Rice large-scale metabolic regulation sub-dgp1 and crop metabolic quality design

Abstract

The application discloses a rice large-scale metabolic regulation sub-DGP1 and crop metabolic quality design. The application finds that a rice DGP1 family protein TIGR01589 domain targets multiple metabolic enzymes and promotes the transport of the metabolic enzymes to the nucleus; the domain also interferes with the light respiration pathway by changing the composition of the glycine decarboxylase complex in mitochondria. Based on the molecular mechanism of DGP1, a chimeric protein ST containing the domain and coupled with a chloroplast localization signal is designed to realize the genetic engineering modification of Chinese cabbage. The modification strategy can not only guide the substrate flow of the central glycolytic pathway to flavonoid biosynthesis, but also promote the accumulation of photosynthetic product-derived sugars in leaves.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the molecular mechanism of the rice large-scale metabolic regulator DGP1, and an artificial ST relocation system designed based on the DGP1 relocation system, which has been successfully applied to the quality improvement of leafy vegetables. Background Technology

[0002] Primary metabolic pathways, such as the tricarboxylic acid cycle (TCA cycle), glycolysis, lipid metabolism, and amino acid (AA) synthesis and degradation, are responsible for producing the essential organic molecules required for plant growth and development. In contrast, secondary metabolic pathways, while not directly determining plant growth, synthesize a wide variety of phytochemicals, and most of these secondary metabolites originate from intermediates of primary metabolism. Plant metabolism is highly dynamic, adjusting to changes in environmental signals and developmental stages, and its regulation involves multiple molecular levels, including transcription, post-transcriptional, and post-translational processes.

[0003] Phosphorus (Pi) is an essential macronutrient for plants, playing a crucial role in photosynthesis, respiration, and the synthesis of nucleic acids, proteins, and phospholipids. Phosphorus deficiency stress (low-Pi, LP) not only restricts plant photosynthesis and growth but also alters the levels of sugars, amino acids, and secondary metabolites. In Arabidopsis thaliana, phosphorus deficiency significantly increases the levels of various phenylpropanoids, flavonoids, and their derivatives in roots and aboveground parts. Light quality (especially wavelength) is another important environmental factor regulating plant metabolic pathways. Recent large-scale data analysis shows that rice (Oryzasativa) exhibits unique transcriptional and metabolic responses under different light conditions. Red light (RL) is particularly important, helping plants mitigate damage from biotic and abiotic stresses. Studies have shown that red light can influence the biosynthesis of key metabolites by regulating phenylpropanoid and amino acid metabolic pathways in tea plants; and in the callus culture of chicory plants, red light can also promote the accumulation of flavonoids and phenolic substances.

[0004] The glycine cleavage system (GCS) plays a central role in amino acid metabolism, converting glycine (Gly) to serine (Ser) and releasing ammonium ions for the synthesis of other amino acids. This process is regulated by the glycine decarboxylase complex (GDC), which contains four main enzyme proteins: P, T, L, and H. Among them, the H protein (GDCH) plays a crucial regulatory role in the overall reaction rate. Studies have found that knockdown of GDCH in rice significantly affects photorespiration and leaf senescence, which may be related to the accumulation of reactive oxygen species; conversely, overexpression of the H protein in tobacco is associated with increased photosynthetic efficiency.

[0005] Previous studies have confirmed that DGP1 (DEEP GREEN PANICLE1) in rice and its Arabidopsis homolog BPG4 (BRZ-INSENSITIVE-PALE GREEN 4) can act as photosynthetic repressors, inhibiting the activity of the transcriptional activator GLK1 / 2 (GOLDEN2-LIKE1 / 2). Although the DGP1 protein contains the plant-specific TIGR01589 domain, its function has remained unclear. Summary of the Invention

[0006] The purpose of this invention is to provide the design of rice metabolic quality by incorporating the large-scale metabolic regulator DGP1. This invention discovers that the rice DGP1 family responds to two environmental signals (red light and phosphorus deficiency) to mediate large-scale cellular metabolic remodeling, leading to a switch between basal and secondary metabolism. Furthermore, by modifying the DGP1 relocation system, an artificial ST chimeric protein was developed. The GLK1 promoter-driven artificial ST relocation system transports two sets of glycolytic enzymes (GAPC1 / 2 / 3 and PK1 / 4) to chloroplasts, promoting the accumulation of two types of flavor metabolites (flavonoids and sugars) in pakchoi leaves.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the application of the rice DGP1 gene in regulating metabolic remodeling in rice cells, the nucleotide sequence of which is shown in SEQ ID NO.1:

[0009] ATGGACGACGGCGGCGGCGGCGGCGGCGGCGACTCGTCGCCGGCTTCG TACATCAGATTGGTGCAGCA TCTGATCGAGAAGTGCATCTGCTACAACATGAACAAGGAGGAATGCATGGAGACGCTGGAGAAGCACGCCAACATC AAGCCCGTCATCACCTCCACCGTGTGGAAGGAGCTTGAGAAGGAGAACAGCGAGThe underlined base sequence in the TTCTTCGCCACGTACAAGAAGGGCCAAGGAGAGGAACCAGCGGAGAGCAAGAGCAGTAGTTCTTCACAGGAAGCTGCTGGTTCCAAGAGATCAGGCGGAGACGACGACTAG. The underlined base sequence represents the TIGR01589 domain.

[0010] The present invention also provides the application of the protein encoded by the rice DGP1 gene in regulating metabolic remodeling in rice cells, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] The present invention also provides the application of biological materials containing the rice DGP1 gene in regulating metabolic remodeling of rice cells, wherein the nucleotide sequence of the rice DGP1 gene is shown in SEQ ID NO.1, and the biological material includes a recombinant vector and recombinant bacteria.

[0012] Preferably, the rice DGP1 gene achieves metabolic remodeling of rice cells under corresponding phosphorus deficiency or red light conditions.

[0013] This invention also provides an artificial chimeric protein, comprising the TIGR01589 domain of rice DGP1 protein, a chloroplast localization sequence at the N-terminus of rice SIG5 protein, and a FLAG tag. The amino acid sequence of the artificial chimeric protein is shown in SEQ ID NO.2.

[0014] MASTVTTPSRPVSAGCHRRSPRRSAPVVLSLGGGPRRRTPSSTSCSALASPAKQGTAKLPPPQPTASR TAAADAERERERTDYNEYIRLVQHLIEKCICYNMNKEECMETLEKHANIKPVITSTVWKELEKENSEDYKDDDDKD YKDDDDKDYKDDDDK .

[0015] In the above sequence, the single underlined base sequence is the chloroplast localization sequence at the N-terminus of the rice SIG5 protein; the double underlined base sequence is the TIGR01589 domain; and the wavy line base sequence is the 3×FLAG tag.

[0016] The present invention also provides a nucleic acid encoding the aforementioned artificial chimeric protein, the sequence of which is shown in SEQ ID NO. 3:

[0017] ATGGCGTCCACTGTGACGACGCCGAGCCGGCCGGTATCCGCCGGGTGCCACCGGCGCTCGCCGCGGCGGTCAGCGCCCGTCGTGCTCTCCCTCGGCGGCGGGCCGCGGCGGCGCACGCCGTCGTCCACCAGCTGCTCGGCGCTCGCGTCGCCGGCGAAGCAGGGCACGGCCAAGCTCCCTCCGCCGCAGCCGACGGCGTCGAGGACCGCGGCGGCCGACGCGGAGCGGGAGCGGGA GCGGACGGACTACAACGAGTACATCAGATTGGTGCAGCATCTGATCGAGAAGTGCATCTGCTACAACATGAACAAGGAGGAATGCATGGAGACGCTGGAAGCACGCCAACATCAAGCCCGTCATCACCTCCACCGTGTGGAAGGAGCTTGAGAAGGAGAACAGCGAGgactacaaagaccatgacggtgattataaagatcatgacatcgactacaaggatgacgatgatgatgacaag.

[0018] The present invention also provides the application of the artificial chimeric protein or the nucleic acid in the improvement of vegetable flavor substances.

