Overexpression of GmPHR1 Promotes Soybean Yield through Global Regulation of Nutrient Acquisition and Root Development

Abstract

MYB-CC transcription factors (TFs) are essential for plant growth and development. Members of the MYB-CC subfamily with long N terminal domains, such as phosphate starvation response 1 (PHR1) or PHR1-like TFs, have well documented functions, while those with short N terminal domains remain less understood. In this study, we identified a nodule specific MYB-CC transcription factor 1 (GmPHR1) in soybean that is different from other canonical PHR family genes in that GmPHR1 harbors a short N terminal ahead of its MYB-CC domain and was highly induced by rhizobium infection. The overexpression of GmPHR1 dramatically increased the ratio of deformed root hairs, enhanced subsequent soybean nodulation, and promoted soybean growth in pot experiments. The growth promotion effects of GmPHR1 overexpression were further demonstrated in field trails in which two GmPHR1-OE lines yielded 10.78% and 8.19% more than the wild type line. Transcriptome analysis suggested that GmPHR1 overexpression led to global reprogramming, with 749 genes upregulated and 279 genes downregulated, especially for genes involved in MYB transcription factor activities, root growth, and nutrient acquisition. Taken together, we conclude that GmPHR1 is a key gene involved in the global regulation of nodulation, root growth, and nutrient acquisition in soybeans, and is thus a promising candidate gene to target for soybean yield enhancement.

Keywords: PHR; growth promotion; nitrogen; nodulation; phosphate.

Figure 1

GmPHR1 and GmPHR16 were highly expressed in nodules and induced by rhizobium infection. (A). Schematic diagram of canonical and non-canonical PHRs with the position of MYB and coiled-coil domains marked in the proteins. (B). Expression levels of non-canonical GmPHRs in different tissues of soybean plants were compared using data downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/pz/ accessed on 1 September 2021). Expression levels of atypical GmPHRs presented in a heatmap. (C). Time course expression analysis of GmPHR1 and GmPHR16 inoculated with Bradyrhizobium elkanii BXYD3. (D). Positive soybean hairy roots carrying GmPHR1pro-GUS vector were generated as described in the methods. Hairy roots were inoculated with Bradyrhizobium elkanii BXYD3 for one month. Tissue expression patterns were determined with GUS histochemical staining and visualized with stereoscopic microscopes. For (C), asterisks represent statistically significantly differences from respective controls in the Student’s t test (*: 0.01 < p ≤ 0.05, **: 0.001 < p ≤ 0.01, ***: p ≤ 0.001).

Figure 2

Overexpression of GmPHR1 promotes soybean growth. (A). Growth performance of wild type and transgenic soybean seedling lines overexpressing GmPHR1 (OE1 and OE2). Bar = 5 cm. (B–D). Plant height (B), biomass (DW: dry weight) (C), and leaf SPAD values (D) were measured after one-month of growth. n = 10. (E–G). Total nitrogen (E), phosphorus (F), and potassium (G) contents of soybean plants in (F). (H–J). Total nitrogen (H), phosphorus (I), and potassium (J) concentrations of soybean plants in (F). For (B–J), asterisks represent statistically significantly differences from respective controls in the Student’s t test (*: 0.01 < p ≤ 0.05, **: 0.001 < p ≤ 0.01). ns: not significant at 0.05 value.

Figure 3

Overexpression of GmPHR1 promotes nodulation in soybeans. (A). Nodules on the roots of wild type and overexpressing transgenic (OE1 and OE2) lines of soybean seedlings. Bar = 1 cm. (B,C). Nodule number and nodule fresh weight of nodules in (A). (D). Observed deformed root hairs on wild type and two GmPHR1-OE lines inoculated with Bradyrhizobium elkanii BXYD3. (E). Comparison of deformed root hairs three days after Bradyrhizobium inoculation. For (B,C,E), asterisks represent statistically significantly differences from respective controls in the Student’s t test (*: 0.01 < p ≤ 0.05, **: 0.001 < p ≤ 0.01).

Figure 4

GmPHR1 overexpression promotes growth and increased yield under field conditions. (A). Growth performance of wild type and GmPHR1-OE transgenic soybean plants grown under field conditions at the R6 and R7 stages. Bars = 10 cm. (B,C). The biomass (DW: dry weight) and number of pods per plant for wild type and GmPHR1-OE transgenic soybean plants grown under field conditions. (D,E). The seed number and seed weight per plant of soybean in (A). For (B–E), asterisks represent statistically significantly differences from respective controls in the Student’s t test (*: 0.01 < p ≤ 0.05, **: 0.001 < p ≤ 0.01, ***: p ≤ 0.001).

Figure 5

Overexpression of GmPHR1 induces global transcriptional reprogramming in soybeans. (A,B). Differentially expressed genes (DEG) compared between wild type and GmPHR1-OE1 transgenic lines in a volcano plot (A) and heatmap (B). Significant expression change threshold was a fold change > 2 and an FDR adjusted p value < 0.05. (C). MYB transcriptional factors identified among DEGs. (D). Significant changes of genes relative to symbiotic signaling genes in soybeans between WT and OE. (E–G), Comparison of genes acting in nitrogen acquisition (E,F) and growth regulation (G) between wild type and GmPHR1-OE1 transgenic plants. For (E–G), asterisks represent statistically significantly differences from respective controls in the Student’s t test (*: 0.01 < p ≤ 0.05, **: 0.001 < p ≤ 0.01).