A Scalable Open-Source Pipeline for Large-Scale Root Phenotyping of Arabidopsis

Large-scale phenotyping of multicellular organisms is one of the current challenges in biology. We present a comprehensive and scalable pipeline that allows for the efficient phenotyping of root growth traits on a large scale. This includes a high-resolution, low-cost acquisition setup as well as the automated image processing software BRAT. We assess the performance of this pipeline in Arabidopsis thaliana under multiple growth conditions and show its utility by performing genome-wide association studies on 16 root growth traits quantified by BRAT each day during a 5-d time-course experiment. The most significantly associated genome region for root growth rate is a locus encoding a calcium sensing receptor. We find that loss of function and overexpression of this gene can significantly alter root growth in a growth condition dependent manner and that the minor natural allele of the Calcium Sensor Receptor locus is highly significantly enriched in populations in coastal areas, demonstrating the power of our approach to identify regulators of root growth that might have adaptive relevance.     

Developmentally equivalent tissue sampling based on growth kinematic profiling of Arabidopsis inflorescence stems

? Directional growth in Arabidopsis thaliana during bolting of the inflorescence stem makes this an attractive system for study of the underlying processes of tissue elongation and cell wall extension. Analysis of local molecular events accompanying Arabidopsis inflorescence stem elongation is hampered by difficulties in isolating developmentally matched tissue samples from different plants. ? Here, we present a novel sampling approach in which specific developmental stages along the developing stem are defined nonintrusively in terms of their relative elemental growth rate by use of time-lapse imagery and subsequent derivation of growth kinematic profiles for individual plants. ? Growth kinematic profiling reveals that key developmental transitions such as the point of maximum elongation rate and the point of cessation of elongation occur over broad and overlapping ranges across individuals within a population of the Columbia (Col-0) ecotype. The position of these transitions is only weakly correlated with overall plant height, which undermines the common assumption that physically similar plants have closely matched growth profiles. ? This kinematic profiling approach provides high-resolution growth phenotyping of the developing stem and thereby enables the harvest, pooling and analysis of developmentally matched tissue samples from multiple Arabidopsis plants.     

Robotic Assay for Drought (RoAD): an automated phenotyping system for brassinosteroid and drought responses

Brassinosteroids (BRs) are a group of plant steroid hormones involved in regulating growth, development, and stress responses. Many components of the BR pathway have previously been identified and characterized. However, BR phenotyping experiments are typically performed in a low-throughput manner, such as on Petri plates. Additionally, the BR pathway affects drought responses, but drought experiments are time consuming and difficult to control. To mitigate these issues and increase throughput, we developed the Robotic Assay for Drought (RoAD) system to perform BR and drought response experiments in soil-grown Arabidopsis plants. RoAD is equipped with a robotic arm, a rover, a bench scale, a precisely controlled watering system, an RGB camera, and a laser profilometer. It performs daily weighing, watering, and imaging tasks and is capable of administering BR response assays by watering plants with Propiconazole (PCZ), a BR biosynthesis inhibitor. We developed image processing algorithms for both plant segmentation and phenotypic trait extraction to accurately measure traits including plant area, plant volume, leaf length, and leaf width. We then applied machine learning algorithms that utilize the extracted phenotypic parameters to identify image-derived traits that can distinguish control, drought-treated, and PCZ-treated plants. We carried out PCZ and drought experiments on a set of BR mutants and Arabidopsis accessions with altered BR responses. Finally, we extended the RoAD assays to perform BR response assays using PCZ in Zea mays (maize) plants. This study establishes an automated and non-invasive robotic imaging system as a tool to accurately measure morphological and growth-related traits of Arabidopsis and maize plants in 3D, providing insights into the BR-mediated control of plant growth and stress responses.     

