001, two-sided Student’s t test). Source data

Extended Data Fig. 9 Numbers of pavement cells across maximum width of fully-expanded leaves of the transgenic Arabidopsis plants.

a, Representative fully-expanded leaves detached from 21-d-old wild-type and the transgenic plants (lines A1 and A3) of Arabidopsis. Leaves were cut along the white dashed-line across maximum width of fully-expanded leaves, and decolorized with ethanol. The number of pavement cells along the cutting line was counted under microscopy. Scale bars=0.5 cm. b, Number of pavement cells along the cutting line in detached leaves of wild-type and the transgenic plants (lines A1 and A3) of Arabidopsis (n=10). Individual values (black-coded dots) and means are shown. Statistical analyses were performed (*P value < 0.05, **P value < 0.01, two-sided Student’s t test). Error bars indicate SD. Source data

Extended Data Fig. 10 Nuclear heat-responsive expression of the chloroplast gene psbA stimulates branching and increases biomass in Arabidopsis and tobacco.

a,c, Number of rosette-leaf branches per mature plant of wild-type and transgenic lines of Arabidopsis (A1 and A3, n=8) and tobacco (T6, T13 and T58, n=10) harboring the pHsfA2::RbcSPTP-psbA cDNA construct. b,d, Comparative analysis of aboveground biomass per plant between wild-type and the transgenic lines of Arabidopsis (A1 and A3, n=8) and tobacco (T6, T13 and T58, n=5). Individual values (black-coded dots) and means are shown. Statistical analyses were performed (*P value < 0.05, **P value < 0.01, ***P value < 0.001, two-sided Student’s t test). Error bars indicate SD. Source data

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Chen, J., Chen, S., He, N. et al. Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield. Nat. Plants 6, 570–580 (2020). https://doi.org/10.1038/s41477-020-0629-z

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