Dynamic growth QTL action in diverse light environments: characterization of light regime-specific and stable QTL in Arabidopsis.

Plant growth is a complex process affected by a multitude of genetic and environmental factors and their interactions. To identify genetic factors influencing plant performance under different environmental conditions, vegetative growth was assessed in Arabidopsis thaliana cultivated under constant or fluctuating light intensities, using high-throughput phenotyping and genome-wide association studies. Daily automated non-invasive phenotyping of a collection of 382 Arabidopsis accessions provided growth data during developmental progression under different light regimes at high temporal resolution. Quantitative trait loci (QTL) for projected leaf area, relative growth rate, and PSII operating efficiency detected under the two light regimes were predominantly condition-specific and displayed distinct temporal activity patterns, with active phases ranging from 2 d to 9 d. Eighteen protein-coding genes and one miRNA gene were identified as potential candidate genes at 10 QTL regions consistently found under both light regimes. Expression patterns of three candidate genes affecting projected leaf area were analysed in time-series experiments in accessions with contrasting vegetative leaf growth. These observations highlight the importance of considering both environmental and temporal patterns of QTL/allele actions and emphasize the need for detailed time-resolved analyses under diverse well-defined environmental conditions to effectively unravel the complex and stage-specific contributions of genes affecting plant growth processes.

Temporal dynamics of QTL effects on vegetative growth in Arabidopsis thaliana.

We assessed early vegetative growth in a population of 382 accessions of Arabidopsis thaliana using automated non-invasive high-throughput phenotyping. All accessions were imaged daily from 7 d to 18 d after sowing in three independent experiments and genotyped using the Affymetrix 250k SNP array. Projected leaf area (PLA) was derived from image analysis and used to calculate relative growth rates (RGRs). In addition, initial seed size was determined. The generated datasets were used jointly for a genome-wide association study that identified 238 marker-trait associations (MTAs) individually explaining up to 8% of the total phenotypic variation. Co-localization of MTAs occurred at 33 genomic positions. At 21 of these positions, sequential co-localization of MTAs for 2-9 consecutive days was observed. The detected MTAs for PLA and RGR could be grouped according to their temporal expression patterns, emphasizing that temporal variation of MTA action can be observed even during the vegetative growth phase, a period of continuous formation and enlargement of seemingly similar rosette leaves. This indicates that causal genes may be differentially expressed in successive periods. Analyses of the temporal dynamics of biological processes are needed to gain important insight into the molecular mechanisms of growth-controlling processes in plants.

Genetic diversity for nitrogen use efficiency in Arabidopsis thaliana.

MAIN CONCLUSION: The plasticity of plant growth response to differing nitrate availability renders the identification of biomarkers difficult, but allows access to genetic factors as tools to modulate root systems to a wide range of soil conditions. Nitrogen availability is a major determinant of crop yield. While the application of fertiliser substantially increases the yield on poor soils, it also causes nitrate pollution of water resources and high costs for farmers. Increasing nitrogen use efficiency in crop plants is a necessary step to implement low-input agricultural systems. We exploited the genetic diversity present in the worldwide Arabidopsis thaliana population to study adaptive growth patterns and changes in gene expression associated with chronic low nitrate stress, to identify biomarkers associated with good plant performance under low nitrate availability. Arabidopsis accessions were grown on agar plates with limited and sufficient supply of nitrate to measure root system architecture as well as shoot and root fresh weight. Differential gene expression was determined using Affymetrix ATH1 arrays. We show that the response to differing nitrate availability is highly variable in Arabidopsis accessions. Analyses of vegetative shoot growth and root system architecture identified accession-specific reaction modes to cope with limited nitrate availability. Transcription and epigenetic factors were identified as important players in the adaption to limited nitrogen in a global gene expression analysis. Five nitrate-responsive genes emerged as possible biomarkers for NUE in Arabidopsis. The plasticity of plant growth in response to differing nitrate availability in the substrate renders the identification of morphological and molecular features as biomarkers difficult, but at the same time allows access to a multitude of genetic factors which can be used as tools to modulate and adjust root systems to a wide range of soil conditions.