[0019] Preferably, the vegetable flavor compounds include flavonoids and sugars.

[0020] The present invention also provides a method for improving the flavor substances of vegetables, including the step of transferring the nucleic acid into vegetables to promote the accumulation of flavonoids and sugars in vegetable leaves.

[0021] Preferably, the vegetable includes bok choy.

[0022] The present invention discloses the following technical effects:

[0023] This invention confirms that members of the DGP1 family can regulate the dynamic reprogramming of primary and secondary metabolic pathways in rice in response to phosphorus deficiency and photoperiodic light conditions. This explains how plant cells rapidly and globally remodel their metabolomes under environmental changes. This invention also enables the genetic engineering of pakchoi by expressing the chimeric protein ST, which contains a conserved TIGR01589 domain and is coupled with a chloroplast localization signal. In pakchoi seedlings, ST expression driven by the GLK1 promoter specifically relocalizes the rate-limiting enzymes of glycolysis (GAPC and PK) into chloroplasts without significantly affecting GLK-mediated photosynthesis or GDC-mediated photorespiration. This strategy not only redirects substrates from the glycolysis pathway to flavonoid biosynthesis but also promotes the accumulation of photosynthetic product-derived sugars in leaves. In summary, the relocalization of artificially designed target proteins holds promise for providing a new method for customized research on flavor improvement in leafy vegetables. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The response of the rice DGP1 family genes to red light and phosphorus deficiency; (a) Phenotypic observations were conducted on representative mutant plants (dgp1, dgp1b, dgp1c, dgp1dgp1b, phr2, phyA, phyB), overexpression plants (DGP1-OE1 and DGP1B-OE1), and wild-type ZH11 plants on day 50 after germination, with a scale bar of 15 cm; (b) Chlorophyll a, chlorophyll b, and total chlorophyll content in leaves of all varieties in Figure a; (c) Photosynthetic parameters (A) in leaves of all varieties in Figure a. sat The measured values ​​of Ci) are shown in **, where P < 0.05, indicating a significant difference. This result is based on one-way ANOVA. (d) RT-qPCR analysis was performed on the DGP1, DGP1B, and DGP1C transcripts in the leaves of the WT line in response to RL and LP conditions. After LP (10 μM KH2PO4) treatment for 0, 6, 12, 24, and 48 h or RL (50 μmol·m -2 ·s -1(e) On day 4 after treatment, total RNA samples were extracted from rice leaves of 3-week-old ZH11 seedlings; normalization was performed using the rice UBQ gene; the experiment was repeated three times; data are presented as mean ± standard error (SE, n = 6); (e) RT-qPCR analysis was performed on the DGP1 gene in wild-type leaves responding to LP under 40 μM, 20 μM and 0 μM KH2PO4 conditions; samples were extracted at 0, 2, 6, 12, 24 and 48 μM KH2PO4 concentration gradients. h, Total RNA samples were extracted from leaves of 3-week-old ZH11 rice seedlings; normalization was performed using the rice UBQ gene, and the experiment was repeated three times; data are presented as mean ± standard error (SE, n=6); (f) Multiple sequence alignment of DGP1 (LOC_Os01g62060), DGP1B (LOC_Os05g38680), and DGP1C (LOC_Os03g17200) proteins; (g) Immunoblotting analysis of DGP1 protein in phr2 mutant and wild-type ZH11 under LP treatment conditions; Total protein samples were extracted from leaves of 3-week-old ZH11 rice seedlings after 72 h of LP treatment (0µM, 10µM, 20µM, 40µM KH2PO4); ACTIN was used as a control, and the immunoblotting experiment was repeated twice with completely consistent results; (h) phyA Immunoblotting analysis was performed on DGP1 protein in phyB mutant and wild-type ZH11 under RL response conditions; total protein samples were extracted from leaves of 3-week-old ZH11 rice seedlings after RL treatment at Rc50 or Rc100 for 72 h; ACTIN was used as a control, and the immunoblotting experiments were repeated twice with completely consistent results; "LP" indicates low phosphate, "RL" indicates red light, and "Rc50" and "Rc100" represent 50 µmol·m30, respectively. -2 ·s -1 100µmol·m -2 ·s -1 Red light;

[0026] Figure 2 Subcellular localization of DGP1, DGP1B, and GDCH in rice; (a) DGP1 signaling in rice protoplasts; co-transformation of rice protoplasts using two constructs, DGP1-GFP and COX11-mCherry; COX11-mCherry labeled mitochondria, chlorophyll fluorescently labeled chloroplasts; scale bar, 10 µm; (b) Co-localization of DGP1 and DGP1B in Nicotiana benthamiana; subcellular localization of GDCH protein in transgenic COX11-mCherry-RFP Nicotiana benthamiana, COX11-mCherry used for mitochondrial localization; scale bar, 100 µm;

[0027] Figure 3DGP1s induce large-scale metabolic remodeling in rice leaves by mediating nuclear translocation of a wide range of metabolic enzymes and altering mitochondrial GDC composition; (a) Immunoblotting analysis of DGP1 protein in leaves of the DGP1-ES2 cell line under control conditions or after 48 h of β-estradiol treatment (2 µM); ACTIN was used as a control, and the immunoblotting experiment was repeated twice with completely consistent results; (b) In the Y2H experiment, the TIGR01589 domain of DGP1 directly interacted with GAPC1, GAPC2, GAPC3, PK1 / 4, PDC1, CS, SCSb, FH, and MDH12.1; DDO, double-deficient medium, QDO, quadruple-deficient medium; GLK1 / GLK2 were positive controls; (c) Nucleoproteome showed WT, dgp1, and dgp1 Heatmap of DGP1-interacting enzyme factors during NP or RL response of dgp1b; experiment repeated three times; ND, not detected; leaves of 3-week-old rice seedlings were collected 3 days after NP or RL treatment; (d) Pull-down experiment showed that DGP1 interfered with the interaction between GDCH and GDCP; MBP fusion protein was immobilized on glutathione agarose gel 4B resin, and HIS or GST fusion protein was added; the pulled-down protein was separated by 10% SDS-PAGE and immunoblotted with anti-HIS, anti-GST or anti-MBP antibodies, respectively; (e) Venn diagram showing the degree of overlap of metabolite accumulation differences between DGP1-OE1 line and β-estradiol-treated DGP1-ES2 line; FC >2, VIP>1.

[0028] Figure 4 To investigate DGP1-mediated nuclear translocation of GAPC1 and PK4; (a) Subcellular localization of PK4 and FH proteins in *Nicotiana benthamiana* cell lines with or without DGP1 protein; Scale bar, 100 µm; The experiment was repeated twice with consistent results; (b) Immunofluorescence staining of nuclear DGP1 in leaf cells isolated from WT plants under control conditions or after NP / RL treatment; Leaves of 3-week-old rice seedlings were collected 3 days after NP or RL treatment; Blue, DAPI-stained nuclei; Red, antibody signal; Scale bar, 10 µm; (c) Immunoblotting analysis of PK4 and FH proteins in β-estradiol-treated DGP1-ES2, DGP1-OE1, RL-treated WT plants, NP-treated WT plants, RL-treated dgp1dgp1b mutant, NP-treated dgp1 mutant, and WT (ZH11) plants; PK4 and FH proteins were collected from 3-week-old WT plants under control conditions and on day 3 after NP / RL treatment. Total protein or nucleoprotein samples were extracted from the leaves of ZH11 rice seedlings; ACTIN (total) and LIG4 (nucleoprotein) were used as controls; the immunoblotting experiment was repeated twice and the results were the same.

