Grain yield is a highly polygenic trait determined by the number of grains per unit area, as well as by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped a grain weight QTL using wild emmer introgressions in a durum wheat background, and showed that grain weight is associated with the GNI-A1 gene, a regulator of floret fertility. In-depth characterization of grain number and grain weight indicated that suppression of distal florets by the wild emmer GNI-A1 allele increase weight of proximal grains in basal and central spikelets due to alteration in assimilate distribution. Re-sequencing of GNI-A1 in tetraploid wheat demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which was present in two gene copies and contained a non-synonymous mutation in the C-terminus of the protein. Using an F2 population generated from a cross between wild emmer accessions Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique polymorphism is associated with grain weight, independent of grain number. Moreover, we showed, for the first time, that GNI-A1 proteins are transcriptional activators and that selection targeted compromised activity of the protein. Our finding expand the knowledge of the genetic basis underlying trade-off between key yield components and may contribute to breeding efforts for enhanced grain yield.
15Grain yield is a highly polygenic trait determined by the number of grains per unit area, as well as 16 by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, 17 the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped 18 a grain weight QTL using wild emmer introgressions in a durum wheat background, and showed 19 that grain weight is associated with the GNI-A1 gene, a regulator of floret fertility. In-depth 20 characterization of grain number and grain weight indicated that suppression of distal florets by 21 the wild emmer GNI-A1 allele increase weight of proximal grains in basal and central spikelets 22 due to alteration in assimilate distribution. Re-sequencing of GNI-A1 in tetraploid wheat 23 demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which 24 was present in two gene copies and contained a non-synonymous mutation in the C-terminus of 25 the protein. Using an F2 population generated from a cross between wild emmer accessions 26 Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique 27 polymorphism is associated with grain weight, independent of grain number. Moreover, we 28 showed, for the first time, that GNI-A1 proteins are transcriptional activators and that selection in 29 domesticated wheat targeted compromised activity of the protein. Our finding expand the 30 knowledge of the genetic basis underlying trade-off between key yield components and may 31 contribute to breeding efforts for enhanced grain yield. 32 33 Recently, the quantitative trait locus Grain Number Increase 1 (GNI1) was identified and 62 characterized as a homeodomain leucine zipper class I (HD-Zip I) transcription factor. Reduced 63 function mutation (N105Y) within the conserved homeodomain of GNI-A1, and knockdown of 64 4 GNI1 in transgenic hexaploid wheat, indicated that it is a suppressor of floret fertility. Transcript 65 abundance of GNI-B1 (orthologous copy on B genome) was negligible in floral organs of tetraploid 66 and hexaploid wheat, and was suggested to be pseudogenized in the genome of ancestral Aegilops 67 species (Sakuma et al. 2019). 68 Potential GW, defined as the intrinsic capacity of the grain to accumulate dry matter 69 (Bremner and Rawson 1978), is relatively low in distal grain positions. Several studies indicated 70 that the negative relationship between average GN and GW is an outcome of the high proportion 71 of low potential GW in high yielding cultivars (Acreche and Slafer 2006; Ferrante et al. 2015; 72 Fischer 2008; Miralles and Slafer 1995). Alternatively, Brenner and Rawson (Bremner and 73 Rawson 1978) showed that distal grains have similar potential GW as proximal grains. In addition, 74 removal of florets prior to grain filling increased weight of the remaining grains (Calderini and 75 Reynolds 2000b; Fischer and HilleRisLambers 1978), suggesting a degree of growth limitation 76 imposed by competition for insufficient source. 77 The physiological...
Developing sensory modules for specific molecules of interest represents a fundamental challenge in synthetic biology and its applications. A somewhat generalizable approach for this challenge is demonstrated here by evolving a naturally occurring chemically induced heterodimer into a genetically encoded sensor for herbicides. The interaction between PYRABACTIN-RESISTANT-like receptors and type-2C protein phosphatases is induced by abscisic acid—a small-molecule hormone in plants. We considered abscisic acid receptors as a potential scaffold for the development of biosensors because of past successes in their engineering, a structurally defined ligand cavity and the availability of large-scale assays for their activation. A panel of 475 receptor variants, mutated at ligand-proximal residues, was screened for activation by 37 herbicides from several classes. Twelve compounds activated at least one member of the mutant panel. To facilitate the subsequent improvement of herbicide receptors through directed evolution, we engineered a yeast two-hybrid platform optimized for sequential positive and negative selection using fluorescence-activated cell sorting. By utilizing this system, we were able to isolate receptors with low nanomolar sensitivity and a broad dynamic range in sensing a ubiquitous group of chloroacetamide herbicides. Aside from its possible applicative value, this work lays down conceptual groundwork and provides infrastructure for the future development of biosensors through directed evolution.
Summary Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 μm, which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.
The yeast two-hybrid (Y2H) assay is widely used for protein–protein interaction characterization due to its simplicity and accessibility. However, it may mask changes in affinity caused by mutations or ligand activation due to signal saturation. To overcome this drawback, we modified the Y2H system to have tunable protein expression by introducing a fluorescent reporter and a pair of synthetic inducible transcription factors to regulate the expression of interacting components. We found that the application of inducers allowed us to adjust the concentrations of interacting proteins to avoid saturation and observe interactions otherwise masked in the canonical Y2H assay, such as the abscisic acid-mediated increase in affinity of monomeric abscisic acid receptors to the coreceptor. When applied in future studies, our modified system may provide a more accurate characterization of protein–protein interactions.
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