Sugars, signalling, and plant development

Like all organisms, plants require energy for growth. They achieve this by absorbing light and fixing it into a usable, chemical form via photosynthesis. The resulting carbohydrate (sugar) energy is then utilized as substrates for growth, or stored as reserves. It is therefore not surprising that modulation of carbohydrate metabolism can have profound effects on plant growth, particularly cell division and expansion. However, recent studies on mutants such as stimpy or ramosa3 have also suggested that sugars can act as signalling molecules that control distinct aspects of plant development. This review will focus on these more specific roles of sugars in development, and will concentrate on two major areas: (i) cross-talk between sugar and hormonal signalling; and (ii) potential direct developmental effects of sugars. In the latter, developmental mutant phenotypes that are modulated by sugars as well as a putative role for trehalose-6-phosphate in inflorescence development are discussed. Because plant growth and development are plastic, and are greatly affected by environmental and nutritional conditions, the distinction between purely metabolic and specific developmental effects is somewhat blurred, but the focus will be on clear examples where sugar-related processes or molecules have been linked to known developmental mechanisms.     

A novel 3D mouse embryo atlas based on micro-CT

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.     

An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat

Mutagenesis is a powerful tool used for studying gene function as well as for crop improvement. It is regaining popularity because of the development of effective and cost efficient methods for high-throughput mutation detection. Selection for semi-dwarf phenotype during green revolution has reduced genetic diversity including that for agronomically desirable traits. Most of the available mutant populations in wheat (Triticum aestivum L.) were developed in post-green revolution cultivars. Besides the identification and isolation of agronomically important alleles in the mutant population of pre-green revolution cultivar, this population can be a vital resource for expanding the genetic diversity for wheat breeding. Here we report an Ethylmethane Sulfonate (EMS) generated mutant population consisting of 4,180 unique mutant plants in a pre-green revolution spring wheat cultivar ‘Indian’. Released in early 1900s, ‘Indian’ is devoid of any known height-reducing mutations. Unique mutations were captured by proceeding with single M₂ seed from each of the 4,180 M₁ plants. Mutants for various phenotypic traits were identified by detailed phenotyping for altered morphological and agronomic traits on M₂ plants in the greenhouse and M₃ plants in the field. Of the 86 identified mutants, 75 (87%) were phenotypically stable at the M₄ generation. Among the observed phenotypes, variation in plant height was the most frequent followed by the leaf morphology. Several mutant phenotypes including looped peduncle, crooked plant morphology, ‘gritty’ coleoptiles, looped lower internodes, and burnt leaf tips are not reported in other plant species. Considering the extent and diversity of the observed mutant phenotypes, this population appears to be a useful resource for the forward and reverse genetic studies. This resource is available to the scientific community.     

amp1 - a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering

amp1 , a mutant of Arabidopsis thaliana has a phenotype altered in three different aspects of plant development; spatial pattern, photomorphogenetic growth, and initiation of flowering. While fewer than 0.1% of the seedlings of wild-type plants are non-dicot as many as 20% of the seedlings of the amp1 mutant are tricot or tetracot. The rate of leaf initiation is faster and vegetative phyllotaxy is altered in amp1 . When grown in the dark amp1 seedlings show morphogenetic properties similar to light-grown wild-type plants: they do not form an apical hook, have hypocotyls shorter than wild-type plants and form etiolated true leaves. amp1 mutant flowers significantly earlier than congenic Amp1 plants. The mutant has six times more cytokinin than wild-type suggesting that endogenous cytokinin levels might play an important role in mediating these different developmental processes. AMP1 might code for a negative regulator of cytokinin biosynthesis, or may be required for the degradation of cytokinin.     

