A Novel High-Throughput Phenotyping Hydroponic System for Nitrogen Deficiency Studies in Arabidopsis thaliana.

High-throughput phenotyping enables the temporal detection of subtle changes in plant plasticity and adaptation to different conditions, such as nitrogen deficiency, in an accurate, nondestructive, and unbiased way. Here, we describe a protocol to assess the contribution of nitrogen addition or deprival using an image-based system to analyze plant phenotype. Thousands of images can be captured throughout the life cycle of Arabidopsis, and those images can be used to quantify parameters such as plant growth (area, caliper length, diameter, etc.), in planta chlorophyll fluorescence, and in planta relative water content.

Characterization of the response to abiotic stresses of high ascorbate Arabidopsis lines using phenomic approaches.

L-Ascorbic acid (AsA, vitamin C) is a key antioxidant and enzyme cofactor in plants. Ascorbate controls cell division, affects cell expansion, and plays an important role in modulating plant senescence. It protects plants against reactive oxygen species that are produced in response to abiotic and biotic stresses. Manual phenotyping indicated that Arabidopsis lines over-expressing enzymes in the myo-inositol pathway have elevated AsA, accumulate more biomass of both aerial and root tissues and are tolerant to abiotic stresses including salt, cold, heat, and environmental pollutants. However, manual phenotyping is time consuming, low throughput, subjective, and limited to the resolution of the human eye. In contrast, high throughput phenotyping technologies are accurate, non-destructive, and more sensitive, allowing the detection of subtle phenotypes. Therefore, we used a phenomics platform to phenotype our high AsA Arabidopsis lines with visible, fluorescence, and near infrared cameras. Based on this approach, high AsA lines grew faster, accumulated more biomass, and displayed healthier chlorophyll fluorescence and water content profiles than controls. By studying abiotic stress in a high throughout fashion using optimized protocols, we have also shown that these high AsA lines are tolerant to salt and water limitation stresses. In addition, we developed open source algorithms to analyze images and by comparing results obtained with a widely used commercial software against our algorithms, here we show that our method achieved good accuracy for all phenotypic parameters of interest including projected leaf area, rosette diameter (caliper length), compactness, and color classification.

Mechanisms underlying the enhanced biomass and abiotic stress tolerance phenotype of an Arabidopsis MIOX over-expresser.

Myo-inositol oxygenase (MIOX) is the first enzyme in the inositol route to ascorbate (L-ascorbic acid, AsA, vitamin C). We have previously shown that Arabidopsis plants constitutively expressing MIOX have elevated foliar AsA content and displayed enhanced growth rate, biomass accumulation, and increased tolerance to multiple abiotic stresses. In this work, we used a combination of transcriptomics, chromatography, microscopy, and physiological measurements to gain a deeper understanding of the underlying mechanisms mediating the phenotype of the AtMIOX4 line. Transcriptomic analysis revealed increased expression of genes involved in auxin synthesis, hydrolysis, transport, and metabolism, which are supported by elevated auxin levels both in vitro and in vivo, and confirmed by assays demonstrating their effect on epidermal cell elongation in the AtMIOX4 over-expressers. Additionally, we detected up-regulation of transcripts involved in photosynthesis and this was validated by increased efficiency of the photosystem II and proton motive force. We also found increased expression of amylase leading to higher intracellular glucose levels. Multiple gene families conferring plants tolerance/expressed in response to cold, water limitation, and heat stresses were found to be elevated in the AtMIOX4 line. Interestingly, the high AsA plants also displayed up-regulation of transcripts and hormones involved in defense including jasmonates, defensin, glucosinolates, and transcription factors that are known to be important for biotic stress tolerance. These results overall indicate that elevated levels of auxin and glucose, and enhanced photosynthetic efficiency in combination with up-regulation of abiotic stresses response genes underly the higher growth rate and abiotic stresses tolerance phenotype of the AtMIOX4 over-expressers.

Three NPF genes in Arabidopsis are necessary for normal nitrogen cycling under low nitrogen stress.

Internal nitrogen (N) cycling is crucial to N use efficiency. For example, N may be remobilized from older, shaded leaves to young leaves near the apex that receive more direct sunlight, where the N can be used more effectively for photosynthesis. Yet our understanding of the mechanisms and regulation of N transport is limited. To identify relevant transporters in Arabidopsis, fifteen transporter knockout mutants were screened for defects in leaf N export using nitrogen-13 (13N) administered as 13NH3 gas to leaves. We found that three nitrate/peptide transporter family (NPF) genes were necessary for normal leaf N export under low N but not adequate soil N availability, including AtNPF7.1, which has not been previously characterized. High-throughput phenotyping revealed altered leaf area and chlorophyll fluorescence relative to wild-type plants. High AtNPF7.1 expression in flowers and large flower stalks of Atnpf7.1 mutants in low N suggests that AtNPF7.1 influences leaf N export via sink-to-source feedback, perhaps via a role in sensing plant internal N-status. We also identified previously unreported phenotypes for the mutants of the other two NPF transporters that indicate possible roles in N sensing networks.

Moderate to severe water limitation differentially affects the phenome and ionome of Arabidopsis.

Food security is currently one of the major challenges that we are facing as a species. Understanding plant responses and adaptations to limited water availability is key to maintain or improve crop yield, and this is even more critical considering the different projections of climate change. In this work, we combined two high-throughput -'omic' platforms ('phenomics' and 'ionomics') to begin dissecting time-dependent effects of water limitation in Arabidopsis leaves and ultimately seed yield. As proof of concept, we acquired high-resolution images with visible, fluorescence, and near infrared cameras and used commercial and open source algorithms to extract the information contained in those images. At a defined point, samples were also taken for elemental profiling. Our results show that growth, biomass and photosynthetic efficiency were affected mostly under severe water limitation regimes and these differences were exacerbated at later developmental stages. The elemental composition and seed yield, however, changed across the different water regimes tested and these changes included under- and over- accumulation of elements compared with well-watered plants. Our results demonstrate that the combination of phenotyping techniques can be successfully used to identify specific bottlenecks during plant development that could compromise biomass, yield, and the nutritional quality of plants.