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Root system architecture in cereals: progress, challenges and perspective

We report roots are essential multifunctional plant organs involved in water and nutrient uptake, metabolite storage, anchorage, mechanical support, and interaction with the soil environment. Understanding of this ‘hidden half’ provides potential for manipulation of root system architecture (RSA) traits to optimize resource use efficiency and grain yield in cereal crops. Unfortunately, root traits are highly neglected in breeding due to the challenges of phenotyping, but could have large rewards if the variability in RSA traits can be fully exploited. Until now, a plethora of genes have been characterized in detail for their potential role in improving RSA. The use of forward genetics approaches to find sequence variations in genes underpinning desirable RSA would be highly beneficial. Advances in computer vision applications have allowed image-based approaches for high-throughput phenotyping of RSA traits that can be used by any laboratory worldwide to make progress in understanding root function and dissection of the genetics. At the same time, the frontiers of root measurement include non-invasive methods like X-ray computer tomography and magnetic resonance imaging that facilitate new types of temporal studies. Root physiology and ecology are further supported by spatiotemporal root simulation modeling. The discovery of component traits providing improved resilience and yield advantage in target environments is a key necessity for mainstreaming root-based cereal breeding. The integrated use of pan-genome resources, now available in most cereals, coupled with new in-field phenotyping platforms has the potential for precise selection of superior genotypes with improved RSA.

59 BASIC BIOLOGICAL SCIENCES↗

Phenome‐to‐genome insights for evaluating root system architecture in field studies of maize

Abstract Understanding the genetic basis of root system architecture (RSA) in crops requires innovative approaches that enable both high‐throughput and precise phenotyping in field conditions. In this study, we evaluated multiple phenotyping and analytical frameworks for quantifying RSA in mature, field‐grown maize in three field experiments. We used forward and reverse genetic approaches to evaluate >1700 maize root crowns, including a diversity panel, a biparental mapping population, and maize mutant and wild‐type alleles at two known RSA genes,DEEPER ROOTING 1(DRO1) andRootless1(Rt1). We show the utility of increasing the dimensionality of traditional two‐dimensional (2D) techniques, referred to as the “2D multi‐view” method, to improve the capture of whole root system information for mapping genetic variation influencing RSA. Comparison of univariate and multivariate genome‐wide association study (GWAS) approaches revealed that multivariate traits were effective at dissecting complex RSA phenotypes and identifying pleiotropic quantitative trait loci (QTLs). Overall, three‐dimensional (3D) root models generated from X‐ray computed tomography and digital phenotyping captured a larger proportion of RSA trait variations compared to other methods of root phenotyping, as evidenced by both genome‐wide and single‐gene analyses. Among the individual root traits, root pulling force emerged as a highly heritable estimate of RSA that identified the largest number of shared QTLs with 3D phenotypes. Our study shows that integrating complementary phenotyping technologies helps to provide a more comprehensive understanding of the genetic architecture of RSA in field‐grown maize.

Genetics & Heredity↗

Uncovering natural variation in root system architecture and growth dynamics using a robotics-assisted phenomics platform

The plant kingdom contains a stunning array of complex morphologies easily observed above-ground, but more challenging to visualize below-ground. Understanding the magnitude of diversity in root distribution within the soil, termed root system architecture (RSA), is fundamental in determining how this trait contributes to species adaptation in local environments. Roots are the interface between the soil environment and the shoot system and therefore play a key role in anchorage, resource uptake, and stress resilience. Previously, we presented the GLO-Roots (Growth and Luminescence Observatory for Roots) system to study the RSA of soil-grown Arabidopsis thaliana plants from germination to maturity (Rellán-Álvarez et al., 2015). In this study, we present the automation of GLO-Roots using robotics and the development of image analysis pipelines in order to examine the temporal dynamic regulation of RSA and the broader natural variation of RSA in Arabidopsis , over time. These datasets describe the developmental dynamics of two independent panels of accessions and reveal highly complex and polygenic RSA traits that show significant correlation with climate variables of the accessions’ respective origins.