Non-canonical structure, function and phylogeny of the Bsister MADS-box gene OsMADS30 of rice (Oryza sativa).

Bsister MADS-box genes play key roles in female reproductive organ and seed development throughout seed plants. This view is supported by their high conservation in terms of sequence, expression and function. In grasses, there are three subclades of Bsister genes: the OsMADS29-, the OsMADS30- and the OsMADS31-like genes. Here, we report on the evolution of the OsMADS30-like genes. Our analyses indicate that these genes evolved under relaxed purifying selection and are rather weakly expressed. OsMADS30, the representative of the OsMADS30-like genes from rice (Oryza sativa), shows strong sequence deviations in its 3' region when compared to orthologues from other grass species. We show that this is due to a 2.4-kbp insertion, possibly of a hitherto unknown helitron, which confers a heterologous C-terminal domain to OsMADS30. This putative helitron is not present in the OsMADS30 orthologues from closely related wild rice species, pointing to a relatively recent insertion event. Unlike other Bsister mutants O. sativa plants carrying a T-DNA insertion in the OsMADS30 gene do not show aberrant seed phenotypes, indicating that OsMADS30 likely does not have a canonical 'Bsister function'. However, imaging-based phenotyping of the T-DNA carrying plants revealed alterations in shoot size and architecture. We hypothesize that sequence deviations that accumulated during a period of relaxed selection in the gene lineage that led to OsMADS30 and the alteration of the C-terminal domain might have been a precondition for a potential neo-functionalization of OsMADS30 in O. sativa.

Dynamics of Maize Vegetative Growth and Drought Adaptability Using Image-Based Phenotyping Under Controlled Conditions.

Changes in climate are likely to have a negative impact on water availability and soil fertility in many maize-growing agricultural areas. The development of high-throughput phenotyping platforms provides a new prospect for dissecting the dynamic complex plant traits such as abiotic stress tolerance into simple components. The growth phenotypes of 20 maize (Zea mays L.) inbred lines were monitored in a non-invasive way under control, nitrogen, and water limitation as well as under combined nitrogen and water stress using an automated phenotyping system in greenhouse conditions. Thirteen biomass-related and morphophysiological traits were extracted from RGB images acquired at 33 time points covering developmental stages from leaf count 5 at the first imaging date to leaf count 10-13 at the final harvest. For these traits, genetic differences were identified and dynamic developmental trends during different maize growth stages were analyzed. The difference between control and water stress was detectable 3-10 days after the beginning of stress depending on the genotype, while the effect of limited nitrogen supply only induced subtle phenotypic effects. Phenotypic traits showed different response dynamics as well as multiple and changing interaction patterns with stress progression. The estimated biovolume, leaf area index, and color ratios were found to be stress-responsive at different stages of drought stress progression and thereby represent valuable reference indicators in the selection of drought-adaptive genotypes. Furthermore, genotypes could be grouped according to two typical growth dynamic patterns in water stress treatments by c-means clustering analysis. Inbred lines with high drought adaptability across time and development were identified and could serve as a basis for designing novel genotypes with desired, stage-specific growth phenotypes under water stress through pyramiding. Drought recovery potential may play an equal role as drought tolerance in plant drought adaptation.

Image Phenotyping of Spring Barley (Hordeum vulgare L.) RIL Population Under Drought: Selection of Traits and Biological Interpretation.

