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

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.

TbMYC4A Is a Candidate Gene Controlling the Blue Aleurone Trait in a Wheat-Triticum boeoticum Substitution Line

Triticum boeoticum Boiss (AbAb, 2n = 2x = 14) is one of the sources of the blue grain trait controlled by blue aleurone layer 2 (Ba2). However, the underlying genes have not been cloned. In this study, a transcriptomic comparison between a blue-grained wheat-T. boeoticum substitution line and its wheat parent identified 41 unigenes related to anthocyanin biosynthesis and 29 unigenes related to transport. The bHLH transcription factor gene TbMYC4A showed a higher expression level in the blue-grained substitution line. TbMYC4A contained the three characteristic bHLH transcription factor domains (bHLH-MYC_N, HLH and ACT-like) and clustered with genes identified from other wheat lines with the blue grain trait derived from other Triticeae species. TbMYC4A overexpression confirmed that it was a functional bHLH transcription factor. The analysis of a TbMYC4A-specific marker showed that the gene was also present in T. boeoticum and T. monococcum with blue aleurone but absent in other Triticeae materials with white aleurone. These results indicate that TbMYC4A is a candidate gene of Ba2 controlling the blue aleurone trait. The isolation of TbMYC4A is helpful for further clarifying the genetic mechanism of the blue aleurone trait and is of great significance for breeding blue-grained wheat varieties.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Factors influencing physical dormancy and its elimination in two legumes

Introduction: Prosopis laevigata (Humb. & Bonpl. ex Willd.) M. C. Johnst, P. glandulosa Torr., Vachellia schaffneri (S. Watson) Seigler & Eibinger, V. pennatula (S. Watson) Seigler & Eibinger and V. farnesiana (L.) Wight & Arn. are characteristic species of semi-arid areas. Their seeds show physical dormancy and are naturally scarified by chewing, trampling, digestive tract of fauna, fire, or washing away during rains. Objective: To describe the morphology of the seed coat of three species of Vachellia and two of Prosopis, and to assess the chemical, mechanical and thermal scarification of seeds. Materials and methods: Chemical (HCl for 30, 120, 150 and 180 min), thermal (80, 100, 120 and 140 °C for 3 min) and mechanical (sanding) scarification were applied. Resistance to breaking by compression was measured. The experimental design was randomized complete blocks per species. Results and discussion: Seeds showed a layer of lignified and impermeable macrosclereids, but the aleurone layer could not be detected. Sanding allowed germination from 81.2 to 100 %. Chemical and thermal treatments showed no differences, only in the case of P. laevigata, chemical scarification for 180 min caused higher germination (72.5 %) compared to the control. Vachellia schaffneri seeds were more resistant to compression (669 N) and had more intense dormancy (0.83), while P. glandulosa had the lowest dormancy (0.42). Conclusions: Mechanical scarification was the best method to eliminate dormancy in Vachellia and Prosopis seeds.

Laser Microdissection Transcriptome Data Derived Gene Regulatory Networks of Developing Rice Endosperm Revealed Tissue- and Stage-specific Regulators Modulating Starch Metabolism

Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Laboratory germinability and germination energy of spelt grain depending on fertilization and storage

Spelt is a promising crop for processing, as valuable micronutrients are evenly distributed in its grain, while in modern wheat bread varieties they are concentrated in the hull, aleurone layer and germ. Its proteins contain more gliadins and fewer glutenins, making gluten weak, but better digestible in the human body. Purpose and objectives: to assess the germinability and germination energy of spelt depending on the types, doses and timing of nitrogen fertilization and storage period. Materials and methods. The study was conducted in the Laboratory of Evaluation of Grain and Grain Product Quality of the Chair of Grain Storage and Processing Technologies of Uman National University of Horticulture. We studied winter spelt variety Yevropa created by hybridization of Triticum aestivum L. with Triticum spelta L. Fertilizers were ammonium nitrate, ammonium sulfate, granular superphosphate, and potassium chloride. Results and discussion. The study found that before storage the germination energy of grain was 87–90% depending on fertilization. Nitrogen fertilizers significantly increased this indicator. When grain was stored for 30 and 90 days, the germination energy was the highest and amounted to 98–99%. Longer storage significantly decreased this parameter. It was the lowest after P60K60 application: 95%, 92% and 87% when grain was stored for 180, 270 and 360 days, respectively. Before and after 30- and 90-days storage of spelt grain, this parameter amounted to 99% and regardless of fertilizers. The lowest laboratory germinability was observed after 1-year storage of grain (81–84% depending on fertilizers). This indicator was the highest with P60 + N120, amounting to 84%, and the lowest without fertilizers or with N60S70 + N60. Conclusions. The germination energy of spelt grain was found to be the highest after 30- to 180-day storage (96-99%). This indicator was most affected by nitrogen fertilizers. After 360-day storage, the germination energy reduced to 87–91% depending on the type and timing of nitrogen fertilization. The laboratory germinability of spelt grain of 97–99% maintained from harvesting to storage day 180. When spelt grain was stored in sealed containers without temperature control, this indicator gradually decreased to 81–84% for 360 days of storage, depending on fertilizers