[0029] Figure 5(a) Ion chromatograms of phenylalanine and three flavonoids (cherrocin, 3'-methoxyquercetin, and arbutin) extracted from leaves of pal6 pal8, DGP1-OE1, pal6 pal8 DGP1-OE1, and WT seedlings; (b) Phenotypes of pal6 pal8, DGP1-OE1, pal6 pal8 DGP1-OE1, glk1 glk2, and WT ZH11 plants 30 days after germination; Scale bar, 10 cm; (c) Mechanistic model of DGP1 triggering the phenylalanine pathway via PAL6 / 8 in rice leaves; “NP” indicates phosphate-free (0 µM KH2PO4), “RL” indicates red light (50 µmol·m -2 ·s -1 );

[0030] Figure 6 Establishment of the ST-relocation system for Chinese cabbage; (a) Amino acid sequences of rice DGP1 and artificial protein ST, with binding residues of PK4 and GAPC1 calculated on AlfaFold3, and subdomains represented above the sequences; (b) Schematic diagrams of the structures of GLK1::ST-FLAG and GLK1::DGP1-FLAG, immunoblotting analysis of leaf FLAGs in Chinese cabbage lines (ST-1 / 2 / 3 / 4 and DGP1-1 / 2 / 3 / 4); ACTIN was used as an internal control; the immunoblotting experiment was repeated twice, and the results were identical; (c) Schematic diagram of AlfaFold3 molecular docking of ST protein with metabolic enzymes (GAPC1 and PK4); (d) Subcellular localization of ST-FLAG-GFP; Transformation of Chinese cabbage leaf protoplasts using ST-FLAG-GFP; Chlorophyll fluorescence used to label chloroplasts; Scale bar, 10µm; (e) Photosynthetic parameters A of leaves of each transgenic line in Figure b. sat Measurement of photorespiration parameter GOR; ** indicates p < 0.01, and the difference was statistically significant using one-way ANOVA;

[0031] Figure 7Mechanism diagrams of the DGP1 and ST relocation systems in pak choi; (a) heatmaps of several flavonoids and sugars in the leaves of DGP1-3, ST-2, and WT pak choi seedlings under field growing conditions; (b) ion chromatography of sugars (sucrose, galactose, and fructose) and flavonoids (cherishin) extracted from the leaves of DGP1-3 / 4, ST-1 / 2, and WT pak choi seedlings; (c) differences between DGP1 and the artificial protein ST in targeted metabolic regulation; (d) Representative phenotypes of ST-1 / 2, DGP1-3 / 4 and WT Chinese cabbage seedlings; photographs taken on day 25 after germination; scale bar, 7 cm; (e) Phenotypes of ST-2 and WT Chinese cabbage seedlings under field growing conditions; photographs taken on day 40 after germination; (f) Mechanism model of the DGP1 or ST relocation system; “DGP1-3 / 4” means GLK1::DGP1-FLAG-3 / 4, and “ST-1 / 2” means GLK1::ST-FLAG-1 / 2;

[0032] Figure 8 (a) Immunofluorescence staining of PK4 and FH in mesophyll cells of WT and DGP1-OE1 lines; leaves of 3-week-old rice seedlings were collected under normal conditions; blue, DAPI-stained nuclei; red, antibody signal; scale bar, 10µm; (b) Immunoblotting analysis of DGP1, PK4 and FH proteins in WT plants and DGP1-OE1 lines; total protein samples were extracted from leaves of 3-week-old rice seedlings; ACTIN was used as an internal control; the immunoblotting experiment was repeated twice, and the results were the same; (c) Immunofluorescence staining of DGP1 in WT and DGP1-OE1 lines; leaves of 3-week-old rice seedlings were collected under normal conditions; blue, DAPI-stained nuclei; red, antibody signal; scale bar, 10µm; (d) Quantification of immunostaining signals in Figures a and c; ND indicates not detected; (e) Heatmap of DGP1-interacting enzyme factors in the nuclei of DGP1-OE1 cell lines and WT plant cells; the experiment was repeated three times; ND indicates not detected.

[0033] Figure 9To demonstrate the nuclear translocation of cytoplasmic enzymes (PK4 and FH) controlled by DGP1s under NP or RL conditions; (a) Immunofluorescence staining of PK4 and FH in the nuclei of NP-treated WT, NP-treated dgp1 mutant, RL-treated WT, RL-treated dgp1-dgp1b mutant, and untreated WT leaf cells; Leaves of 3-week-old rice seedlings were collected 3 days after NP or RL treatment under normal conditions; Blue, DAPI-stained nuclei; Red, antibody signal; Scale bar, 10µm; (b) Quantification of the immunostaining signal in figure a; ND, indicating not detected; “NP” indicates phosphate-free (0µM KH2PO4), “RL” indicates red light (50µmol·m -2 ·s -1 );

[0034] Figure 10 Large-scale metabolic remodeling of rice leaves mediated by DGP1; (a) Volcano plots of total metabolites in DGP1-OE1 / WT and DGP1-ES2 / control treated with β-estradiol; VIP >1 indicates significant differences in metabolite levels; green, low accumulation; red, high accumulation; leaf samples were collected from normally growing DGP1-OE1 and DGP1-ES2 after 2 days of treatment with 2µM β-estradiol; (b) Volcano plots of flavonoids and diterpenes in figure a; VIP >1 indicates significant differences in metabolite levels. “F + D” indicates flavonoids and diterpenoids; (c) Overlapping of DGP1 and PK4 immunosignals in the nuclei of leaf cells from β-estradiol-treated DGP1-ES2 plants; Leaves of 3-week-old rice DGP1-ES2 seedlings were collected under normal conditions or on day 2 after β-estradiol (2µM) treatment; Blue, DAPI-stained nuclei; Red, PK4 signal; Green, DGP1 signal; Scale bar, 10µm;

[0035] Figure 11 Heatmaps of total metabolic changes in rice leaves induced by DGP1; (a) Heatmap of total DAMs in DGP1-ES2 lines between control and β-estradiol treatment (n = 3); total metabolite levels (1994 metabolites) were collected from leaves of 3-week-old rice DGP1-ES2 seedlings on day 2 after control and 2 μM β-estradiol treatment; (b) Heatmap of total DAMs between WT ZH11 and DGP1-OE1 (n = 3); broad-target metabolite library (1994 metabolites) was collected from leaves of 3-week-old rice seedlings under control conditions.

[0036] Figure 12Comparative analysis of the leaf metabolome of the core components of this invention; (a) Venn diagram showing the overlap between DGP1-OE1 and DGP1B-OE1 (FC > 2 and p < 0.05); DGP1-OE1 line and glk1 glk2 mutant; NP-treated WT plants and RL-treated WT plants; DGP1-OE1 line, NP-treated WT plants and RL-treated WT plants; leaf samples collected 3 days after NP or RL treatment; (b) Venn diagram showing the overlap between RL-treated WT plants and RL-treated dgp1 dgp1b mutant (FC > 2 and p < 0.05); RL-treated WT plants and RL-treated phyA phyB mutant; NP-treated WT plants and NP-treated dgp1 mutant; NP-treated WT plants and NP-treated phr2 mutant; leaf samples collected 3 days after NP or RL treatment; “NP” indicates phosphate-free (0 µM KH2PO4), “RL” indicates red light (50 µmol·m -2 ·s -1 );

[0037] Figure 13 To illustrate the activation of phenylpropane-derived secondary metabolomes by DGP1s in rice leaves treated with NP or RL; (a) Heatmap of the most upregulated flavonoids and phenolamines in DGP1-OE1 compared to the glk1-glk2 mutant (FC>4 and p<0.05); (b) Heatmap of the most upregulated flavonoids and aniline content in NP-treated WT plants compared to the dgp1 mutant and the NP-treated dgp1 mutant (FC>4 and p<0.05); (c) Heatmap of the most upregulated flavonoids and benzamides in RL-treated WT lines compared to the dgp1-dgp1b mutant and the RL-treated dgp1-dgp1b mutant (FC>4 and p<0.05); (d) Phenotype of WT ZH11 plants on day 3 after RL, NP, and Phe treatments; scale bar, 10 cm; (e) Ion chromatogram of phenylalanine extracted from plants in figure d; (f) Heatmaps of the most upregulated flavonoids and phenolic amines in WT plants treated with phenylalanine (FC > 4 and p < 0.05); "NP" indicates no phosphate (0 µM KH₂PO₄), and "RL" indicates red light (50 µmol·m⁻¹). -2 ·s -1 “Phe” represents phenylalanine (3µM);

