The auxin efflux carrier PIN1a regulates vascular patterning in cereal roots.

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.

ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana.

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

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 ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Determination of wheat spike and spikelet architecture and grain traits using X-ray Computed Tomography imaging.

BACKGROUND: Wheat spike architecture is a key determinant of multiple grain yield components and detailed examination of spike morphometric traits is beneficial to explain wheat grain yield and the effects of differing agronomy and genetics. However, quantification of spike morphometric traits has been very limited because it relies on time-consuming manual measurements. RESULTS: In this study, using X-ray Computed Tomography imaging, we proposed a method to efficiently detect the 3D architecture of wheat spikes and component spikelets by clustering grains based on their Euclidean distance and relative positions. Morphometric characteristics of wheat spikelets and grains, e.g., number, size and spatial distribution along the spike can be determined. Two commercial wheat cultivars, one old, Maris Widgeon, and one modern, Siskin, were studied as examples. The average grain volume of Maris Widgeon and Siskin did not differ, but Siskin had more grains per spike and therefore greater total grain volume per spike. The spike length and spikelet number were not statistically different between the two cultivars. However, Siskin had a higher spikelet density (number of spikelets per unit spike length), with more grains and greater grain volume per spikelet than Maris Widgeon. Spatial distribution analysis revealed the number of grains, the average grain volume and the total grain volume of individual spikelets varied along the spike. Siskin had more grains and greater grain volumes per spikelet from spikelet 6, but not spikelet 1-5, compared with Maris Widgeon. The distribution of average grain volume along the spike was similar for the two wheat cultivars. CONCLUSION: The proposed method can efficiently extract spike, spikelet and grain morphometric traits of different wheat cultivars, which can contribute to a more detailed understanding of the sink of wheat grain yield.

Preparation, Scanning and Analysis of Duckweed Using X-Ray Computed Microtomography.

Quantification of anatomical and compositional features underpins both fundamental and applied studies of plant structure and function. Relatively few non-invasive techniques are available for aquatic plants. Traditional methods such as sectioning are low-throughput and provide 2-dimensional information. X-ray Computed Microtomography (µCT) offers a non-destructive method of three dimensional (3D) imaging in planta, but has not been widely used for aquatic species, due to the difficulties in sample preparation and handling. We present a novel sample handling protocol for aquatic plant material developed for µCT imaging, using duckweed plants and turions as exemplars, and compare the method against existing approaches. This technique allows for previously unseen 3D volume analysis of gaseous filled spaces, cell material, and sub-cellular features. The described embedding method, utilizing petrolatum gel for sample mounting, was shown to preserve sample quality during scanning, and to display sufficiently different X-ray attenuation to the plant material to be easily differentiated by image analysis pipelines. We present this technique as an improved method for anatomical structural analysis that provides novel cellular and developmental information.

Roots-eye view: Using microdialysis and microCT to non-destructively map root nutrient depletion and accumulation zones.

Improvement in fertilizer use efficiency is a key aspect for achieving sustainable agriculture in order to minimize costs, greenhouse gas emissions, and pollution from nutrient run-off. To optimize root architecture for nutrient uptake and efficiency, we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling, such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values that can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non-invasive, continuous method for sampling available nutrients in the soil. Here, for the first time, we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots whereas ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root-available nutrients non-destructively and in almost real time.