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Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed

The root growth angle defines how roots grow towards the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience and as a consequence yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin independent higher responsiveness to gravity compared to wild type plants. We cloned the EGT2 gene by a combination of bulked segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. Subcellular localization experiments revealed that EGT2 localizes to the nucleus and cytoplasm. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA-seq, we observed that seven expansin genes were transcriptionally downregulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.Significance StatementTo date the potential of utilizing root traits in plant breeding remains largely untapped. In this study we cloned and characterized the ENHANCED GRAVITROPISM2 (EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain containing protein. We demonstrated that EGT2 is a key gene of root growth angle regulation in response to gravity which is conserved in barley and wheat and could be a promising target for crop improvement in cereals.

In this study, the targetting-induced local lesions in genomes approach was used to identify mutants for genes related to starch metabolism in barley. Starch is the major reserve of plants and serves as primary carbohydrate component in human and livestock diets and has also numerous industrial applications. Mutants for biosynthetic or regulatory genes of starch metabolism often produce starch granules with abnormal morphological and molecular features that could be of interest for technological applications. We report the identification of 29 mutations in five starch-related barley genes (Bmy1,GBSSI,LDA1,SSIandSSII) through the molecular screening of TILLMore, a sodium azide-mutagenized population. Almost all the mutations detected were CG–TA transitions and several (c.60%) implied a change in amino-acid sequence and therefore possible phenotypic effects. Four mutants showed non-sense or splice-junction alterations, which could drastically affect the protein function.

A sodium azide-mutagenized population of barley (cv. 'Morex') was developed and utilized to identify mutants at target genes using the 'targeting induced local lesions in genomes' (TILLING) procedure. Screening for mutations at four agronomically important genes (HvCO1, Rpg1, eIF4E and NR) identified a total of 22 new mutant alleles, equivalent to the extrapolated rate of one mutation every 374 kb. All mutations except one were G/C to A/T transitions and several (approximately 68%) implied a change in protein amino acid sequence and therefore a possible effect on phenotype. The high rate of mutation detected through TILLING is in keeping with the high frequency (32.7%) of variant phenotypes observed amongst the M(3) families. Our results indicate the feasibility of using this resource for both reverse and forward genetics approaches to investigate gene function in barley and related crops.

The amylose/amylopectin ratio has a major influence over the properties of starch and determines its optimal end use. Here, high amylose durum wheat has been bred by combining knock down alleles at the two homoelogous genes encoding starch branching enzyme IIa (SBEIIa-A and SBEIIa-B). The complete silencing of these genes had a number of pleiotropic effects on starch synthesis: it affected the transcriptional activity of SBEIIb, ISA1 (starch debranching enzyme) and all of the genes encoding starch synthases (SSI, SSIIa, SSIII and GBSSI). The starch produced by grain of the double SBEIIa mutants was high in amylose (up to ∼1.95 fold that of the wild type) and contained up to about eight fold more resistant starch. A single nucleotide polymorphism adjacent to the splice site at the end of exon 10 of the G364E mutant copies of both SBEIIa-A and SBEIIa-B resulted in the loss of a conserved exonic splicing silencer element. Its starch was similar to that of the SBEIIa double mutant. G364E SBEIIa pre-mRNA was incorrectly processed, resulting in the formation of alternative, but non-functional splicing products.

In this study, a forward-genetics analysis was performed on a portion of TILLMore, a chemically mutagenized population of barley cv. ‘Morex’ (http://www.distagenomics.unibo.it/TILLMore/), to identify root morphology alterations by comparison with ‘Morex’ wild-type. For this purpose, a simple paper-roll approach was performed to identify phenotypic variants at the seedling stage. The analysis of c. 1000 M4 families allowed us to identify c. 70 lines with altered root morphology. A more accurate phenotypic characterization of a portion of the mutant lines has been performed using stereomicroscopy and a scanning electron microscopy approach.

In this study, the targetting-induced local lesions in genomes approach was used to identify mutants for genes related to starch metabolism in barley. Starch is the major reserve of plants and serves as primary carbohydrate component in human and livestock diets and has also numerous industrial applications. Mutants for biosynthetic or regulatory genes of starch metabolism often produce starch granules with abnormal morphological and molecular features that could be of interest for technological applications. We report the identification of 29 mutations in five starch-related barley genes (Bmy1, GBSSI, LDA1, SSI and SSII) through the molecular screening of TILLMore, a sodium azide-mutagenized population. Almost all the mutations detected were CG‐TA transitions and several (c. 60%) implied a change in amino-acid sequence and therefore possible phenotypic effects. Four mutants showed non-sense or splice-junction alterations, which could drastically affect the protein function.

A durum wheat TILLING (targeting induced local lesions in genomes) population of 2601 M3 families was developed from cv. Svevo using ethyl methanesulfonate as a chemical mutagen. The entire M3 population was field-grown for phenotypic evaluations. Despite the polyploid nature of the wheat genome, a preliminarily phenotypic screening showed a high frequency of morphological alterations (~22%); specific phenotyping for seed morphology was undertaken. Furthermore, a reverse-genetics experiment was performed on DNA collected from M2 leaves for the homoeologous genes SBEIIa-A and SBEIIa-B involved in starch metabolism. One non-sense mutation for both genes was identified; specific crosses are planned in order to pyramid the two mutations.

Significance To date, the potential of utilizing root traits in plant breeding remains largely untapped. In this study, we cloned and characterized the ENHANCED GRAVITROPISM2 (EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain–containing protein. We demonstrated that EGT2 is a key gene of root growth angle regulation in response to gravity, which is conserved in barley and wheat and could be a promising target for crop improvement in cereals., The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain–containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.