[0038] Figure 14The response of DGP1 / DGP1B to GDC-mediated photorespiration pathway in response to NP or RL; (a) In Y2H analysis, the DGP1B, DGP1, or DGP1-TIGR01589 domains interact with GDCH; DDO, double-deficient medium; QDO, quadruple-deficient medium; (b) Pull-down experiments showing the interaction of DGP1 or DGP1B with GDCH; MBP fusion protein was immobilized on glutathione agarose gel 4B resin, and HIS fusion protein was added; the pulled-down protein was separated by 10% SDS-PAGE and immunoblotting was performed with anti-HIS or anti-MBP antibodies, respectively; (c) Firefly luciferase complementarity imaging experiment, showing the interaction of DGP1 and GDCH in Nicotiana benthamiana leaves; Agrobacterium colonies with different structures infiltrated Arabidopsis thaliana leaves; after 2 days of infiltration, luciferase activity was recorded using a CCD camera; cps: counts per second; (d) The GDCHc fragment is essential for interaction with DGP1, DGP1B, and GDCP; specific deletions of full-length and truncated GDCH constructs (GDCHa, GDCHb, GDCHc, and GDCHd) are shown in the figure; the growth capacity of yeast cells on QDO medium indicates the interaction; empty pGADT7 vector was used as a negative control; (e) Measurement of the relative oxidation rate of glycine in leaves of mutants (dgp1, dgp1, dgp1b, and phr2) and WT seedlings after 3 days of phosphorus deficiency stress. “NP” indicates phosphate-free (0 µM KH2PO4); (f) Measurement of the relative oxidation rate of glycine in leaves of mutants (dgp1, dgp1, dgp1b, and phyA, phyB) and WT seedlings after 3 days of RL conditions; “Rc50” represents red light (50 µmol·m⁻¹). -2 ·s -1 );

[0039] Figure 15 Mechanism model for DGP1s-triggered metabolic reprogramming in rice leaves; (a) DGP1s mediates the subcellular migration of a wide range of metabolic enzymes into the nucleus; the repositioning of the PK3-PK1 / 4 complex mediated by dgp1 redirects its substrate PEP to phenylalanine, constitutively activating the biosynthesis of phenylalanine-derived metabolites via PAL6 / 8; “PEP” represents phosphoenolpyruvate, and “Phe” represents phenylalanine; (b) DGP1s modulate the photorespiration cycle by altering the composition of the GDC through interaction with the acyl-binding domain of the H subunit, thereby blocking the recruitment of the P subunit; (c) Multiple sequence alignment of DGP1 family proteins in monocotyledons; the top row represents the TIGR01589 domain; (d) Multiple sequence alignment of DGP1 family proteins in dicotyledons. The top row represents the TIGR01589 domain;

[0040] Figure 16Establishment of transgenic lines GLK1::ST-FLAG and GLK1::DGP1-FLAG for Chinese cabbage; (a) Representative phenotypes of ST-1 / 2 / 3 / 4, DGP1-1 / 2 / 3 / 4 and WT Chinese cabbage seedlings on day 20 after germination; Scale bar, 5 cm; (b) Immunoblot analysis of FLAG in leaves of ST-1 / 2, DGP1-3 / 4 and WT lines; total protein, chloroplast or nucleoprotein samples were extracted from leaves of transgenic seedlings that had grown normally for 3 weeks; ACTIN (total), LIG4 (nucleus) and RBCL (chloroplast) were used as controls; the immunoblotting experiment was repeated twice and the results were the same; (c) Immunoblot analysis of PK and GAPC in leaves of ST-1 / 2, DGP1-3 / 4 and WT lines; total protein, chloroplast or nucleoprotein samples were extracted from leaves of transgenic seedlings that had grown normally for 3 weeks; ACTIN (total), LIG4 (nucleus) and RBCL (chloroplast) were used as controls; the immunoblotting experiment was repeated twice and the results were the same; "DGP1" indicates DGP1-flag, "ST" indicates ST-flag. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Example 1

[0047] 1. Genetic material and growth conditions

[0048] To construct transgenic rice plants overexpressing DGP1 and DGP1B, the coding regions of these genes were amplified from first-strand cDNA, and the PCR products were inserted into the pCAMBIA1300 vector containing the CaMV35S promoter. T3 generation homozygous lines (DGP1-OE1 and DGP1B-OE1) were used for experimental analysis. An estradiol-inducible vector was constructed via an LR reaction between the introductory vector and the target vector pMDC7, and the T3 generation homozygous line DGP1-ES2 was used for experimental analysis. CRISPR mutants (phr2, phyA, phyB, dgp1, dgp1b, dgp1c, pal6, pal8, glk1, and glk2) were purchased from Weimi Biotechnology Co., Ltd. Double mutants (phyA phyB, dgp1 dgp1b, pal6 pal8, and glk1 glk2) were obtained through hybridization with the corresponding single mutants.

[0049] Transgenic plants of Pak-choi (Brassica rapa ssp. chinensis) (GLK1::ST-FLAG-1 / 2 and GLK1::DGP1-FLAG-3 / 4) were constructed using genetic transformation methods [refer to "Establishment of an Agrobacterium-mediated genetic transformation and CRISPR / Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)"]. The coding regions of ST-FLAG or DGP1-FLAG were inserted into the WMV025 vector, and expression was driven by the GLK1 promoter. All plant materials used in this invention were grown in paddy fields under normal conditions. Detailed information on all rice lines is shown in Table 1, and primers used for genotyping are shown in Tables 2-3.

[0050] Table 1. Detailed information on rice lines

[0051]

[0052] Table 2 Primers used for genotype identification

[0053]

[0054] Table 3

[0055]

[0056] 1.1 Real-time quantitative PCR (RT-qPCR)

[0057] Total RNA was extracted from rice leaves using the Trelief® RNApre Pure Plant Kit (tsingke, catalog number: TSP411) according to the manufacturer's instructions. The RNA samples, free of genomic DNA residue, were reverse transcribed into first-strand cDNA using the Goldenstar® RT6 cDNA Synthesis Kit (tsingke, catalog number: TSK301S). The total volume of the qPCR reaction system was 20 μL, containing 2 μL of cDNA template, 2 μL of forward and reverse primer mixture (R+F, 10 μM), and 10 μL of 2×TSINGKE® MasterqPCR Mix (tsingke, catalog number: TSE201). ddH2O was added to bring the total volume to 20 μL, and the reaction was performed on a Qtower 3.0 real-time PCR instrument (Analytik Jena). Fluorescence signals were quantified according to a standard curve. PCR reaction conditions were: 95℃ pre-denaturation for 5 min, followed by 40 cycles (95℃ for 10 s, 52℃ for 30 s, and 72℃ for 30 s), and the melting temperature of each gene product was measured. UBQ (Os03g0234200) was used as an internal control gene for expression level normalization, and 2... -ΔΔCT Relative expression levels were calculated using the qPCR method. Each gene was subjected to three biological replicates. The primer sequences for qPCR are shown in Table 2. All reagents used for RT-qPCR analysis were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0058] 1.2 Deep Transcriptome Sequencing

[0059] Total RNA was extracted from leaf tissues of 14-day-old plants (ZH11 and DGP1-OE1) using an RNA extraction kit (Huayueyang, China). RNA integrity was assessed by 1% (w / v) agarose gel electrophoresis and a NanoPhotometer spectrophotometer (IMPLEN, California, USA). Total RNA concentration was determined using the Qubit RNA Assay Kit on a Qubit 2.0 fluorometer (LifeTechnologies, California, USA). RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, California, USA). RNA-seq libraries were constructed according to the manufacturer's instructions using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, Massachusetts, USA), and 100 bp single-end sequencing was performed on an Illumina HiSeq-2000 sequencer. Library sequencing was performed by Qingdao Best Mass Spectrometry Technology Co., Ltd.

