GmNF-YC4-2

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This entry is adapted from the peer-reviewed paper 10.3390/ijms22073586

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.

NF-YC4 transcription factor early maturation disease resistance seed protein

1. Introduction

Effectively feeding an ever-growing global population begins with nutrient rich, resilient crops. Over one in seven people in the world do not have access to a sufficient protein supply in their diets ^[1]. In addition, the majority of the population relies on a plant-based diet for their protein uptake ^[2]. As most livestock also requires plant-based diets for their protein, the dietary needs of the world can mostly be attributed to plants. Thus, generating plants with increased protein content can help efficiently feed the global population.

Crop plants such as Glycine max (soybean) are grown in a wide range of environments and face a multitude of both biotic and abiotic challenges. While plants have evolved a complex immune system ^[3]^[4]^[5]^[6]^[7], crops across the world suffer yield loss due to diseases ^[8]^[9]^[10]^[11] and environmental factors ^[12]^[13]. Many genetic engineering methods have been used to combat these factors ^[8]^[14]. However, many of these induce constitutively active defense responses that can in turn negatively affect the growth and yield of the plant ^[15]^[16]. For example, silencing mitogen-activated protein kinase MAPK4 in soybean plants severely stunts the plants when providing a resistance to pathogens ^[17]. Therefore, genetic engineered resistance that allows for an enhanced pathogen resistance while avoiding major growth defects is important.

While plants have evolved a complex immune system to deal with minor biotic and abiotic perturbations, they have also evolved a more drastic mechanism to deal with seasonal environmental changes. As plants are sessile organisms, rather than maintaining full function throughout a harsh season, they instead undergo programmed cell death called senescence ^[18]^[19]^[20]. This process allows for nutrient re-localization, such as nitrogen remobilization from leaves to seeds, and it is important for seed quality and nitrogen use efficiency ^[21]^[22]^[23]^[24]. With the looming threat of climate change and the growing global population, the challenge set before us is to improve crop nitrogen use efficiency in order to allow for less reliance on nitrate fertilizers ^[22]. It has been speculated that stimulating autophagy during stress response could improve crops’ resistance to the effects of climate change ^[22].

2. GmNF-YC4-2-OE Plants Have High Transcript Levels of GmNF-YC4-2 in Leaves and Seeds

To confirm the efficacy of our OE vectors, the transcript level of GmNF-YC4-2 was assessed in leaves and seeds. The transcript level of GmNF-YC4-2 in leaves of GmNF-YC4-2-OE lines ranged from 12.3-16.4 average relative quantification (RQ) compared to wild type (WT) (Figure 1A). That magnitude was increased in seeds where the average RQ ranged from 31.0-69.3 (Figure 1A). The validity of the 35S driven OE vectors used in this study was assessed by GUS (β-glucuronidase) staining of 35S-QQS-GUS constructs in soybean. Moderate to high levels of GUS signal were present in leaves, flowers, pods and seeds, indicating 35S activity in these tissues (Figure 1B–D) and confirming previous studies demonstrating no tissue specificity for the CaMV 35S promoter ^[25].

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.

3. GmNF-YC4-2 Is Involved in Regulation of Plant Composition

Similar to previous studies in GmNF-YC4-1-OE plants ^[26], GmNF-YC4-2-OE plants also showed altered leaf and seed composition. GmNF-YC4-2-OE plants had a decrease in leaf starch (Figure 2A,B, p < 0.05 for GmNF-YC4-2-OE 1,2 and p < 0.1 for GmNF-YC4-2-OE 3) and leaf protein content was increased (Figure 2C, p < 0.01 for GmNF-YC4-2-OE 1 and p < 0.05 for GmNF-YC4-2-OE 2,3).

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.

Metabolic analysis on GmNF-YC4-2-OE seeds was performed to determine if this metabolic effect in leaf tissue was unique. Near Infrared Spectroscopy (NIRS) analysis on seeds showed a significant increase in protein levels and decreased oil levels in three independent GmNF-YC4-2-OE lines compared to WT plants (Figure 3A, p < 0.01 for all three lines). Chemical analysis on crushed seeds was performed to test levels of ash, crude fat, crude fiber, crude protein, and total sugars. Of these metabolites, ash (p < 0.01, < 0.1, and < 0.05, respectively), crude fat (p < 0.01 for all), and crude fiber (p < 0.05 for all) showed a significant decrease in OE lines, while protein (p < 0.01 for all) and total sugar (p < 0.05 for GmNF-YC4-2-OE 1 and 3) levels were increased (Figure 3B). In addition, GmNF-YC4-2-OE plants had an even higher seed protein content, approximately 5–10%, when compared to GmNF-YC4-1-OE plants (Figure S1, p < 0.01). Interestingly, there was no significant difference in seed yield per plant between WT and OE plants (Figure 3C). Therefore, GmNF-YC4-2-OE plants display similar metabolic alterations in both leaf and seed tissue.

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.

4. GmNF-YC4-2 Confers Broad Disease Resistance

To test if GmNF-YC4-2-OE could affect interactions between soybean and pathogens, WT and transgenic plants were inoculated with a virus, bacterium, and fungus. GmNF-YC4-2-OE plants inoculated with a recombinant Bean pod mottle virus (BPMV) expressing green fluorescent protein (GFP) had reduced numbers of green fluorescent infection foci when compared to WT plants at both 11 and 13 days post inoculation (DPI) (Figure 4A, p < 0.01). The GmNF-YC4-2-OE plants were also infected with Pseudomonas syringae pv. glycinea Race 4 (PsgR4), the cause of bacterial blight. The growth of PsgR4 was decreased by 96.3%, 96.8%, and 92.8% in GmNF-YC4-2-OE 1, GmNF-YC4-2-OE 2, and GmNF-YC4-2-OE 3 plants compared to Williams 82 control plants (Figure 4B, all p < 0.01).

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.

The plants were planted in the field and inoculated with the fungus that leads to sudden death syndrome (SDS)-Fusarium virguliforme. OE plants also displayed less severe symptoms of SDS in a field inoculation trial with a 67.8%, 67.8%, and 88.1% decrease in foliar disease index for GmNF-YC4-2-OE 1, GmNF-YC4-2-OE 2, and GmNF-YC4-2-OE 3 respectively (Figure 4C, all p < 0.01). These data show that overexpression of GmNF-YC4-2 in soybean plants confers enhanced disease resistance to the viral, bacterial, and fungal pathogens tested along with the altered leaf and seed composition.

5. GmNF-YC4-2 Regulates Plant Maturation

GmNF-YC4-2-OE plants displayed several aspects of expedited growth. In the field, GmNF-YC4-2-OE plants showed significantly faster pod development compared to the WT plants (Figure 5A). OE plants of all three lines also showed earlier senescence compared to WT (Figure 5B). As the date of flowering was similar for both WT and OE (Figure 5C), the faster maturation was seen mainly in the transition from flowering stage to pod development. GmNF-YC4-2-OE plants contained more pods than WT at 73 Days After Planting (DAP), while WT contained more flowers (Figure 5D, flowers: p < 0.01 for GmNF-YC4-2-OE 2, <0.05 for GmNF-YC4-2-OE 3; pods: p < 0.01 GmNF-YC4-2-OE 1 and GmNF-YC4-2-OE 3, < 0.1 for GmNF-YC4-2-OE 2). Thus, GmNF-YC4-2-OE plants transited quickly from flowering to pod development and this likely impacted senescence independent of flowering time. OE plants fully matured around two weeks prior to WT plants.

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.

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