Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Plant Science
Non Technical Summary
High grain yield and high grain quality are the two most important and desirable traits in cereal crop production. Grain size is a major determinant of grain yield in wheat and also affects the quality and market values of wheat grain products. This proposal is focused on characterization of genes that affect grain size and grain quality in wheat and barley. We will characterize a Growth-Regulating Factor 4 (GRF4) gene, which may play a key role in grain size regulation in wheat. The project was proposed on the basis of the recent studies on the rice GRF4 gene, which is one of the most important genes regulating grain size in rice. Wheat has three copies of the GRF4 gene in its hexaploid genome. We will determine how each of them regulate grain development in wheat. A better understanding of grain size formation will facilitate wheat breeders in development of new wheat cultivars with increased grain yield and improved grain quality. Enhanced grain productivity and improved grain quality will eventually help the agricultural industry to compete in the international wheat grain market.We will also perform biochemical studies on a barley mutant that produces high beta-glucan in grain and attempt to clone the gene underlying this mutation in barley. Development of new cultivars with elevated beta-glucan in the grain has been a highly pursued goal of barley breeding programs in Idaho and across the nation. Beta-glucan is a dietary fiber found in some cereal crops such as oats and barley. It is of great beneficial effects on human health. It is a soluble fiber and can be digested partially and absorbed slowly in the human intestines. Supplementation of beta-glucan in diets can reduce blood sugar and cholesterol levels and is highly recommended for patients with heart and diabetic conditions. Wild type barley cultivars contain about 8% beta-glucan in grain, whereas our high beta-glucan mutant CM1 contains as high as 18% beta-glucan in grain. Molecular cloning of the high beta-glucan gene will provide a useful tool for breeders in development of high beta-glucan cultivars. The biochemical information generated from this study will be very beneficial in food barley cultivar development by better manipulating the metabolic pathways related to food barley quality. Development of new barley cultivars with elevated beta-glucan in grains will help Idahoan barley growers in expansion of improved barley grain products in the international barley market.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Seed yield and grain quality are two of the most important factors affecting grain production in cereal crops. Understanding the genetic bases and molecular mechanisms underlying flower formation and grain development will eventually help breeders in selection of new cultivars with elevated grain yield potentials and improved grain quality. Our current knowledge on flower and grain development has come mostly from intense studies of the model laboratory plant Arabidopsis thaliana, which has no direct farming application. In addition, Arabidopsis is a dicot, and is very different in anatomy and regulatory mechanisms of flowers and grains compared to monocots. The molecular mechanisms underlying flower and grain development in wheat and barley are poorly understood. The long-term goal of this research is to understand how flower and seed development in cereal crops is regulated at the molecular level. The immediate objectives of this Hatch project will be focused on the identification and characterization of the GRF4 gene that may regulate grain size in wheat. We will also perform biochemical characterization of a barley mutant that accumulates beta-glucan in the grain, and provide biochemical evidence for molecular cloning of the causal gene underlying the high beta-glucan trait in barley. We will pursue the following specific objectives in next five years:(1) Evaluation of the natural variation in the GRF4 DNA sequence on grain size in wheat. We will select representative wheat germplasm lines that develop either large grains or small grains, and investigate if the natural grain size variation is correlated with polymorphisms in GRF4. Primers corresponding to each of the three GRF4 cDNAs will be used to amplify GRF4 cDNAs from the 10 wheat representative lines. The total 30 GRF4 cDNAs will be sequenced and analyzed in comparison with the control cultivar. It is expected that the 5 germplasms with large grains may contain some characteristic changes in DNA sequence in GRF4 genes.