Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12414-12419.

Increasing seed size and quality by manipulating BIG SEEDS 1 in legume species

Liangfa Gea, Jianbin Yub,c, Hongliang Wanga,d, Diane Luthe, Guihua Baib, Kan Wange, Rujin Chena,1

aNoble Research Institute, Ardmore, Oklahoma 73401; bUSDA/ARS/Hard Winter Wheat Genetics Research Unit, Manhattan, KS 66506; cPresent address: Pioneer Hi-Bred International, Inc., Willmar, MN 56201-9608; dPresent address: USDA/ARS/Crop Genetics and Breeding Research, Tifton, GA 31793; eCenter for Plant Transformation, Plant Sciences Institute, and Department of Agronomy, Iowa State University, Ames, IA 50011;

1Corresponding author: Rujin Chen, Noble Research Institute, Ardmore, Oklahoma 73401. Tel: (580) 224-6730, rchen@noble.org

 

Abstract

Plant organs such as seeds are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that has been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a novel and conserved role for BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight and amino acid content. Our results provide a new strategy for the increase of yield and seed quality in legumes.

PMID: 27791139

 

Supplement 

Medicago truncatula big seeds1 (mtbs1) mutants exhibit enlarged organs including seeds, seed pods and leaves

Medicago truncatula big seeds1-1 (bs1-1) mutant was isolated from a fast neutron bombardment (FNB)-induced deletion mutant collection of Medicago truncatula (cv. Jemalong A17) (1). The mtbs1-1 mutant exhibited larger seeds, seed pods and leaves than wild-type plants, suggesting a key role of BS1 in determining lateral organ size (Figures 1, 2) (1). Time-course experiments show that developing seeds at stages before 9 days post anthesis (DPA) were indistinguishable between wild-type and the mtbs1-1 mutant. After 9 DPA, mtbs1-1 seeds were significantly larger than wild-type seeds (1). Seed pods were also larger in the mutant, even at stages before 9 DPA (Figure 1). Mature seeds of the mtbs1-1 mutant were 49% heavier than wild-type seeds (1).

 

In the mtbs1-1 mutant, both terminal and lateral leaflets of trifoliate leaves were dramatically increased in length, width, perimeter and area compared with wild-type counterparts (Figure 2). In addition, the shape of leaflets was also altered. In contrast to the flat leaflets of wild-type plants, mtbs1-1 leaflets were dome-shaped with a positive Gaussian curvature (2) and could be flattened only by cutting the edges, suggesting uneven lamina growth (Figure 2).

 

An increase in organ size may result from an increase in cell proliferation and/or cell expansion, two successive processes contributing to the final organ size (3, 4). Measurements show that epidermal cells of fully expanded cotyledons were indistinguishable in size between mtbs1-1 and wild-type (A17) plants, consistent with the observation that cell proliferation rather than cell expansion is altered in the mtbs1-1 mutant compared with wild type plants (Figure 3).

 

Cell division rate measurements show that the first, newly-emerged leaf (L1) in both wild type (A17) and mtbs1-1 was similarly undergoing rapid cell proliferation with the highest cell division rate (Figure 4). In the second and third leaves (L2 and L3), cell division rate was rapidly decreased in A17. By contrast, cell division rate was gradually decreased in L2 and L3 in the mtbs1-1 mutant, supporting an extended cell proliferation activity in developing leaves in the mtbs1-1 mutant compared with wild type (Figure 4). In agreement with the observed cell division rates, the size of epidermal cells in L1 was not different between A17 and mtbs1-1 (Figure 4). By contrast, epidermal cells in L2 were larger in wild type than the mtbs1-1 mutant, consistent with the notion that a majority of epidermal cells undergo cell proliferation in L2 in the mtbs1-1 mutant and a majority of epidermal cells already undergo cell expansion in L2 in wild type (Figure 4). Collectively, these results show that the enlarged organ phenotype was caused by a prolonged cell proliferation activity in the mtbs1-1 mutant.

 

 

Figure 1. Development of seed pods in wild type (A17, top) and the mtbs1-1 mutant (bottom). Numbers denote days post anthesis (DPA); M, mature stage. Two representative seed pods are shown for each time point. Scale bar, 1 cm. Adapted from Ge et al., (2016) Proc Natl Acad Sci U S A 113, 12414-12419.

 

 

Figure 2.M. truncatula bs1-1 mutant exhibited increased leaf size. (A, B) Shoot apices of two-month-old wild type (A17; A) and mtbs1-1 mutant (B). (C-H) The fourth visible leaf from the shoot apex in A17 (C, D) and mtbs1-1 (E-H). (I-N) The sixth visible leaf from the shoot apex in A17 (I, J) and mtbs1-1 (K-N). (C, E, G, I, K, M), adaxial view; (D, F, H, J, L, N), abaxial view. (G, H, M, N) leaflet margin was cut to flatten leaf blades. Scale bars, 1 cm. Adapted from Ge et al., (2016) Proc Natl Acad Sci U S A 113, 12414-12419.

 

 

Figure 3. Enlarged cotyledon size is caused by an increase of cell number but not cell size in the mtbs1-1 mutant. (A) Morphology of fully expanded cotyledons in three-week-old wild type (A17) and mtbs1-1. (B) Quantification of cotyledon area in A17 and mtbs1-1. Shown are means ± s.d. for n=12. (C, D) SEM images f abaxial epidermal cells of fully expanded cotyledons in A17 (C) and mtbs1-1 (D). (E, F) Quantification of abaxial epidermal cell area (E) and cell number (F) of fully expanded cotyledons in A17 and mtbs1-1. Shown are means ± s.d. for n=60. Adapted from Ge et al., (2016) Proc Natl Acad Sci U S A 113, 12414-12419.

 

 

Figure 4. Cell division rate in A17 and mtbs1-1. (A, B) Morphology and growth of the first (L1), second (L2), third (L3) and fourth (L4) leaf in A17 (A) and mtbs1-1 (B) in 24 hours. Top panels, leaves at t = 0 h; bottom panels, leaves at t = 24 h. (C-F) SEM images of abaxial epidermal cell of the first leaf (C, E) and second leaf (D, F) in A17 (C, D) and mtbs1-1 (E, F). (G) Measurements of cell division rates of the first four leaves (L1-L4) in A17 and mtbs1-1. (H) Measurements of cell areas of the first two leaves in A17 and mtbs1-1. Shown are means ± s.d. for n≥ 60. **, Student’s t-test, p<0.001. Scale bars, 1 cm (A, B); 100 μm (C-F). Adapted from Ge et al., (2016) Proc Natl Acad Sci U S A 113, 12414-12419.

 

 

Reference:

1. Ge L, et al. (2016) Increasing seed size and quality by manipulating BIG SEEDS1 in legume species. Proc Natl Acad Sci U S A 113(44):12414-12419.
2. Nath U, Crawford BCW, Carpenter R, & Coen E (2003) Genetic Control of Surface Curvature. Science 299(5611):1404-1407.
3. Hepworth J & Lenhard M (2014) Regulation of plant lateral-organ growth by modulating cell number and size. Curr Opin Plant Biol 17:36-42.
4. Gonzalez N, et al. (2010) Increased leaf size: different means to an end. Plant Physiol 153(3):1261-1279.