GmNF-YC4-2 Increases Protein, Exhibits Broad Disease Resistance and Expedites Maturity in Soybean

Abstract

The NF-Y gene family is a highly conserved set of transcription factors. The functional transcription factor complex is made up of a trimer between NF-YA, NF-YB, and NF-YC proteins. While mammals typically have one gene for each subunit, plants often have multigene families for each subunit which contributes to a wide variety of combinations and functions. Soybean plants with an overexpression of a particular NF-YC isoform GmNF-YC4-2 (Glyma.04g196200) in soybean cultivar Williams 82, had a lower amount of starch in its leaves, a higher amount of protein in its seeds, and increased broad disease resistance for bacterial, viral, and fungal infections in the field, similar to the effects of overexpression of its isoform GmNF-YC4-1 (Glyma.06g169600). Interestingly, GmNF-YC4-2-OE (overexpression) plants also filled pods and senesced earlier, a novel trait not found in GmNF-YC4-1-OE plants. No yield difference was observed in GmNF-YC4-2-OE compared with the wild-type control. Sequence alignment of GmNF-YC4-2, GmNF-YC4-1 and AtNF-YC1 indicated that faster maturation may be a result of minor sequence differences in the terminal ends of the protein compared to the closely related isoforms.

Keywords: NF-YC4 transcription factor; disease resistance; early maturation; seed protein.

Figure 1

GmNF-YC4-2 transcript level was increased in leaf and seed tissue in GmNF-YC4-2-OE plants. (A) Quantitative reverse transcription PCR of GmNF-YC4-2 transcript level. GUS staining of 35S-QQS-GUS soybean plants shows 35S drove expression of GUS in leaves (B), flowers (C), and pods/seeds (D). All data in bar chart shows mean ± SE (Standard Error), n = 3. A two-tailed Student’s t-test was used to compare GmNF-YC4-2-OE and WT, ** p < 0.01; * p < 0.05; ^• p < 0.1.

Figure 2

Leaf starch and protein composition was altered in GmNF-YC4-2-OE plants. Leaf starch content in GmNF-YC4-2-OE plants was decreased when compared to WT, indicated by starch staining (A) and quantification (B). (C) Leaf protein content was increased in GmNF-YC4-2-OE plants. All data in bar charts show mean ± SE, n = 3 or 4 (in C). A two-tailed Student’s t-test was used to compare GmNF-YC4-2-OE and WT, ** p < 0.01; * p < 0.05; ^• p < 0.1.

Figure 3

Seed composition was altered in GmNF-YC4-2-OE plants. (A) Seed protein content was significantly increased in all lines while oil content was significantly decreased. Composition was analyzed by near infrared spectroscopy (NIRS). (B) Ash content, crude fat and crude fiber were decreased compared to WT, while crude protein and total sugars were increased. Composition was analyzed by chemical methods. (C) No significant difference in seed yield was found. All data in bar charts show mean ± SE, (in A) n = 235 (WT), 125 (GmNF-YC4-2-OE 1), 155 (GmNF-YC4-2-OE 2), 81 (GmNF-YC4-2-OE 3), (in B) n = 3, (in C) n = 20 (WT), 24 (GmNF-YC4-2-OE 1), 17 (GmNF-YC4-2-OE 2), and 26 (GmNF-YC4-2-OE 3). A two-tailed Student’s t-test was used to compare GmNF-YC4-2-OE and WT; ** p < 0.01, * p < 0.05, ^• p < 0.1.

Figure 4

GmNF-YC4-2-OE plants showed enhanced disease resistance. (A) The BPMV viral foci rate was decreased at both 11 and 13 DPI in GmNF-YC4-2-OE plants compared to WT. (B) Growth of PsgR4 bacteria was reduced in GmNF-YC4-2-OE plants. CFU, colony forming units. (C) GmNF-YC4-2-OE plants also showed enhanced resistance to SDS. All data in bar charts show mean ± SE, (in A and B) n = 3, (in C) n = 5 (WT, GmNF-YC4-2-OE 1,2), 6 (GmNF-YC4-2-OE 3). A two-tailed Student’s t-test was used to compare GmNF-YC4-2-OE and WT; ** p < 0.01.

Figure 5

GmNF-YC4-2-OE plants transited from flowering stage to podding stage faster than WT plants. (A) GmNF-YC4-2-OE plants showed seeding pods while WT plants were still in the flowering stage at 77 DAP. (B) GmNF-YC4-2-OE plants senesced earlier than WT plants at 96 DAP. (C) The onset of flowering (DAP of first observed open flower) was slightly faster for GmNF-YC4-2-OE 1 plants but similar in GmNF-YC4-2-OE 2 and 3 plants compared to WT. (D) At 73 DAP, pod development for GmNF-YC4-2-OE plants was advanced compared to WT plants while the number of flowers was decreased. All data in bar charts show mean ± SE, (in C) n = 24 (WT), 46 (GmNF-YC4-2-OE 1), 39 (GmNF-YC4-2-OE 2), 26 (GmNF-YC4-2-OE 3); (in D) 15 (WT), 9 (GmNF-YC4-2-OE 1), 19 (GmNF-YC4-2-OE 2), 11 (GmNF-YC4-2-OE 3). A two-tailed Students t-test was used to compare GmNF-YC4-2-OE and WT; ** p < 0.01, * p < 0.05, ^• p < 0.1.

Figure 6

Protein sequence alignment of GmNF-YC4-2 with closest homolog in soybean (GmNF-YC4-1) and Arabidopsis (AtNF-YC1), neither of which has been shown to control maturation as we saw with GmNF-YC4-2. It is likely that sequence differences in the terminal ends (outlined in red) may be involved in GmNF-YC4-2′s function in early maturation. The ‘*’ at the end of the GmNF-YC4-1 and GmNF-YC4-2 sequences signifies a stop codon.