[0060] 1.3 Firefly luciferase complementation imaging (LCI)

[0061] The coding sequence of DGP1 (Os01g0837600) was cloned into the pCAMBIA1300-nLUC vector, and the coding sequence of GDCH (Os02g0170100) was cloned into the pCAMBIA1300-cLUC vector. Agrobacterium tumefaciens GV3101 strain carrying the corresponding vectors was cultured in LB medium until OD2000. 600 =0.5, after static incubation at room temperature for 3 hours, the bacterial suspension was injected into tobacco leaves. Two days after injection, luciferase activity was detected using the LB 985 NightSHADE system (Berthold Technologies).

[0062] 1.4 Immunofluorescence staining

[0063] Leaf samples were collected from mutants (dgp1 and dgp1 dgp1b), DGP1-OE1 overexpressing lines, and wild-type plants under control conditions or after 3 days of phosphorus deficiency / red light treatment. Rice leaves were fixed with 4% (w / v) paraformaldehyde at 4°C for 30 min using a vacuum pump, and then stored in 10% (v / v) phosphate-buffered saline (PBS) at -80°C. Cell nuclei were extracted for immunostaining analysis; slide preparation and immunoassay procedures followed the guidelines in the literature "The MRE11-ATM-SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae". Commercial antibodies for PK4 and FH were purchased from Shanghai Ouyi Biotechnology Co., Ltd. (Table 4). Cell nuclei were counterstained using Vector-shield mounting medium containing 4',6-diamidindole-2-phenylindole (DAPI; Vector Laboratory, catalog number: H-1200), and digital images of specific immune signals were captured using a fluorescence microscope (Olympus DP80).

[0064] Table 4 Antibody Information Related to This Invention

[0065]

[0066] 1.5 Subcellular localization

[0067] The coding sequences for DGP1, DGP1B, and GDCH were cloned into the pJIT163-GFP vector, forming fusion proteins with the GFP sequence. Agrobacterium GV3101 strain carrying the vectors (DGP1-GFP + DGP1B-GFP, GDCH-GFP + COX11-mCherry) were cultured to OD200. 600 After incubation at room temperature for 3 h, the culture medium was 0.5 μL, and the cells were injected into the epidermal leaf cells of *Nicotiana benthamiana*. GFP (excitation wavelength 488 nm, emission wavelength 500-540 nm) and RFP (excitation wavelength 561 nm, emission wavelength 600-650 nm) signals were observed using a confocal laser scanning microscope (Carl Zeiss LSM 710). Furthermore, rice protoplasts were co-transfected with DGP1-GFP, DGP1B-GFP, and DGP1C-GFP along with the mitochondrial marker protein COX11-mCherry. After incubation at 28°C in the dark for 20 h, GFP and RFP fluorescence signals were observed using a confocal laser scanning microscope (Carl Zeiss LSM 710).

[0068] 1.6 Bimolecular Fluorescence Complementary (BiFC)

[0069] The coding sequences of PK4 (Os12g0145700) and FH (Os03g0337900) were cloned into the pFGC-nYFP vector, and the coding sequence of DGP1 was cloned into the pFGC-cYFP vector. The vectors were transformed with Agrobacterium EHA105 and injected into leaves of *Nicotiana benthamiana*. Forty-eight hours after injection, the expression of fluorescent proteins in the infected leaves was observed using a confocal laser scanning microscope (Carl Zeiss LSM 710).

[0070] 1.7 Western blot

[0071] 20 mg of leaf tissue was ground into powder in liquid nitrogen and homogenized with 3 volumes of extraction buffer [50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 2% polyvinylpyrrolidone (PVPP)]. The homogenate was centrifuged at 15,000 g for 20 min at 4 °C, and the supernatant was used for Western blot analysis. Protein concentration was determined using a BCA protein quantification kit (Pierce, Rockford, Illinois, USA). 100 μg of protein was loaded onto each well, separated by 12% SDS-PAGE, and transferred to a Hybond-P membrane (GE Healthcare UK Ltd.). The blot membrane was treated with affinity-purified primary antibodies (PK4, FH, DGP1, and LIG4) (Table S3), followed by the addition of horseradish peroxidase-conjugated rabbit anti-IgG secondary antibody (Sigma Corp, USA). Immuno-Blot detection was performed using an Immuno-Blot assay kit (Bio-Rad, Hercules, California, USA) according to the manufacturer's instructions.

[0072] 1.8 Yeast two-hybrid (Y2H) and pull-down experiments

[0073] In the yeast two-hybrid experiment, the coding sequences of relevant genes were amplified and cloned into pGADT7 and pGBKT7 vectors, respectively. The corresponding vector combinations were transformed into the yeast strain Y2HGOLD, and positive colonies were screened on leucine- and tryptophan-deficient synthesis-deficient medium (SD-Leu-Trp). Protein-protein interactions were detected by observing growth on leucine-, tryptophan-, histidine-, and adenine-deficient synthesis-deficient medium (SD-Leu-Trp-His-Ade). Primers used to construct the Y2H vector are shown in Tables 2-3.

[0074] In the pull-down assay, recombinant purified MBP-GDCH or negative control MBP was incubated with glutathione agarose 4B (GE Healthcare) in pull-down buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% (v / v) glycerol, 0.5 mM EDTA, 0.1% (v / v) Triton X-100, 5 mM β-mercaptoethanol and a mixture of protease inhibitors] at 4°C for 4 h. Then, recombinant DGP1-HIS or DGP1B-HIS was added, and incubation continued at 4°C for 3 h. After washing five times with pull-down buffer, agarose beads were collected by brief centrifugation (2,000 g, 3 min) and resuspended in protein extraction buffer. Proteins were separated by SDS-PAGE and analyzed by Western blotting with the corresponding antibodies.

[0075] 1.9 Nucleoprotein Extraction

[0076] Using Minute TM Plant Cytoplasmic and Nucleoprotein Extraction Kit (Beijing Invent Biotechnology Co., Ltd.) extracts nucleoproteins from leaf tissues of 4-week-old rice seedlings.

[0077] 1.10 Metabolite LC-MS / MS Analysis

[0078] Samples were prepared from leaves of 3-week-old rice seedlings. A targeted metabolomics approach based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was employed, utilizing two extensive targeted libraries (containing 1994 and 1116 metabolites) to analyze the lyophilized and ground samples. Metabolite targeting was performed using LC-ESI-Q TRAP-MS / MS (AB Sciex 6500, Applied Biosystems, Waltham, MA, USA) in multiple reaction monitoring (MRM) mode. ESI source operating parameters were as follows: temperature 500℃; GSI, GSII, and CUR 50, 60, and 35 psi, respectively; IS 5500 V in positive ion mode and -4500 V in negative ion mode; collision gas high. The total sMRM cycle time was set to 1.0 s, and the residence time of each MRM transition was automatically adjusted based on the total cycle time to ensure at least 10 data points were obtained for each peak.

[0079] SCIEX OS 3.1.5.3945 software (AB Sciex, Framingham, Massachusetts, USA) was used to align peaks by retention time (RT), and metabolite quantification was performed by calculating peak area. In metabolomics data analysis, principal component analysis (PCA) was performed using OmicStudio (www.omicstudio.cn / tool); hierarchical cluster analysis and sample repeatability assessment were performed using R software (http: / / www.r-project.org / ). Log2 transformation was applied to the data to improve standardization, followed by sample repeatability assessment; after Z-score standardization, hierarchical cluster analysis and PCA analysis were performed. Two-tailed t-tests were used to compare the absolute content of metabolites in the three tissues, with p < 0.05 considered statistically significant. Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using Metware Cloud (a free online data analysis platform, https: / / cloud.metware.cn / # / home). The data used for calculation and analysis in this invention are the average of three biological replicates.

