The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stress tolerance

Abstract

Drought and salt stresses impose major constraints on soybean production worldwide. However, improving agronomically valuable soybean traits under drought conditions can be challenging due to trait complexity and multiple factors that influence yield. Here, we identified a nuclear factor Y C subunit (NF-YC) family transcription factor member, GmNF-YC14, which formed a heterotrimer with GmNF-YA16 and GmNF-YB2 to activate the GmPYR1-mediated abscisic acid (ABA) signalling pathway to regulate stress tolerance in soybean. Notably, we found that CRISPR/Cas9-generated GmNF-YC14 knockout mutants were more sensitive to drought than wild-type soybean plants. Furthermore, field trials showed that overexpression of GmNF-YC14 or GmPYR1 could increase yield per plant, grain plumpness, and stem base circumference, thus indicating improved adaptation of soybean plants to drought conditions. Taken together, our findings expand the known functional scope of the NF-Y transcription factor functions and raise important questions about the integration of ABA signalling pathways in plants. Moreover, GmNF-YC14 and GmPYR1 have potential for application in the improvement of drought tolerance in soybean plants.

Keywords: CRISPR/Cas9; NF-Y transcription factor; drought; interaction identification; salt stress; signal transduction; transgenic soybeans.

Figure 1

Stress tolerance of different soybean plants at seedling stage. (a, b) Identification of CRISPR/Cas9‐Gmnf‐yc14 soybean mutant plants. (c–f) Phenotypic analysis of different plants under drought stress (c); 3 days after re‐watering (d); 3 days after salt treatment (e); 5 days after salt treatment (f); fresh weight of the aerial parts and roots, as well as chlorophyll content under drought (g–i) and salt (j–l) stresses. The green bar indicates the average of the two soybean mutant lines; the orange bar indicates the average of the two overexpressing lines in the histogram

Figure 2

Stress resistance of different soybean plants at the flowering and adult stage. (a–e) Phenotypic analysis of the different plants at the flowering stage under conditions of drought and salt stresses (a); MDA and proline content under conditions of drought (b, c) and salt (d, e) stresses. Blue and orange bars indicate the average of the two Gmnf‐yc14‐KO lines and two GmNF‐YC14‐OE lines, respectively. (f) Phenotypic analysis of the plants at the adult stage under drought stress conditions. (g–k) Number of pods per plant (g); plant height (h); grain weight per plant (i); stem base circumference (j); and grain number per plant (k). (l, m) Phenotypic analysis of seeds under conditions of drought stress at the adult stage (l) and the relative proportions of different seed types (m)

Figure 3

Interaction between candidate proteins and GmNF‐YC14. (a–c) Interaction analysis of GmNF‐YA16, GmNF‐YC14, and GmNF‐YB2 proteins by yeast two‐hybrid (a) and three‐hybrid (b, c) assays. (d–f) Luciferase complementation assay to verify the interaction. YA16, GmNF‐YA16; YB2, GmNF‐YB2; YC14, GmNF‐YC14. (g–i) GST‐pulldown assay of the three proteins. (j, k) Stress tolerance analysis of GmNF‐YA16 and GmNF‐YB2 candidate protein genes. (l–o) Physiological and biochemical index analysis of GmNF‐YA16‐ and GmNF‐YB2‐overexpressing soybean hairy root composite plants under drought and salt stress conditions

Figure 4

Differentially expressed genes in GmNF‐YC14‐overexpressing and WT plants. (a) Biological process analysis of differentially expressed genes (DEGs). (b) Pathway enrichment analysis of the DEGs. The red stars indicate the stress‐responsive genes were enriched, and which were related to response to stimulus and hormone signal transduction. (c) qPCR of the functional genes identified from de novo transcriptome data under drought conditions. (d–g) LUC activity assay of the interactions between GmNF‐YC14 and potential target gene promoters. (h) Vector construction map. (i, j) Relative LUC activity assay of the interactions between GmNF‐YC14 and potential target gene promoters. proNAC4, GmNAC4 gene promoter; proDHN15, GmDHN15 gene promoter; proMYB84, GmMYB84 gene promoter; proRD22, GmRD22 gene promoter; proABF2, GmABF2 gene promoter; proABF3, GmABF3 gene promoter; and proABF4, GmABF4 gene promoter

Figure 5

Stress tolerance of candidate proteins and interactions between the NF‐Y transcription factor and the potential target gene GmPYR1. (a, b) GmPYR1 expression in GmNF‐YA16‐ and GmNF‐YB2‐overexpressing soybean hairy roots under drought and salt stresses. (c) Vector construction diagram. (d–f) Relative LUC activity assay (d), LUC activity assay (e, f), and EMSA (g) of the interactions between NF‐Y transcription factor subunit proteins and GmPYR1 target gene promoters. C14, GmNF‐YC14; B2, GmNF‐YB2; and A16, GmNF‐YA16. (h, i) Phenotypic analysis of GmPYR1‐OE soybean plants at the seedling stage under drought (h) and salt (i) stress conditions. (j–m) Fresh weight of the aerial parts of GmPYR1‐OE plants and the chlorophyll content of GmPYR1‐OE seedlings under drought (j–k) and salt (l–m) stresses

Figure 6

Drought tolerance of GmPYR1‐overexpressing soybean plants. (a) Phenotypic analysis of GmPYR1‐OE adult soybean plants under drought stress, (b–f) Number of pods per plant (b); plant height (c); stem base circumference (d); grain number per plant (e); and grain weight per plant (f) of GmPYR1‐OE and WT soybean plants under drought stress conditions. (g) Phenotypic analysis of GmPYR1‐OE seeds under conditions of drought stress at the adult stage. (h) Relative content of different seed types of the GmPYR1‐OE and WT soybean plants under drought conditions at the adult stage

Figure 7

Interaction between the soybean PYR1 protein and ABF transcription factors. (a) EMSA identified the interaction between the ABF transcription factor and four stress‐responsive genes. (b) ABF proteins expressed in tobacco leaves. (c) Empty vector GFP‐tag proteins were expressed in tobacco leaves and lacked phosphorylation activity as evidenced in the Phos‐tag assay. (d‐f) Phos‐tag assay of ABF proteins after 2 h (d, e) and 4 h (f) of ABA treatment. (g, h) Expression of ABF proteins in tobacco leaves after 2 and 4 h of ABA treatment. (i, j) Phosphorylation activity of ABF proteins after 2 h and 4 h of ABA treatment when soybean PYR1 was transformed into tobacco leaves. (k, l) Expression of ABF proteins after co‐transformation in tobacco leaves with soybean PYR1 protein. (m–p) Relative LUC activity of the interactions between soybean PYR1 and ABFs under ABA treatment. PP: λ protein phosphatase

Figure 8

Diagram summarizing soybean tolerance to drought and salinity stresses through NF‐Y‐mediated regulation of the ABA signal transduction pathway