Screening and functional verification of drought resistance-related genes in castor bean seeds

Abstract

Drought is one of the natural stresses that greatly impact plants. Castor bean (Ricinus communis L.) is an oil crop with high economic value. Drought is one of the factors limiting castor bean growth. The drought resistance mechanisms of castor bean have become a research focus. In this study, we used castor germinating embryos as experimental materials, and screened genes related to drought resistance through physiological measurements, proteomics and metabolomics joint analysis; castor drought-related genes were subjected to transient silencing expression analysis in castor leaves to validate their drought-resistant functions, and heterologous overexpression and backward complementary expression in Arabidopsis thaliana, and analysed the mechanism of the genes' response to the participation of Arabidopsis thaliana in drought-resistance.Three drought tolerance-related genes, RcECP 63, RcDDX 31 and RcA/HD1, were obtained by screening and analysis, and transient silencing of expression in castor leaves further verified that these three genes corresponded to drought stress, and heterologous overexpression and back-complementary expression of the three genes in Arabidopsis thaliana revealed that the function of these three genes in drought stress response.In this study, three drought tolerance related genes, RcECP 63, RcDDX 31 and RcA/HD1, were screened and analysed for gene function, which were found to be responsive to drought stress and to function in drought stress, laying the foundation for the study of drought tolerance mechanism in castor bean.

Keywords: RcA/HD1; RcDDX31; RcECP63; Castor bean; Drought stress.

Fig. 1

Results of CAT activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 2

Results of SOD activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 3

Results of POD activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 4

Results of GST activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 5

Results of T-AOC activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 6

Results of MDA activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 7

Results of H₂O₂ activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 8

Results of Pro activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 9

Results of Pro activity assay of castor embryos with different treatments. Note: Different lower case letters represent significant differences (p < 0.05)

Fig. 10

Venn diagram of differentially expressed proteins

Fig. 11

Volcano plots of differentially expressed proteins. Note: A-D show the differentially expressed proteins screened out in the four comparison groups: P48_VS_P60, S48_VS_P48, S60_VS_P60, and S48_VS_S60, respectively. The green dots represent the differentially expressed proteins with downregulated expression, the red dots represent the differentially expressed proteins with upregulated expression, and the gray dots represent the detectable proteins with no significant changes in expression

Fig. 12

Hierarchical clustering analysis of differentially expressed proteins. Note: A-D show the results of hierarchical clustering analysis of the differentially expressed proteins in the four comparison groups P48_VS_P60, S48_VS_P48, S60_VS_P60, and S48_VS_S60, respectively

Fig. 13

Gene Ontology functional annotation analysis of differentially expressed proteins. Note: A-D show the annotation results of differentially expressed proteins in the four comparison groups P48_VS_P60, S48_VS_P48, S60_VS_P60, and S48_VS_S60, respectively

Fig. 14

Volcano plots of differentially expressed metabolites. Note: A-D show the volcano plots of the differentially expressed metabolites in the four comparison groups P48_VS_P60, S48_VS_P48, S60_VS_P60, and S48_VS_S60, respectively. VIP represents the significance of the difference in a given metabolite between groups. Log₂FC represents the fold change of the differentially expressed metabolite. The green dots represent the downregulated metabolites, the red dots represent the upregulated metabolites, and the gray dots represent the detectable metabolites with no significant changes in concentration

Fig. 15

Venn diagram of differentially expressed metabolites in groups P48_VS_P60, S48_VS_P48, S60_VS_P60, and S48_VS_S60

Fig. 16

KEGG enrichment analysis of the differentially expressed metabolites in group P48_VS_P60

Fig. 17

KEGG enrichment analysis of the differentially expressed metabolites in group P48_VS_S48

Fig. 18

KEGG enrichment analysis of the differentially expressed metabolites in group P60_VS_S60

Fig. 19

KEGG enrichment analysis of the differentially expressed metabolites in group S48_VS_S60

Fig. 20

Common metabolic pathways of the differentially expressed proteins and differentially expressed metabolites in group P48_VS_P60. Note: The abscissa represents the metabolic pathways, and the ordinate represents the number of differentially expressed proteins and differentially expressed metabolites simultaneously involved in each metabolic pathway

Fig. 21

Common metabolic pathways for the differentially expressed proteins and differentially expressed metabolites of group P48_VS_S48. Note: The abscissa represents the metabolic pathways, and the ordinate represents the number of differentially expressed proteins and differentially expressed metabolites simultaneously involved in each metabolic pathway

