Silencing of an Ubiquitin Ligase Increases Grain Width and Weight in indica Rice

Abstract

Many quantitative trait loci (QTLs) have been identified by molecular genetic studies which control grain size by regulating grain width, length, and/or thickness. Grain width 2 (GW2) is one such QTL that codes for a RING-type E3 ubiquitin ligase and increases grain size by regulating grain width through ubiquitin-mediated degradation of unknown substrates. A natural variation (single-nucleotide polymorphism at the 346th position) in the functional domain-coding region of OsGW2 in japonica rice genotypes has been shown to cause an increase in grain width/weight in rice. However, this variation is absent in indica rice genotypes. In this study, we report that reduced expression of OsGW2 can alter grain size, even though natural sequence variation is not responsible for increased grain size in indica rice genotypes. OsGW2 shows high expression in seed development stages and the protein localizes to the nucleus and cytoplasm. Downregulation of OsGW2 by RNAi technology results in wider and heavier grains. Microscopic observation of grain morphology suggests that OsGW2 determines grain size by influencing both cell expansion and cell proliferation in spikelet hull. Using transcriptome analysis, upregulated genes related to grain size regulation have been identified among 1,426 differentially expressed genes in an OsGW2_RNAi transgenic line. These results reveal that OsGW2 is a negative regulator of grain size in indica rice and affects both cell number and cell size in spikelet hull.

Evaluation of Factors Affecting In Planta Gene Editing Efficiency in Wheat (Triticum aestivum L.)

“…Three gRNAs previously shown to demonstrate on-target cleavage activity in wheat were selected and cloned into pBUN421 plasmid using the Golden Gate assembly system. , gRNAs #1 and #2 target the first and last exons of the TaGW2 gene (Figure ), respectively, involved in suppression of the seed size in monocots (rice, wheat, etc. ). − Whereas gRNA#1 targets three homeologs (TraesCS6A02G189300, TraesCS6B02G215300, and TraesCS6D02G176900), gRNA#2 is specific for subgenomes A and D (Figure A). gRNA#3 targets the first exon of the B and D copies of the TaLPX-1 gene (TraesCS4B02G037700 and TraesCS4D02G035200, respectively), a susceptibility gene for Fusarium graminearum .…”

Cytological, transcriptome and miRNome temporal landscapes decode enhancement of rice grain size

“…A recent example for this is GW2 , which has been well characterized to be a negative regulator of GS in japonica rice [ 79 ]. Despite the regulatory SNP being absent in indica rice, this gene functions in a similar manner [ 80 ].…”

“…Total RNA was isolated from five seed developmental stages of the two rice genotypes LGR and SN, namely, S1 (0–2 DAP), S2 (3–4 DAP), S3 (5–10 DAP), S4 (11–20 DAP), and S5 (21–29 DAP) using a seed-specific protocol [ 176 ] with few modifications, as previously described [ 37 , 52 , 80 , 177 ]. Briefly, 100 mg tissue was taken by pooling equal amounts of seed tissue from the respective DAP constituting a stage.…”

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

“…The involvement of E3 Ubiquitin Ligases in plant development and the hormone signalling processes were well documented both in dicots and monocots 29 . Recently, using transcriptome analysis revealed about 1426 differentially expressed genes in an OsGW2 RNAi transgenic line 59 . Among these, 115 genes were identified with specific functions including seed, leaf, root, and shoot development, cell cycle regulation and hormone signaling pathway.…”