[0080] 1.11 Measurement of photosynthetic parameters

[0081] Photosynthetic parameters were measured on the middle part of fully expanded leaves of two-month-old rice plants grown in the field. Mutants (dgp1 and dgp1dgp1b) and wild-type plants were measured under control conditions or under red light (50 μmol·m⁻¹). -2 ·s -1 The photosynthetic parameters of mutants (dgp1b, dgp1c, phr2, and phyA / phyB) and overexpression lines (DGP1-OE1 and DGP1B-OE1) under normal conditions were measured for 4 days after phosphorus deficiency (5 μM KH2PO4) treatment. In addition, photosynthetic parameters of 25-day-old pak choi seedlings (wild type, GLK1::DGP1-FLAG-3 / 4, and GLK1::ST-FLAG-1 / 2) grown in the field were measured after leaf expansion. All experiments were conducted between 9:00 AM and 11:30 AM using a portable photosynthetic system (Li-COR Inc., Lincoln, Nebraska, USA), with the leaf chamber temperature and CO2 concentration maintained at 25°C and 400 ppm, respectively, and using 1000 μmol·m³ / min. -2 ·s -1 The light intensity is used to determine the photosynthetic rate under light saturation conditions (A). sat Each strain was subjected to four biological replicates.

[0082] 2. Results and Analysis

[0083] 2.1 Rice DGP1 mediates the nuclear localization of multiple metabolic enzymes and regulates the photorespiration pathway by altering the composition of the GDC complex.

[0084] The typical characteristic of the rice dgp1 mutant is a dark green panicle. Through a BLASTP search in the NCBI (National Center for Biotechnology Information) database, two similar genes to DGP1 (LOC_Os01g62060) were identified in rice, named DGP1B (LOC_Os05g38680, sequence similarity 72.8%) and DGP1C (LOC_Os03g17200, sequence similarity 64.5%). Figure 1 To investigate whether these genes regulate chlorophyll (Chl) synthesis and photosynthetic-related traits, this invention used the CRISPR-Cas9 gene editing system to knock out or overexpress these genes in wild-type rice (ZH11 variety). The results showed that the chlorophyll content of the dgp1 dgp1b double mutant was significantly increased (6.5 ± 0.8 mg / g fresh weight), while the chlorophyll content of the DGP1 overexpressing line DGP1-OE1 (3.2 ± 0.7 mg / g fresh weight) and the DGP1B overexpressing line DGP1B-OE1 (2.65 ± 0.7 mg / g fresh weight) was significantly lower than that of the wild-type control (5.44 ± 0.8 mg / g fresh weight). Figure 1 (a and b, Table 5). Furthermore, the light-saturated photosynthetic rate (A... sat Differences also exist among different strains: dgp1 mutant (33.1±6.2 μmol CO2·m -2 ·s -1 ), dgp1 dgp1b double mutant (33.5±6.2 μmol CO2·m -2 ·s -1 The light-saturated photosynthetic rate of the α-type was higher than that of the wild type (28.5 ± 6.1 μmol CO2·m⁻¹). -2 ·s -1 ), while DGP1-OE1 (23.5±4.4 μmol CO2·m -2 ·s -1 ) and DGP1B-OE1 (23.1±5.1 μmol CO2·m -2 ·s -1 The light-saturated photosynthetic rate of the ) is lower than that of the wild type ( Figure 1 (c; Table 5) Figure 1 In contrast, the intercellular CO2 concentration (Cᵢ) showed the opposite trend (c). Figure 1 (c; Table 6) Figure 1 c): dgp1 mutant (300.4±15.8 μmol CO2·mol) -1 ), dgp1 dgp1b double mutant (295.4±13.8 μmol CO2·mol) -1 The intercellular CO2 concentration was lower in the α-type than in the wild type (340 ± 23 μmol CO2·mol⁻¹). -1 ), while DGP1-OE1 (374.7±15.7 μmol CO2·mol -1 ) and DGP1B-OE1 (376.8±17.1 μmol CO2·mol -1 The intercellular CO2 concentration in the ) was higher than that in the wild type.

[0085] Table 5. Results of chlorophyll content determination in rice leaves (unit: mg·g FW)

[0086]

[0087] Note: ** indicates P < 0.01 compared to wild type.

[0088] Table 6. Leaf photosynthetic parameters (A) sat and Ci) measurement results

[0089]

[0090] Note: ** indicates P < 0.01 compared to wild type.

[0091] RT-qPCR and WB assays showed that DGP1 strongly responded to phosphorus deficiency (LP) and red light (RL) conditions, while DGP1B was strongly induced under RL conditions. Figure 1 Phosphate starvation response regulator 2 (PHR2) regulates the phosphate starvation response. Phytochromes (PHYs) play a key role in sensing the red / far-red (R / FR) light ratio. Phytochromes in rice are encoded by the PHYA, PHYB, and PHYC gene families. Western blotting analysis showed that the PHR2 and PHYA / PHYB signaling pathways regulate the high expression of the DGP1 family genes in rice. Figure 1 (g and h). Furthermore, in rice protoplasts, the DGP1-GFP protein is localized in the nucleus, cytoplasm, and mitochondria, but not in chloroplasts (g and h). Figure 2 (a). In other experiments with benthamiana leaves, DGP1-RFP and DGP1B-GFP localized to the nucleus and mitochondria, with signaling overlapping in these two organelles. Figure 2 (b)

[0092] 2.2 DGP1 family proteins mediate the nuclear translocation of multiple metabolic enzymes

[0093] Yeast two-hybrid screening identified glycolytic enzymes (GAPC1 / 2 / 3 and PK1 / 4) and tricarboxylic acid cycle enzymes (PDC1, CS, SCSb, FH, and MDH12.1). Yeast two-hybrid experiments confirmed that DGP1 interacts explicitly with proteins encoded by 10 full-length genes through this domain. Figure 3 (b)

[0094] Subsequently, bimolecular fluorescence complementation (BiFC) experiments were performed in *Nicotiana benthamiana*. Co-expression of DGP1-cYFP with either PK4-nYFP or FH-nYFP resulted in detected fluorescence signals in the cell nucleus, indicating that DGP1 can mediate the nuclear translocation of PK4 and FH through direct interaction. Figure 4 (a). Conversely, when PK4-YFP or FH-YFP is expressed alone, the YFP signal is mainly localized in the perimembrane region (a). Figure 4 (a) confirms the DGP1-mediated nuclear localization function.

[0095] This invention further compared and analyzed the leaf nuclear proteomes of DGP1-OE1 overexpressing lines and wild-type plants, identifying 149 differentially accumulated proteins (DAPs) (FC>1.5 and p<0.05). In the three wild-type samples, PDC1 and PK4 were not detected, and FH and CS were not fully detected either; however, compared to wild-type, DGP1 overexpression significantly promoted the accumulation of most target factors (GAPC1, GAPC2, GAPC3, PK1, PK4, CS, SCSb, FH, and MDH12.1) in the nucleus. Figure 8 (e). Following NP or RL treatment, DGP1-interacting enzymes were also enriched in the nuclei of wild-type plants, but not in NP-treated dgp1 mutants or RL-treated dgp1-dgp1b mutants. Figure 3 (c) Immunostaining experiments detected DGP1 signaling in the nuclei of mesophyll cells from DGP1-OE1 line, NP, or RL-treated wild-type plants, and partial nuclear accumulation of PK4 and FH was observed. Figure 4 b, Figure 8 ad, Figure 9 (a and b). In contrast, NP-treated dgp1 mutants and RL-treated dgp1 dgp1b mutants showed significantly reduced PK4 / FH signaling (a and b). Figure 9(a and b). Furthermore, Western blotting (WB) confirmed this process; DGP1 protein was detected in the β-estradiol-treated DGP1-ES2 line, indicating that DGP1 was successfully transiently induced to express (a and b). Figure 3 (a) PK4 and FH were detected in the nuclear components of leaves from β-estradiol-treated DGP1-ES2, DGP1-OE1, RL-treated, and NP-treated wild-type plants, but not in the NP-treated dgp1 mutant and the RL-treated dgp1 dgp1b mutant. Figure 4 (c). Therefore, the DGP1-TIGR01589 domain acts as a transport vector, mediating the nuclear translocation of target metabolic enzymes in response to environmental signals.