Fig. 22

Common metabolic pathways for the differentially expressed proteins and differentially expressed metabolites of group P60_VS_S60. Note: The abscissa represents the metabolic pathways, and the ordinate represents the number of differentially expressed proteins and differentially expressed metabolites simultaneously involved in each metabolic pathway

Fig. 23

RT-qPCR detection results of the genes encoding some differentially expressed proteins in castor plants in response to drought stress. Note: Different lowercase letters represent significant differences (P<0.05)

Fig. 24

Phenotypic changes of castor bean leaves after transient infection. Note: A1-A4 are the leaf states of wild-type castor leaves treated with 15% PEG 6000 for 0 h, 0 h, 24 h and 48 h respectively; B1-B4 are the leaf states treated with 15% PEG 6000 for 0 h, 24 h and 48 h before and after PTRV-RcECP 63 bacterial solution; C1-C4 are the leaf states treated with 15% PEG 6000 for 0 h, 24 h and 48 h before impregnation with PTRV-RcDDX 31 and after injection of PTRV-RcDDX 31 bacterial solution; D1-D4 are the leaf states treated with 15% PEG 6000 for 0 h, 24 h and 48 h before impregnation with PTRV-RcA/HD1 and after injection of PTRV-RcA/HD1 bacterial solution

Fig. 25

Expression analysis of RcECP 63 gene transiently infected castor leaves under drought stress. Note: Different lowercase letters represent significant differences(P < 0.05)

Fig. 26

Expression analysis of RcDDX 31 gene transiently infected castor leaves under drought stress. Note: Different lowercase letters represent significant differences (P < 0.05)

Fig. 27

Expression analysis of RcA/HD1 gene transiently infected castor leaves under drought stress. Note: Different lowercase letters represent significant differences (P < 0.05)

Fig. 28

Results of PCR identification of the resistant Arabidopsis thaliana plants with overexpression and complementary expression of RcECP63. Note: M1 and M2: DL 5000 Marker; 1-8: PCR identification results of the resistant complementary-expression A. thaliana plant ECP63-GR. 9-15: PCR identification results of the resistant overexpression A. thaliana plant ECP63-OE

Fig. 29

Results of PCR identification of the resistant Arabidopsis thaliana plants with overexpression and complementary expression of RcDDX31. Note: M1, M2, and M3: DL 5000 Marker; 1-7: PCR identification results of the resistant complementary-expression A. thaliana plant DDX31-GR1; 8-15: PCR identification results of the resistant complementary-expression A. thaliana plant DDX31-GR2; 16-23: PCR identification results of the resistant overexpression A. thaliana plant DDX31-OE

Fig. 30

Results of PCR identification of the resistant Arabidopsis thaliana plants with overexpression and complementary expression of RcA/HD1. Note: M1 and M2: DL 5000 Marker; 1-6: PCR identification results of the resistant complementary-expression A. thaliana plant A/HD1-GR; 7-13: PCR identification of the resistant overexpression A. thaliana plant A/HD1-OE

Fig. 31

RT-qPCR results of positive overexpression Arabidopsis thaliana plants. Note: *: 0.01 <P≤0.05, **: 0<P≤0.01

Fig. 32

RT-qPCR results of positive complementary-expression Arabidopsis thaliana plants. Note: *: 0.01 <P≤0.05, **: 0<P≤0.01

Fig. 33

Effect of different concentrations of mannitol on atecp63, ECP 63-GR, Col-0, and ECP 63-OE. Note: A-D represent the mannitol medium containing 0 mmol/L, 100 mmol/L, 200 mmol/L, and 300 mmol/L mannitol, respectively

Fig. 34

Germination rate, root length, and number of lateral roots of the four types of Arabidopsis thaliana plants by RcECP63 genotype. Note: Figure A shows the germination rate of A. thaliana under drought stress treatments with different mannitol concentrations; Figure B shows the root lengths of A. thaliana plants treated with different mannitol concentrations; Figure C shows the numbers of lateral roots of A. thaliana plants treated with different mannitol concentrations. Different lowercase letters represent significant differences (P<0.05)

Fig. 35

Effect of different mannitol concentration on atddx31-1, DDX31-GR1, Col-0, and DDX31-OE. Note: A-D represents the mannitol medium containing 0 mmol/L, 100 mmol/L, 200 mmol/L, and 300 mmol/L mannitol, respectively