Image-based phenotyping is a non-invasive method that permits the dynamic evaluation of plant features during growth, which is especially important for understanding plant adaptation and temporal dynamics of responses to environmental cues such as water deficit or drought. The aim of the present study was to use high-throughput imaging in order to assess the variation and dynamics of growth and development during drought in a spring barley population and to investigate associations between traits measured in time and yield-related traits measured after harvesting. Plant material covered recombinant inbred line population derived from a cross between European and Syrian cultivars. After placing the plants on the platform (28th day after sowing), drought stress was applied for 2 weeks. Top and side cameras were used to capture images daily that covered the visible range of the light spectrum, fluorescence signals, and the near infrared spectrum. The image processing provided 376 traits that were subjected to analysis. After 32 days of image phenotyping, the plants were cultivated in the greenhouse under optimal watering conditions until ripening, when several architecture and yield-related traits were measured. The applied data analysis approach, based on the clustering of image-derived traits into groups according to time profiles of statistical and genetic parameters, permitted to select traits representative for inference from the experiment. In particular, drought effects for 27 traits related to convex hull geometry, texture, proportion of brown pixels and chlorophyll intensity were found to be highly correlated with drought effects for spike traits and thousand grain weight.

Image-Derived Traits Related to Mid-Season Growth Performance of Maize Under Nitrogen and Water Stress.

Phenotypic measurements under controlled cultivation conditions are essential to gain a mechanistic understanding of plant responses to environmental impacts and thus for knowledge-based improvement of their performance under natural field conditions. Twenty maize inbred lines (ILs) were phenotyped in response to two levels of water and nitrogen supply (control and stress) and combined nitrogen and water deficit. Over a course of 5 weeks (from about 4-leaf stage to the beginning of the reproductive stage), maize phenology and growth were monitored by using a high-throughput phenotyping platform for daily acquisition of images in different spectral ranges. The focus of the present study is on the measurements taken at the time of maximum water stress (for traits that reflect plant physiological properties) and at the end of the experiment (for traits that reflect plant architectural and biomass-related traits). Twenty-five phenotypic traits extracted from the digital image data that support biological interpretation of plant growth were selected for their predictive value for mid-season shoot biomass accumulation. Measured fresh and dry weights after harvest were used to calculate various indices (water-use efficiency, physiological nitrogen-use efficiency, specific plant weight) and to establish correlations with image-derived phenotypic features. Also, score indices based on dry weight were used to identify contrasting ILs in terms of productivity and tolerance to stress, and their means for image-derived and manually measured traits were compared. Color-related traits appear to be indicative of plant performance and photosystem II operating efficiency might be an importance physiological parameter of biomass accumulation, particularly under severe stress conditions. Also, genotypes showing greater leaf area may be better adapted to abiotic stress conditions.

Quantitative monitoring of Arabidopsis thaliana growth and development using high-throughput plant phenotyping.

With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments.

AtRD22 and AtUSPL1, members of the plant-specific BURP domain family involved in Arabidopsis thaliana drought tolerance.

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response.

Optimizing experimental procedures for quantitative evaluation of crop plant performance in high throughput phenotyping systems.

Detailed and standardized protocols for plant cultivation in environmentally controlled conditions are an essential prerequisite to conduct reproducible experiments with precisely defined treatments. Setting up appropriate and well defined experimental procedures is thus crucial for the generation of solid evidence and indispensable for successful plant research. Non-invasive and high throughput (HT) phenotyping technologies offer the opportunity to monitor and quantify performance dynamics of several hundreds of plants at a time. Compared to small scale plant cultivations, HT systems have much higher demands, from a conceptual and a logistic point of view, on experimental design, as well as the actual plant cultivation conditions, and the image analysis and statistical methods for data evaluation. Furthermore, cultivation conditions need to be designed that elicit plant performance characteristics corresponding to those under natural conditions. This manuscript describes critical steps in the optimization of procedures for HT plant phenotyping systems. Starting with the model plant Arabidopsis, HT-compatible methods were tested, and optimized with regard to growth substrate, soil coverage, watering regime, experimental design (considering environmental inhomogeneities) in automated plant cultivation and imaging systems. As revealed by metabolite profiling, plant movement did not affect the plants' physiological status. Based on these results, procedures for maize HT cultivation and monitoring were established. Variation of maize vegetative growth in the HT phenotyping system did match well with that observed in the field. The presented results outline important issues to be considered in the design of HT phenotyping experiments for model and crop plants. It thereby provides guidelines for the setup of HT experimental procedures, which are required for the generation of reliable and reproducible data of phenotypic variation for a broad range of applications.