(2) Creation of a dominant mutation in one of the wheat GRF4 gene. The rice GRF4 mutant contains a change of two base-pairs (from TT to AA) in the third exon of the GRF gene. The mutated gene encodes a peptide that replaces a highly conserved amino acid residue Serine with a Lysine, which is likely to serve as a constitutively active GRF4. In addition, the mutant mRNA of GRF4 becomes more stable because the mutation also eliminates a microRNA binding site. The dual effects of the mutation allow the mutated GRF4 behalves in a dominant manner. We will clone the wheat GRF4 cDNA and create an identical mutation using the PCR-based mutagenesis approach.(3) Evaluation of the dominant active allele of GRF4 on grain size formation in wheat. The mutated GRF4 cDNA will be expressed under the GRF4 gene promoter. This expression construct will be made in a T-DNA vector and used for wheat transformation via Agrobacterium-mediated approach. Alternatively, gene-editing of the GRF4 genes via the CRISPR-Cas9 method could also be carried out. Because all three wheat GRF4 genes are conserved in the target region of gene-editing, this CRISPR-Cas9 approach could potentially change the two base-pairs from TT to AA in all three GRF4 genes simultaneously. (4) Evaluation of AGPase enzyme activity in the high beta-glucan CM1 mutant in barley. AGPase enzyme activities will be assayed in seedling leaves and developing seeds in CM1 in comparison with the wildtype control CDC Alamo. The AGPase activity is expected to be reduced in CM1 mutant, which will support the conclusion that the mutation in the AGPase gene causes the elevated beta-glucan content in CM1.(5) Determination of the contents of starch and key sugar intermediates in developing barley seeds. The contents of starch and ADP-glucose are expected to decrease; the contents of glucose-1-phosphate and UDP-glucose are expected to rise in CM1, which will support the hypothesis that both starch and beta-glucan compete for the same sugar source for their biosynthesis.(6) Determination of the expression levels of the AGPase gene during vegetative growth and seed development in the barley CM1 mutant. The mRNA transcripts of both the large and small subunits of AGPase will be measured by qRT-PCR. The levels of these transcripts are not expected to vary significantly in CM1, which would suggest the mutation in CM1 reduces the AGPase enzyme activity, but does not affect the stability of the transcripts.
Project Methods
A combination of genetic, molecular biology and biochemical approaches will be applied to this project to investigate the potential biological functions of GRF4 genes in grain size regulation in wheat, and roles of an AGPase gene in control of beta-glucan biosynthesis and accumulation in barley.(1) Evaluation of the natural variation in the GRF4 DNA sequence on grain size in wheat. Seeds of wheat lines with large and small grains will be chosen from The National Small Grains Collection at USDA-ARS, Aberdeen ID. We will select a total of 20 representative wheat germplasm lines, 10 lines having large grains and 10 lines with small grains. We will investigate if the natural grain size variation is correlated with polymorphisms in GRF4. Wheat seedlings will be grown in a greenhouse. Total RNA will be isolated and cDNA will be prepared. Primers specific to each of the three wheat GRF4 genes will be synthesized and used for amplification of the full-length GRF4 coding regions from the 20 wheat representative lines. The GRF4 cDNAs will be sequenced and compared. Potential correlations between the grain sizes and GRF4 DNA sequence variations will be analyzed. It is expected that the 10 germplasms with large grains may contain some characteristic changes in DNA sequence in GRF4 genes. To verify if the characteristic changes in GRF4 are indeed associated with grain size, we may use the CRISPR-Cas9 gene editing approach to create identical changes and observe if these changes in GRF4 can cause grain size change in wheat. We have the reagents, experience and facilities necessary to carry out the proposed work (Xie et al., 2011; Wang et al., 2012; 2013; Wang et al 2016). (2) Creation of a dominant mutation in one of the wheat GRF4 gene. The rice GRF4 mutant contains a change of two base-pairs (from TT to AA) in the third exon of the GRF4 gene. The mutated gene encodes a peptide that replaces a highly conserved amino acid residue Serine with a Lysine, which is likely to serve as a constitutively active GRF4. In addition, the mutant GRF4 mRNA also becomes more stable because the mutation eliminates a microRNA binding site. The dual effects of the mutation allow the mutated GRF4 to behave genetically in a dominant manner. We