Fig. 36

Analysis results of the germination rates, root lengths, and numbers of lateral roots of the four types of Arabidopsis thaliana corresponding to the RcDDX31 mutant atddx31-1. Note: Figure A shows the germination rates of A. thaliana drought stress treatments with different mannitol concentrations. Figure B shows the root lengths of A. thaliana plants under drought stress treatments with different mannitol concentrations. Figure C shows the numbers of lateral roots of A. thaliana plants under drought stress treatments with different mannitol concentrations. Different lowercase letters represent significant differences (P<0.05)

Fig. 37

Effects of different mannitol concentrations on atddx31-2, DDX31-GR2, Col-0, and DDX31-OE. Note: A-D represent the mannitol medium containing 0 mmol/L, 100 mmol/L, 200 mmol/L, and 300 mmol/L mannitol, respectively

Fig. 38

Analysis results of germination rates, root lengths, and numbers of lateral roots of the four types of Arabidopsis thaliana plants corresponding to the RcDDX31 mutant atddx31-2. Note: Figure A shows the germination rates of A. thaliana plants under drought stress treatments with different mannitol concentrations. Figure B shows the root lengths of A. thaliana plants under drought stress treatments with different mannitol concentrations. Figure C shows the number of lateral roots of A. thaliana plants under drought stress treatments with different mannitol concentrations. Different lowercase letters represent significant differences (P<0.05)

Fig. 39

Effect of different mannitol concentrations on ata/hd1, A/HD1-GR, Col-0, and A/HD1-OE. Note: A-D represents the mannitol medium containing 0 mmol/L, 100 mmol/L, 200 mmol/L, and 300 mmol/L, respectively

Fig. 40

Analysis results of the germination rates, root lengths, and number of lateral roots of the four types of Arabidopsis thaliana corresponding to RcA/HD1. Note: Figure A shows the germination rates of A. thaliana plants under drought stress treatments with different mannitol concentrations. Figure B shows the root lengths of A. thaliana plants under drought stress treatments with different mannitol concentrations. Figure C shows the numbers of lateral roots of A. thaliana plants under drought stress treatments with different mannitol concentrations. Different lowercase letters represent significant differences (P<0.05)

Fig. 41

Physiological indicators of four types of Arabidopsis thaliana plants with different RcECP63 genotypes under drought stress treatment with PEG 6000. Note: A: MDA measurement results of atecp63, ECP63-GR, Col-0, and ECP63-OE at 0 h, 24 h, and 48 h. B: PRO measurement results of atecp63, ECP63-GR, Col-0, and ECP63-OE at 0 h, 24 h, and 48 h. C: T-AOC measurement results of atecp63, ECP63-GR, Col-0, and ECP63-OE at 0 h, 24 h, and 48 h. D: Measurement results of hydroxyl radical scavenging ability of atecp63, ECP63-GR, Col-0, and ECP63-OE at 0 h, 24 h, and 48 h. different lowercase letters indicate significant differences (P<0.05)

Fig. 42

Physiological indicators of four types of Arabidopsis thaliana plants corresponding to the RcDDX31 gene under PEG 6000 stress. Note: A: MDA concentrations of atddx31-1, DDX31-GR1, atddx31-2, DDX31-GR2, Col-0, and DDX31-OE at 0 h, 24 h, and 48 h. B: Proline concentrations of atddx31-1 DDX31-GR1, atddx31-2, DDX31-GR2, Col-0, and DDX31-OE at 0 h, 24 h, and 48 h. C: T-AOC of atddx31-1, DDX31-GR1, atddx31-2, DDX31-GR2, Col-0, and DDX31-OE at 0 h, 24 h, and 48 h. D: Hydroxyl radical scavenging ability of atddx31-1, DDX31-GR1, atddx31-2, DDX31-GR2, Col-0, and DDX31-OE at 0 h, 24 h, and 48 h. Lowercase letters represent significant differences (P<0.05)

Fig. 43

Physiological indicators of four types of Arabidopsis thaliana according to the RcA/HD1 gene under PEG 6000 stress. Note: A: MDA concentration of ata/d1, A/HD1-GR, Col-0, and A/HD1-OE at 0 h, 24 h, and 48 h. B: Proline concentration of ata/d1, A/HD1-GR, Col-0, and A/HD1-OE at 0 h, 24 h, 48 h. C: T-AOC of ata/d1, A/HD1-GR, Col-0, and A/HD1-OE at 0 h, 24 h, and 48 h. D: Hydroxyl radical-scavenging ability of ata/d1, A/HD1-GR, Col-0, and A/HD1-OE at 0 h, 24 h, and 48 h. different lowercase letters represent significant differences (P<0.05)