[0096] 2.3 Regulation of leaf metabolome by DGP1 or DGP1B

[0097] Given that DGP1 can target multiple metabolic enzymes, it may have an impact on cellular metabolism. To verify this hypothesis, this invention employs a broad-targeted metabolomics approach (covering 1994 metabolites) to quantitatively analyze the leaf metabolomes of wild-type ZH11, DGP1-OE1 overexpression lines, and β-estradiol-treated DGP1-ES2 seedlings. Figure 10 a, Figure 11 (ab). Volcano plots showed that, compared to the control, in the DGP1-ES2 strain treated with β-estradiol for 48 hours, 246 differentially accumulated metabolites (DAMs) were upregulated and 257 were downregulated (FC>2 and VIP>1.0). Figure 10 In the DGP1-ES2 line treated with β-estradiol (a), nuclear immunofluorescence was used to detect DGP1 and PK4. The results are shown in [the table below]. Figure 10 In DGP1-OE1 seedlings, compared with wild type, 368 differentially accumulated metabolites were upregulated and 241 were downregulated (FC>2 and VIP>1.0). Figure 10 (a). Importantly, the DGP1-OE1 strain and the β-estradiol-treated DGP1-ES2 strain shared 338 differentially accumulated metabolites ( Figure 3 (e). In DGP1-OE1 plants, secondary metabolites (flavonoids and diterpenoids) were consistently upregulated (160 differentially accumulated metabolites); in DGP1-ES2 plants, these secondary metabolites were selectively upregulated (53 differentially accumulated metabolites). Figure 10 (b)

[0098] Further analysis of key metabolic pathways revealed significant changes in amino acid biosynthesis, the tricarboxylic acid cycle, and glycolysis. In β-estradiol-treated DGP1-ES2 seedlings, 15 differentially accumulated metabolites were downregulated and 5 were upregulated in the amino acid pathway; 6 differentially accumulated metabolites were downregulated and 3 were upregulated in the tricarboxylic acid cycle; and 5 differentially accumulated metabolites were downregulated and 3 were upregulated in the glycolysis pathway.

[0099] Furthermore, this invention constructed a core metabolite library of ZH11 leaves (containing 1116 metabolites) for high-throughput analysis. In the DGP1-OE1 and DGP1B-OE1 overexpression lines, 200 upregulated and 214 downregulated differentially accumulated metabolites were identified in DGP1-OE1 plants (FC>2, p<0.05), while 222 upregulated and 184 downregulated differentially accumulated metabolites were identified in DGP1B-OE1 plants. Venn diagrams showed significant overlap between the DGP1-OE1 and DGP1B-OE1 lines (319 differentially accumulated metabolites). Figure 12 (a) indicates that DGP1 and DGP1B have functional redundancy in regulating metabolism. In summary, overexpression of DGP1 or DGP1B can lead to a wide range of metabolic changes in rice leaves, including interference with basal metabolic processes, but promotes the biosynthesis of phenylpropane derivatives (such as flavonoids and diterpenoids).

[0100] 2.4 DGP1 family proteins control PHR2 or PHYA / PHYB-induced metabolic remodeling in rice leaves

[0101] This invention further investigates whether DGP1 signaling is involved in short-term metabolic responses induced by phosphorus deficiency or red light. Core targeted metabolomics analysis was performed on leaves of phr2 mutant, dgp1 mutant, and wild-type plants treated for 4 days with phosphorus deficiency. Venn diagrams showed that, under NP conditions, wild-type and dgp1 mutants shared only 137 differentially expressed metabolites, while wild-type and phr2 mutants shared only 96 differentially expressed metabolites. Figure 12 (b)

[0102] Subsequently, this invention analyzed red light (50 μM·m). -2 ·s -1 PhyA / PyB mutants, dgp1 / dgp1b mutants, and wild-type seedlings were treated for 4 days. Venn diagram analysis showed that, under RL conditions, wild-type and PhyA / PyB mutants shared only 108 differentially expressed metabolites, while wild-type and dgp1 / dgp1b mutants shared only 114 differentially expressed metabolites. Figure 12 (b)

[0103] This invention further focuses on the most significantly upregulated differentially accumulated metabolites (FC>4, p<0.05) in the flavonoid and phenolic amine subgroups of the phenylpropane derivative metabolome in phosphorus-deficient and red light-treated wild-type seedlings, and compares them with the phosphorus-deficient dgp1 mutant and the red light-treated dgp1 dgp1b mutant. The heatmap shows that the biosynthesis of these metabolites induced by phosphorus deficiency and red light was significantly impaired in the dgp1 and dgp1 dgp1b mutants, respectively. Figure 13 (b and c).

[0104] Under phosphorus deficiency and red light conditions, 197 differentially expressed metabolites in wild-type plants were co-regulated. The DGP1-OE1 line shared 222 differentially expressed metabolites (56.5%) with phosphorus-deficient wild-type plants, 204 differentially expressed metabolites (52%) with red light-treated wild-type plants, and only 87 differentially expressed metabolites (22.1%) with the glk1 glk2 mutant. Further comparison revealed the most significant upregulation of flavonoids and phenolic amines (FC>4, p<0.05) in the DGP1-OE1 line and the glk1 glk2 mutant. Heatmaps showed that these metabolites did not accumulate significantly in the glk1 glk2 mutant compared to the wild type. Figure 13 (a). Therefore, DGP1 signaling can significantly remodel the leaf metabolome in response to phosphorus deficiency and red light conditions, and this process is independent of GLK1 / 2 activity.

[0105] 2.5 PAL6 and PAL8 are essential for DGP1 activation of the general phenylpropanoid pathway in rice leaves.

[0106] Phenylalanine acts as a feedback inhibitor to suppress pyruvate kinase (PK) activity and is also a precursor to various secondary metabolites. Interestingly, compared to control conditions, both phosphorus deficiency and red light treatment increased phenylalanine levels in wild-type plants. Figure 13 (d and e). To investigate whether exogenous phenylalanine treatment triggers metabolic responses in rice leaves, this invention performed core-targeted metabolomics analysis on leaves of wild-type seedlings treated with 3 μM phenylalanine for 3 days. The results showed that 307 differentially accumulated metabolites (FC>2, p<0.05) were identified in the phenylalanine-treated wild-type leaves, with a pattern similar to the metabolic changes in the DGP1-OE1 line under control conditions. The heatmap shows the upregulated differentially accumulated metabolites (FC>4, p<0.05) in the flavonoid and phenolic amine subgroups of the phenylalanine-treated wild-type plants. Figure 13 (f)

[0107] Phenylalanine ammonia-lyases (PALs) are key enzymes in the universal phenylpropanoid pathway, catalyzing the conversion of phenylalanine to trans-cinnamic acid. Nine PAL genes have been identified in rice, of which only PAL6 and PAL8 are highly expressed in leaf tissues. The inventors hypothesized that PAL6 and PAL8 play a role in the DGP1-activated phenylpropanoid pathway in rice leaves, and therefore constructed a pal6-pal8 double mutant and a pal6-pal8 DGP1-OE1 line for metabolic-related genetic analysis. Figure 5 (b). EICs results showed that the phenylalanine content was slightly higher in the pal6 pal8 mutant compared with wild-type seedlings, while the phenylalanine content was significantly accumulated in the pal6 pal8 DGP1-OE1 line. Figure 5 (a). Sakuranetin, 3'-methoxyquercetin, and kumatakenin were the most significantly upregulated flavonoids in the DGP1-OE1 strain, while no significant accumulation of these substances was detected in the pal6 and pal8 DGP1-OE1 strains. Figure 5 (a). Therefore, double mutations in PAL6 and PAL8 lead to the accumulation of the substrate phenylalanine and inhibit DGP1-activated flavonoid biosynthesis. These results provide genetic evidence that DGP1 redirects glycolytic flux from the tricarboxylic acid cycle to the PEP-phenylalanine-phenylpropane pathway and activates the biosynthesis of phenylpropane derivative secondary metabolites. Figure 5 (c)