will create an identical mutation (from TC to AA) in the wheat GRF4 cDNA using the PCR-based mutagenesis approach. This substitution would create a mutated peptide product in which a highly conserved Serine (S) residue (at amino acid position 163) would be replaced with a Lysine (K), generating a constitutively active dominant allele of GRF4. We will also create a mutant allele with synonymous codons, in which the miR396-priming site is disrupted, but protein sequence is not changed. This would allow us to differentiate the importance of the miR396-priming site from that of the change in the key residue (S163K). Rice GRF4 forms a heterodimer with OsGIF, and acts as a transcription factor required for expression of a set of genes that are implicated in grain development (Chen et al., 2019). We will develop an in vivo system in yeast cells, in which the transcription activity of the GRF4 variants will be tested for their ability to drive expression of a GUS reporter under the promoter of a GRF4-responsive gene identified in our previous work (Chen et al., 2019).(3) Evaluation of the dominant active allele of GRF4 on grain size formation in wheat. We will create an expression construct in which the mutated GRF4 cDNA will be driven by the GRF4 gene promoter. This expression construct will be made in a T-DNA vector and used for wheat transformation via Agrobacterium-mediated approach. Alternatively, gene-editing of the GRF4 genes via the CRISPR-Cas9 method could also be carried out. Because all three wheat GRF4 genes are conserved in the target region of gene-editing, this CRISPR-Cas9 approach could potentially change the two base-pairs from TT to AA in all three GRF4 genes simultaneously. Transgenic wheat seedlings will be analyzed for expression of the GRF4 mutant allele, and grain size changes will be scored. The mRNA levels of the GRF4 mutant allele will also be assayed using real-time PCR. Transgenic wheat plants are expected to express and maintain an elevated level of the GRF4 mutant allele, and the grain size is expected to increase. If time permits, we may initiate direct genome editing via CRISPR-Cas9, to create transgenic wheat plants expressing the Serine-163-Lysine (S163K) mutation from all three GRF4 genes. (4) Evaluation of AGPase enzyme activity in the high beta-glucan CM1 mutant in barley. Seeds of the high beta-glucan mutant CM1 and its wild-type control CDC Alamo will be obtained from our collaborator Dr. G. Hu, USDA-ARS, Aberdeen ID. Barley plants will be grown in a greenhouse. Developing seeds will be harvested 14, 21, 28 and 32 days post anthesis (DPA). Seedling leaves will be sampled 10, 15, 20 and 25 days post germination (DPG). Samples will be frozen in liquid nitrogen and stored in -80°C. Crude extract will be used for assays of AGPase activity as described previously (Smith, 1988; 1990; Johnson et al., 2003). AGPase enzyme activities will be assayed in seedling leaves and developing seeds in CM1 in comparison with the wild-type control CDC Alamo. The AGPase activity is expected to be reduced in CM1 mutant, which will support the conclusion that the mutation in the AGPase gene causes the elevated beta-glucan content in CM1.(5) Determination of the contents of starch and key sugar intermediates in developing seeds. For starch quantification, pellet of endosperm extract will be washed by ethanol and acetone, and starch content will be measured as described (Smith, 1988). Glucose content will be assayed using Megazyme D-glucose GOPOD Assay Kit (Christensen and Scheller, 2012). The contents of starch and ADP-glucose are expected to decrease; the contents of glucose-1-phosphate and UDP-glucose are expected to rise in CM1, which will support the hypothesis that both starch and beta-glucan compete for the same sugar source for their biosynthesis.(6) Determination of the expression levels of the AGPase gene in CM1. For evaluating AGPase gene expression during seed development, total RNA will be extracted using the RNAase mini kit. cDNA will be synthesized from mRNA using random primers (SuperScript, Invitrogen). Quantitative real-time PCR will be performed using real-time TaqMan technology (Derveaux et al., 2010; Bustin et al., 2010). Real-time PCR probes and PCR primers will be designed using the primer express software (Cheng et al. 2016). Actin gene will be used as an internal control. The mRNA transcripts of both the large and small subunits of AGPase will be measured by qRT-PCR. The levels of these transcripts are not expected to vary significantly in CM1, which would suggest the mutation in CM1 reduces the AGPase enzyme activity, but does not affect the stability of the transcripts.