Morphological features of rye grain with low pentosan content

Background. The grain coat plays a major role in the development of rye cultivars with low levels of water-soluble pentosans (WSP). Grain coat thinness is a diagnostic trait for low WSP (arabinoxylans) content. To improve the technology of low-pentosan rye breeding, it becomes important to study the effect of changes in the anatomy of low-pentosan grains on morphological characteristics.Materials and methods. Grains with thin coats (transparent) were identified with the LFS-1 diaphanoscope in the populations of 7 commercial winter rye cultivars grown in Russian Federation. Anatomical and morphological features of seed coats were studied on thick- and thin-coated grains identified in the rye cultivars ‘Era’ and ‘Vyatka’. The thickness of grain coats and the aleurone layer were assessed according to L. N. Lyubarsky.Results and conclusions. In thin-coated rye grains, we found a reduction in the thickness of the pericarp and seed coats by 50–70% and in the aleurone layer by 32.1–39.6%, compared to thick-coated grains. With a constant cell size in “transparent” grains, a decrease in the thickness of the aleurone layer and coat occurs at the expense of the reduction in not only the cell walls, but also the intercellular space. The pericarp in the tissues of the second multicellular layer contains the bulk of the protective biologically passive WSP. It was proved that there were no significant changes in grain parameters, such as length, width and relative volume, depending on coat thinness. Significant differences were found in the 1000 grain weight. In “transparent” grains the index increased by 5.2–19.7%, compared with “nontransparent” ones.

Red Wheat Alters Colon Cancer Risk, Oxidative Stress, and the Gut Microbiome

Objectives Red wheat, the class of wheat used to make yeast bread products, is associated with reductions in colon cancer biomarkers, regardless of refinement state. We hypothesized that red wheat as well as the phenol-rich aleurone and testa layers of red wheat would reduce colonic precancerous lesions and oxidative stress, and beneficially modulate the gut microbiome in rats with diet-induced obesity. Methods Rats were divided into seven groups (12/group) and fed a normal fat diet (NFD), high-fat diet (50% of total kcal as fat, HFD), whole red wheat + HFD (whR + HFD), refined red wheat + HFD (refR + HFD), refined white wheat + HFD (refW + HFD), aleurone layer + HFD (AL + HFD), or testa layer + HFD (TL + HFD). After a 14-day adaptation period, rats received two i.p. injections of the colon-specific carcinogen 1,2-dimethylhydrazine (DMH), administered one week apart. Sixty-three days after the second injection, colons were harvested and precancerous lesions (aberrant crypt foci, ACF) were enumerated. Staining intensity of 3-nitrotyrosine (3-NT) was determined immunohistochemically in distal colon tissue. Microbial DNA from cecal contents was sequenced using a 16S rRNA metagenomic approach. Differences in alpha and beta diversity, and microbial abundances were determined. Results Compared to the NFD, the HFD had a greater number of ACF, regardless of size (i.e., AC/ACF). The refR + HFD had significant reductions in medium ACF (3–5 AC/ACF; 2.62 vs. 4.28), large ACF (≥6 AC/ACF; 0.06 vs. 0.45), ACF multiplicity (1.58 vs. 2.01) and 3-NT (% positivity per ACF; 2.06% vs. 4.51%) compared to the HFD. All diets containing wheat reduced large ACF number. The TL + HFD and AL + HFD demonstrated trends for reducing ACF with 8 AC (0.06 vs. 0.18) and 3-NT (2.22% vs. 4.51%), respectively, compared to the HFD. Beta diversity significantly differed between diet groups (R2 = 0.27, P = 0.001), and there was greater abundance of Faecalitalea, Fusicatenibacter, and Lactobacillus in the cecal contents of rats fed wheat-containing diets. Conclusions Red wheat reduces precancerous lesions, oxidative stress, and beneficially modulates the gut microbiome relative to a non-wheat diet. The phenol-rich testa and aleurone layers alone had little influence on these outcomes. Funding Sources NDSU Collaborative Seed Grant Program.