CRISPR-Cas9 mediated mutation in GRAIN WIDTH and WEIGHT2 (GW2) locus improves aleurone layer and grain nutritional quality in rice

Abstract

Enhancing crop productivity and their nutritional quality are the key components and primary focus of crop improvement strategy for fulfilling future food demand and improving human health. Grain filling and endosperm development are the key determinants of grain yield and nutritional quality. GRAIN WIDTH and WEIGHT2 (GW2) gene encodes a RING-type E3 ubiquitin ligase and determines the grain weight in cereal crops. Here we report GW2 knockout (KO) mutants in Indica (var. MTU1010) through CRISPR/Cas9 genome editing. The endosperm of GW2-KO mutant seed displays a thick aleurone layer with enhanced grain protein content. Further the loss of function of OsGW2 results in improved accumulation of essential dietary minerals (Fe, Zn, K, P, Ca) in the endosperm of rice grain. Additionally, the mutants displayed an early growth vigour phenotype with an improved root and shoot architecture. The hull morphology of GW2-KO lines also showed improved, grain filling thereby promoting larger grain architecture. Together, our findings indicate that GW2 may serve as a key regulator of improved grain architecture, grain nutritional quality and an important modulator of plant morphology. The study offers a strategy for the development of improved rice cultivars with enriched nutritional quality and its possible implementation in other cereals as well.

Figure 1

Structural organization of OsGW2, eSpCas9 expression cassette and mutation analysis. (A) Rice GW2 gene consisting of 8 exons interrupted by 7 introns and RING-U box domain highlighted blue. The structural organization of OsGW2 gene was graphically represented using exon–intron graph maker (http://wormweb.org/exonintron). The 20nt-sgRNA target site was selected at RING/U box domain. (B) Specific 20-nt sgRNA target sequence along with BsaI cloning sites. (C) sgRNA expression cassette showing rice U3 promoter and polIII terminator consisting two BsaI restriction sites separated by a 718 bp DNA block. (D) T-DNA border having genome editing tools (eSPCas9 and sgRNA) in pMDC99 vector. The lower figure (D) represents different stages of plant development following tissue culture methods. (E) PCR confirmation of T0 plants showing presence of Cas9 (887 bp) and hpt (954 bp) genes. The last figure (E) showing PCR amplification of 746 bp flanked sequence including the 20nt target region from the Cas9 positive plants for identification of INDEL mutations. (F) Genotyping confirmation of GW2-KO mutant lines showing C-insertion (GW2-KO1) and A-deletion (GW-KO2) mutation generated by Cas9 nuclease. The image was created by chromatogram viewer Chromas 2.6.6 software (http://technelysium.com.au/wp/chromas/).

Figure 2

Characterization aleurone density and grain protein content of GW2-KO mutant. Transversally sectioned (1–2 mm size) dehusked mature rice grains from WT and GW2-KO lines, stained with iodine solution for visualization aleurone morphology. The ventrolateral (A) and dorsolateral (B) section showing aleurone layer morphology of the WT and GW2-KO lines. Magnified visualization of ventrolateral (A1) and dorsolateral (B1) sections of rectangular box from (A, B). (C) The aleurone layer density of GW2-KO lines significantly p ≤ 0.01 (**) improved both in ventral and dorsal surface compared to WT seed. Arrowheads indicate the aleurone layer thickness. (D) The transversally sectioned (1–2 mm size) rice grains from WT and GW2-KO lines stained in Bradford reagent. The GW2-KO mutants showed high intense dark blue color compared to WT seed. The lower figure represents the closer view of aleurone layer of WT and GW2-KO lines. The aleurone layer of GW2-KO exhibits more protein bodies compared to WT. The scale bar represents 10 or 20 µm respectively. Data ± SD (n = 15). (E) Transversally sectioned of WT and GW-KO mature grains, stained in 0.05% (w/v) Safranin. Both WT and GW-KO showed single aleurone cell layer morphologies. The GW2-KO mutants exhibited larger cell size in the aleurone layer compare to WT seed. (F) The grain protein content of GW2-KO mutants was substantially increased (p ≤ 0.01) compared to WT (n = 15). (G-H) The UPLC-MS/MS analysis of GW2-KO seeds showed significantly p ≤ 0.01 (**) higher free amino acid accumulation (Ser, Gln, Lys, Asp and Asn) compared to WT. Data ± SD (n = 6).

Figure 3

Assessment and distribution of grain mineral content. (A) ICP-MS quantification mineral content (Fe, Zn, P, Ca and K) in the mature dehusked rice seeds. The seed iron (9–11%), zinc (13–15%), phosphorus (9–11%), calcium (8–10%) and potassium (6–7%) content were significantly p ≤ 0.01 (**) increased in the GW-KO rice lines. Data ± SD (n = 9). (B) The transverse cut section showing variation in the Prussian blue stain intensity among WT and GW2-KO lines. The GW2-KO lines accumulates improved iron content in the rice endosperm compared to WT seed. Scale bar 50 µm. (C) The dithizone stained longitudinally cross section of half seed form WT and GW-KO lines showing different degrees of zinc deposition. The GW-KO lines accumulates higher amount of zinc metal in the endosperm tissue compared to WT seed. Scale bar 100 µm. Microscopy was performed using Carl Zeiss stereoscopic zoom microscope (Discovery V8) attached with cooled digital camera.

Figure 4

Grain phenotype and agronomic performance of OsGW2. GW2-KO mutant lines showed improved hull morphology and seed architecture and grain width compared to WT plant (A). (B) The GW2-KO lines exhibited increased seed length and 100 grain volume corresponding to WT line. (C) Phenotyping of 30-day old GW2-KO lines showing improved root-shoot length and biomass corresponding to WT plant.