[0108] Y2H screening of DGP1 also identified GDCH, a key component of the mitochondrial GDC complex involved in the photorespiration pathway. In yeast systems, co-expression of the full-length sequences of DGP1, DGP1B, or DGP1-TIGR01589 with GDCH resulted in growth on tetra-deficient media, indicating a positive protein-protein interaction between the two. Figure 14 (a) In vitro pull-down experiments using DGP1-HIS or DGP1B-HIS fusion proteins and MBP-GDCH fusion proteins confirmed these interactions. Figure 14 (b) Emissions were detected in regions simultaneously expressing DGP1-nLUC and c-LUC-GDCH, while no signal was detected in tobacco leaves expressing c-LUC alone. Figure 14(c) The glycine cleavage system (GCS) consists of four subunit proteins: H, T, P, and L, among which the H protein GDCH plays a central role in co-regulating GCS activity with other subunits. Since GDCT and GDCP form stable complexes with GDCH in plants, this invention investigated the competitive relationship between DGP1 and GDCT / GDCP in GDCH binding. Pull-down experiments showed that GST-GDCT or GST-GDCP proteins interact with GDCH-HIS (…). Figure 3 (d). After the addition of recombinant MBP-DGP1, the band intensity corresponding to GST-GDCP decreased, while the band intensity corresponding to GST-GDCT remained unchanged. Figure 3 (d). This indicates that DGP1 preferentially competes with GDCP for GDCH binding. Further evidence shows that the acyl-binding domain of GDCH is crucial for DGP1 binding, as is the case for GDCP and DGP1B. Figure 14 (d) This means that DGP1 / DGP1B blocks GDCH, which may inhibit GDCP recruitment. Further analysis of glycine oxidation rate (GOR) in various mutants after NP or RL treatment showed a sharp decrease in GOR in WT seedlings, while the sensitivity of NP-treated mutants (dgp1 and phr2) or RL-treated mutants (dgp1, dgp1b, and phyA, phyB) was reduced. Figure 14 (e and f).

[0109] 2.6 ST-mediated protein repositioning system increases the content of two types of flavor compounds in Chinese cabbage (Brassica chinensis L.)

[0110] "Leafy vegetables" encompass a wide variety of species. Flavonoids offer potential health benefits, thus improving dietary structure by regulating flavonoid content in vegetables is of great significance. DGP1 overexpression can activate the synthesis of phenylpropane-derived flavonoids, but it inhibits photosynthesis and photorespiration by suppressing the activity of GLK and GDC, respectively. Based on these phenomena, this invention attempts to redesign the metabolic flux targeted by DGP1 to achieve genetic improvement of leafy vegetables. SIG5 is a nuclear-encoded chloroplast σ factor, whose N-terminal region mediates protein entry into chloroplasts. This invention constructs a chimeric protein ST-FLAG, which contains the N-terminal region of SIG5, the TIGR01589 domain, and a 3×FLAG tag ( Figure 6 (a and b). The latest version of AlphaFold3 can accurately simulate the structure of biomolecular complexes. Using this tool, three-dimensional models of the interactions between ST and PK4, and ST and GAPC1 were constructed. Figure 6 (c). Transient expression experiments showed that the ST-FLAG-GFP protein specifically localized to chloroplasts in the protoplasts of Chinese cabbage seedlings ( Figure 6 (d). This invention constructed DGP1-FLAG and ST-FLAG expression vectors driven by the GLK1 promoter of the photosynthetic gene, and obtained transgenic seedlings ( Figure 6 b, Figure 16 (a) Unlike the GLK1::ST-FLAG-1 / 2 (ST-FLAG-1 / 2) line, the GLK1::DGP1-FLAG-3 / 4 (DGP1-FLAG-3 / 4) line exhibits yellowish-green leaves under normal growth conditions. Figure 7 (d and e). Western blotting experiments showed that DGP1-FLAG was detected in the nuclei of DGP1-FLAG-3 / 4 seedlings, while ST-FLAG was detected in the chloroplasts of ST-FLAG-1 / 2 seedlings. Figure 16 (b) As expected, GAPC and PK interacting with TIGR01589 were observed in the nuclear portion of DGP1-FLAG-3 / 4 seedlings, while these proteins were observed in the chloroplast portion of ST-FLAG-1 / 2 seedlings. Figure 16 (c)

[0111] This invention compared and analyzed representative flavonoids and sugars in the leaves of DGP1-FLAG-3, ST-FLAG-2, and wild-type seedlings under field growing conditions. The heatmap showed that, compared to wild-type seedlings, the contents of most representative flavonoids in the leaves of both transgenic seedlings were significantly increased. Figure 7 (a). However, compared with wild-type seedlings, the sugar content (fructose, galactose, and sucrose) in ST-FLAG-2 seedlings was significantly increased, while the sugar content in DGP1-FLAG-3 seedlings showed no significant change. Figure 7 (a) Peak signal of EICs for metabolites (sakurain, fructose, galactose, and sucrose) Figure 7 (b) with heat Figure 1 In addition, the photosynthetic physiological parameter light-saturated photosynthetic rate (A) sat ) in DGP1-FLAG-3 (21.06±1.50 μmol CO2·m -2 ·s -1 ) and DGP1-FLAG-4 (20.69±1.76 μmol CO2·m -2 ·s -1 The concentration was significantly reduced in the ST-FLAG-1 strain (22.67 ± 1.52 μmol CO2·m3). -2 ·s -1 ) and ST-FLAG-2 (22.80±1.24 μmol CO2·m -2 ·s -1The strain was compared with the wild type (24.24±1.09 μmol CO2·m). -2 ·s -1 There was no statistically significant difference compared to () Figure 6 (e). The photorespiration parameter glycine oxidation rate (GOR) was significantly decreased in DGP1-FLAG-3 / 4 seedlings, but showed no significant change in ST-FLAG-1 / 2 seedlings. Figure 6 (e).

[0112] In summary, this invention demonstrates that members of the DGP1 family can dynamically reprogram primary and secondary metabolic pathways in rice in response to phosphorus deficiency and photoperiodic light conditions. Figure 15 (a and b) This explains the rapid and global remodeling of cellular metabolism in plant cells under environmental changes. Specifically, DGP1s modulate the photorespiration cycle by isolating a series of cytoplasmic enzymes for direct transport to the nucleus and by altering the composition of the GDC through interaction with the acyl-binding domain of the H subunit, thereby blocking the recruitment of the P subunit. The DGP1-mediated repositioning of the PK3-PK1 / 4 complex redirects its substrate PEP to phenylalanine, constitutively activating the phenylpropane-like pathway via PAL6 / 8.

[0113] The TIGR01589 domain, which is highly conserved in 13 monocots and 5 dicots, Figure 15 (c and d) indicate that the DGP1 family may widely induce environment-triggered metabolic reprogramming in higher plants. The artificial ST system localizes the rate-limiting glycolysis enzymes GAPC and PK to chloroplasts without significantly affecting GLK-mediated photosynthesis or GDC-mediated photorespiration. This strategy directs the substrate from central glycolysis towards flavonoid biosynthesis while simultaneously accumulating photosynthetically produced sugars (…) in the leaves of *Pyracantha fortuneana* seedlings. Figure 7 (f). This invention provides a new approach to flavor improvement in leafy vegetables by rationally designing targeted proteins to reposition and regulate flavor metabolites.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An artificial chimeric protein, characterized in that, The artificial chimeric protein includes the TIGR01589 domain of the rice DGP1 protein, the chloroplast localization sequence at the N-terminus of the rice SIG5 protein, and a FLAG tag. The amino acid sequence of the artificial chimeric protein is shown in SEQ ID NO.

2.

2. The application of the artificial chimeric protein as described in claim 1 in the improvement of vegetable flavor compounds, characterized in that, The flavor compounds in the vegetable are flavonoids and sugars, and the vegetable is bok choy.

3. A method for improving the flavor compounds of vegetables, characterized in that, The method includes constructing an expression vector expressing the artificial chimeric protein of claim 1, transferring it into vegetables, and obtaining transgenic seedlings that promote the accumulation of flavonoids and sugars in vegetable leaves; the vegetable is Chinese cabbage.

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