59 BASIC BIOLOGICAL SCIENCES↗

RhizoVision Explorer: open-source software for root image analysis and measurement standardization

Abstract Roots are central to the function of natural and agricultural ecosystems by driving plant acquisition of soil resources and influencing the carbon cycle. Root characteristics like length, diameter and volume are critical to measure to understand plant and soil functions. RhizoVision Explorer is an open-source software designed to enable researchers interested in roots by providing an easy-to-use interface, fast image processing and reliable measurements. The default broken roots mode is intended for roots sampled from pots and soil cores, washed and typically scanned on a flatbed scanner, and provides measurements like length, diameter and volume. The optional whole root mode for complete root systems or root crowns provides additional measurements such as angles, root depth and convex hull. Both modes support providing measurements grouped by defined diameter ranges, the inclusion of multiple regions of interest and batch analysis. RhizoVision Explorer was successfully validated against ground truth data using a new copper wire image set. In comparison, the current reference software, the commercial WinRhizo™, drastically underestimated volume when wires of different diameters were in the same image. Additionally, measurements were compared with WinRhizo™ and IJ_Rhizo using a simulated root image set, showing general agreement in software measurements, except for root volume. Finally, scanned root image sets acquired in different labs for the crop, herbaceous and tree species were used to compare results from RhizoVision Explorer with WinRhizo™. The two software showed general agreement, except that WinRhizo™ substantially underestimated root volume relative to RhizoVision Explorer. In the current context of rapidly growing interest in root science, RhizoVision Explorer intends to become a reference software, improve the overall accuracy and replicability of root trait measurements and provide a foundation for collaborative improvement and reliable access to all.

59 BASIC BIOLOGICAL SCIENCES↗

DEEPER: An Intergrated Platform for Deeper Roots

Crops with deeper roots would have multiple benefits, including better drought tolerance, reduced requirement for nitrogen fertilizer, and better sequestration of atmospheric CO 2 . DEEPER is an integrated platform of phenomic, genomic, and in silico technologies to generate maize lines with deeper roots. DEEPER is: LEADER (Leaf Elemental Accumulation from Deep Roots) is a breakthrough technology to nondestructively measure rooting depth by using the plant itself as a sensor. LEADER uses handheld X-ray Fluorescence spectrometry to quantify foliar accumulation of elements that are differentially distributed in the soil profile. LEADER is nondestructive and is orders of magnitude cheaper, faster, and more precise than any competing assay of rooting depth in the field. LEADER is able to distinguish deep-rooted from shallow-rooted maize lines in the field without the need for costly and noisy soil coring. RootRobot/DIRT3D, to automatically phenotype root architecture in any field, combining RootRobot, a mechatronics platform to excavate, clean, section, and image mature root crowns, with DIRT3D, software to quantify architectural traits in 3D. Anatomics, a high-throughput platform to phenotype root anatomy, combining LAT 2.0, a technology for 3D imaging of root anatomy and composition, with RootScan3D, software to automatically extract 3D anatomical and cell wall composition metrics from LAT 2.0 output. Using this platform we discovered two novel root traits, parenchyma cell wall thickness and multiseriate cortical sclerenchyma, that improve rooting depth and drought tolerance in maize and wheat. OpenSimRoot/Deep, software to simulate root interaction with hard subsoils. Using this platform we discovered novel concepts regarding how to increase crop rooting depth by modulating how individual root axes respond to hard soil. DeepGenes, a toolkit of genes, parent lines, and genomic selection strategies to enable breeding hybrids with deeper roots. We discovered 3 novel root genes that increase rooting depth in maize and wheat. DEEPER discovered novel root phenotypes for deeper rooting, and delivered validated ideotypes for deeper-rooted maize; novel technologies to rapidly assess root depth, root architecture and anatomy in field-grown plants; novel software tools for root modeling and 3D image analysis of root architecture and anatomy; and validated genes and genomic selection models to deploy traits for deeper rooting in maize breeding. Each DEEPER technology is transformative in its own right, and exceeds existing technologies. They are mutually synergistic, deployable for field-grown plants, and are ready for application. The phenotyping and modeling technologies are readily applicable to many crops, and genetic leads in maize may have utility in other grasses. Taken as a whole they represent a transformative platform to develop deeper-rooted crops, with greater drought tolerance, reduced fertilizer requirement, and greater carbon sequestration.