The Arabidopsis MERISTEM DISORGANIZATION 1 gene is required for the maintenance of stem cells through the reduction of DNA damage

In plants, stem cells reside in apical meristems, and provide the descendants required for post-embryonic growth and development throughout the life of a plant. To identify a novel factor required for the maintenance of stem cells, we isolated an Arabidopsis mutant, named meristem disorganization 1-1 (mdo1-1), that exhibits several developmental defects, such as abnormal phyllotaxy and plastochron, stem fasciation and retarded root growth. We found that the mutant plants fail to maintain stem cells, resulting in the differentiation or death of stem cells. The mutant plants also showed several phenotypes related to DNA damage, suggesting that the mutant cells are exposed constitutively to DNA damage even without external genotoxic stress. The growth defect and the hypersensitivity to DNA-damaging agents of mdo1-1 were enhanced significantly when combined with a lesion of the ATAXIA-TELANGIECTASIA MUTATED (ATM) gene, but not of the ATM/RAD3-RELATED (ATR) gene, suggesting that the function of the MDO1 gene is closely related to that of ATM kinase. The MDO1 gene encodes an unknown protein that is conserved in a wide variety of land plants. The results thus suggested that the MDO1 gene product is required for the maintenance of stem cells through a reduction in DNA damage.     

Genetic variation of growth dynamics in maize (Zea mays L.) revealed through automated non‐invasive phenotyping

Hitherto, most quantitative trait loci of maize growth and biomass yield have been identified for a single time point, usually the final harvest stage. Through this approach cumulative effects are detected, without considering genetic factors causing phase‐specific differences in growth rates. To assess the genetics of growth dynamics, we employed automated non‐invasive phenotyping to monitor the plant sizes of 252 diverse maize inbred lines at 11 different developmental time points; 50 k SNP array genotype data were used for genome‐wide association mapping and genomic selection. The heritability of biomass was estimated to be over 71%, and the average prediction accuracy amounted to 0.39. Using the individual time point data, 12 main effect marker‐trait associations (MTAs) and six pairs of epistatic interactions were detected that displayed different patterns of expression at various developmental time points. A subset of them also showed significant effects on relative growth rates in different intervals. The detected MTAs jointly explained up to 12% of the total phenotypic variation, decreasing with developmental progression. Using non‐parametric functional mapping and multivariate mapping approaches, four additional marker loci affecting growth dynamics were detected. Our results demonstrate that plant biomass accumulation is a complex trait governed by many small effect loci, most of which act at certain restricted developmental phases. This highlights the need for investigation of stage‐specific growth affecting genes to elucidate important processes operating at different developmental phases.     

Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

High‐content imaging using automated microscopy and computer vision allows multivariate profiling of single‐cell phenotypes. Here, we present methods for the application of the CISPR‐Cas9 system in large‐scale, image‐based, gene perturbation experiments. We show that CRISPR‐Cas9‐mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image‐based phenotyping. We developed a pipeline to construct a large‐scale arrayed library of 2,281 sequence‐verified CRISPR‐Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine‐learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in‐depth characterization of gene perturbation effects. This approach enables genome‐scale image‐based multivariate gene perturbation profiling using CRISPR‐Cas9.     

Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth

Actin depolymerizing factor (ADF)/cofilin is important for regulating actin dynamics, and in plants is thought to be required for tip growth. However, the degree to which ADF is necessary has been elusive because of the presence of multiple ADF isoforms in many plant species. In the moss Physcomitrella patens , ADF is encoded by a single, intronless gene. We used RNA interference to demonstrate that ADF is essential for plant viability. Loss of ADF dramatically alters the organization of the F-actin cytoskeleton, and leads to an inhibition of tip growth. We show that ADF is subject to phosphorylation in vivo , and using complementation studies we show that mutations of the predicted phosphorylation site partially rescue plant viability, but with differential affects on tip growth. Specifically, the unphosphorylatable ADF S6A mutant generates small polarized plants with normal F-actin organization, whereas the phosphomimetic S6D mutant generates small, unpolarized plants with a disorganized F-actin cyotskeleton. These data indicate that phosphoregulation at serine 6 is required for full ADF function in vivo , and, in particular, that the interaction between ADF and actin is important for tip growth.