59 BASIC BIOLOGICAL SCIENCES↗

Morphophysiological Plant Phenotyping for the Development of Plant Breeding Under Drought and Heat Conditions: A Practical Approach

ABSTRACT Currently, the breeding programs focus their efforts on identifying and developing tolerant genotypes to adverse conditions, such as drought and high temperatures. In this context, the physiological approach, which involves phenotyping several traits, is useful for breeding programs. Leaf photosynthetic traits have become one of the main objectives to be evaluated for breeders due to their relationship with improving grain yield and biomass production. Gas exchange ( Ge ) and chlorophyll “a” fluorescence ( Chf ) are the main tools to characterize the photosynthetic activity in real time at the leaf level. Consequently, several association studies using proximal and nonproximal sensing (e.g., RGB, thermography) have been developed. However, for the correct application of this breeding approach, it is essential to have a basic knowledge of both the physiological principles involved in the readings and the limitations of phenotyping due to the characteristics of the devices available on the market. This revision also covers other traits, such as the morphological and anatomical characteristics of leaves and roots, and the use of isotopes complementing Ge and Chf measurements.

Estrada, Félix [Instituto de Investigaciones Agrop↗

Systems Analysis of the Physiological and Molecular Mechanisms of Sorghum Nitrogen Use Efficiency, Water Use Efficiency and Interactions with the Soil Microbiome (Final Report for DE-SC0014395)

The specific project objectives were to: 1) Conduct deep census surveys of root microbiomes concurrent with phenotypic characterizations of a diverse panel of sorghum genotypes across multiple years to define the microbes associated with the most productive lines under drought and low nitrogen conditions. 2) Associate systems-level genotypic, microbial, and environmental factors with improved sorghum performance using robust statistical approaches. 3) Develop culture collections of sorghum root/leaf associated microbes that recapitulate root-enriched sequences defined in the census. 4) Perform controlled environment experiments for in-depth characterization and hypothesis testing of G sorghum x G microbe x E interactions . Validate physiological mechanisms, map genetic loci for stress tolerance, and determine the persistence of optimal microbial strains under greenhouse and field conditions.

59 BASIC BIOLOGICAL SCIENCES↗

Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants

Abstract Root nodule symbiosis (RNS) is a complex trait that enables plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. Pinpointing the evolutionary origins of RNS is critical for understanding its genetic basis, but building this evolutionary context is complicated by data limitations and the intermittent presence of RNS in a single clade of ca. 30,000 species of flowering plants, i.e., the nitrogen-fixing clade (NFC). We developed the most extensive de novo phylogeny for the NFC and an RNS trait database to reconstruct the evolution of RNS. Our analysis identifies evolutionary rate heterogeneity associated with a two-step process: An ancestral precursor state transitioned to a more labile state from which RNS was rapidly gained at multiple points in the NFC. We illustrate how a two-step process could explain multiple independent gains and losses of RNS, contrary to recent hypotheses suggesting one gain and numerous losses, and suggest a broader phylogenetic and genetic scope may be required for genome-phenome mapping.

59 BASIC BIOLOGICAL SCIENCES↗

EcoBOT: an AI/ML enabled automated phenotyping capability for model plants

Introduction: Advances in automation and AI/ML offer new opportunities for plant science, including design, modeling, and analysis. This study aimed to develop an automated platform for researching small model plants under axenic conditions and integrate it with AI/ML tools. Methods: The EcoBOT platform was developed, which consists of sterile containers (EcoFABs) for growing plants and imaging for monitoring plant growth and health. Brachypodium distachyon was grown on the EcoBOT, and its response to nutrient limitation and copper stress was evaluated. Results: The results showed that Brachypodium distachyon grown in the EcoBOT maintained sterility and responded to nutrient limitation and copper stress. Analysis of over 6,500 root and shoot images revealed varying sensitivity and response rates to copper. Bayesian Optimization was used to improve model accuracies relating copper concentrations to plant biomass via sequential experiments, resulting in a >30% improvement. Discussion: The findings of this study demonstrate the potential of the EcoBOT platform for researching plant responses to environmental factors. Future experiments could focus on relating other chemical stresses and microbial interactions to create generalized models of plant responses.

AI image analysis↗