An image-based technique for automated root disease severity assessment using PlantCV.

Premise: Plant disease severity assessments are used to quantify plant-pathogen interactions and identify disease-resistant lines. One common method for disease assessment involves scoring tissue manually using a semi-quantitative scale. Automating assessments would provide fast, unbiased, and quantitative measurements of root disease severity, allowing for improved consistency within and across large data sets. However, using traditional Root System Markup Language (RSML) software in the study of root responses to pathogens presents additional challenges; these include the removal of necrotic tissue during the thresholding process, which results in inaccurate image analysis. Methods: Using PlantCV, we developed a Python-based pipeline, herein called RootDS, with two main objectives: (1) improving disease severity phenotyping and (2) generating binary images as inputs for RSML software. We tested the pipeline in common bean inoculated with Fusarium root rot. Results: Quantitative disease scores and root area generated by this pipeline had a strong correlation with manually curated values (R 2 = 0.92 and 0.90, respectively) and provided a broader capture of variation than manual disease scores. Compared to traditional manual thresholding, images generated using our pipeline did not affect RSML output. Discussion: Overall, the RootDS pipeline provides greater functionality in disease score data sets and provides an alternative method for generating image sets for use in available RSML software.

Arabidopsis weep mutants exhibit narrow root angles.

Peach ( Prunus persica ) trees with a mutation in the weep gene exhibit a weeping branch phenotype. In contrast, Arabidopsis ( Arabidopsis thaliana ) weep mutants do not have a shoot architecture phenotype. A recent report revealed that barley ( Hordeum vulgare ) and wheat ( Triticum aestivum ) with mutations in EGT2, a WEEP homolog, have steeper root angles than standard varieties. We investigated the root architecture of three Arabidopsis weep mutant lines. All three lines exhibited steeper root angles and a smaller convex hull area, indicating that the total area explored by the root system is reduced. These results reveal WEEP is important for regulating lateral root angles in a dicot.

Root Crown Response to Fungal Root Rot in Phaseolus vulgaris Middle American × Andean Lines.

Fusarium root rot (FRR) is a global limiter of dry bean (Phaseolus vulgaris L.) production. In common bean and other legumes, resistance to FRR is related to both root development and root architecture, providing a breeding strategy for FRR resistance. Here, we describe the relationships between root traits and FRR disease symptoms. Using "shovelomics" techniques, a subset of recombinant inbred lines was phenotyped for root architecture traits and disease symptoms across three Michigan fields, including one field with artificially increased Fusarium brasiliense disease pressure. At the early growth stages, stem diameter, basal root number, and distribution of hypocotyl-borne adventitious roots were all significantly related to FRR disease scores. These results demonstrate that root architecture is a component of resistance to FRR in the field at early growth stages (first expanded trifoliate) complementing previous studies that evaluated root traits at later developmental stages (flowering, pod fill, etc.). Correlation matrices of root traits indicate that resistant and susceptible lines have statistically different root systems and show that basal root number is a key feature in resistant root systems while adventitious root distribution is an important feature in susceptible root systems. Based on the results of this study, selection for increased basal root number, increased adventitious root number, and even distribution of adventitious roots in early growth stages (first expanded trifoliate) would positively impact resistance to FRR.

Long-Distance and Trans-Generational Stomatal Patterning by CO2 Across Arabidopsis Organs.

Stomata control water loss and carbon dioxide uptake by both altering pore aperture and developmental patterning. Stomatal patterning is regulated by environmental factors including atmospheric carbon dioxide (p[CO2]), which is increasing globally at an unprecedented rate. Mature leaves are known to convey developmental cues to immature leaves in response to p[CO2], but the developmental mechanisms are unknown. To characterize changes in stomatal patterning resulting from signals moving from mature to developing leaves, we constructed a dual-chamber growth system in which rosette and cauline leaves of Arabidopsis thaliana were subjected to differing p[CO2]. Young rosette tissue was found to adjust stomatal index (SI, the proportion of stomata to total cell number) in response to both the current environment and the environment experienced by mature rosette tissue, whereas cauline leaves appear to be insensitive to p[CO2] treatment. It is likely that cauline leaves and cotyledons deploy mechanisms for controlling stomatal development that share common but also deploy distinctive mechanisms to that operating in rosette leaves. The effect of p[CO2] on stomatal development is retained in cotyledons of the next generation, however, this effect does not occur in pre-germination stomatal lineage cells but only after germination. Finally, these data suggest that p[CO2] affects regulation of stomatal development specifically through the development of satellite stomata (stomata induced by signals from a neighboring stomate) during spacing divisions and not the basal pathway. To our knowledge, this is the first report identifying developmental steps responsible for altered stomatal patterning to p[CO2] and its trans-generational inheritance.

Application of Optical Topometry to Analysis of the Plant Epidermis.

The plant epidermis regulates key physiological functions contributing to photosynthetic rate, plant productivity, and ecosystem stability. Yet, quantitative characterization of this interface between a plant and its aerial environment is laborious and destructive with current techniques, making large-scale characterization of epidermal cell parameters impractical. Here, we present our exploration of optical topometry (OT) for the analysis of plant organ surfaces. OT is a mature, confocal microscopy-based implementation of surface metrology that generates nanometer-scale digital characterizations of any surface. We report epidermal analyses in Arabidopsis (Arabidopsis thaliana) and other species as well as dried herbarium specimens and fossilized plants. We evaluate the technology's analytical potential for identifying an array of epidermal characters, including cell type distributions, variation in cell morphology and stomatal depth, differentiation of herbarium specimens, and real-time deformations in living tissue following detachment. As applied to plant material, OT is very fast and nondestructive, yielding richly mineable data sets describing living tissues and rendering a variety of their characteristics accessible for statistical, quantitative genetic, and structural analysis.

Leaf-galling phylloxera on grapes reprograms host metabolism and morphology.

Endoparasitism by gall-forming insects dramatically alters the plant phenotype by altering growth patterns and modifying plant organs in ways that appear to directly benefit the gall former. Because these morphological and physiological changes are linked to the presence of the insect, the induced phenotype is said to function as an extension of the parasite, albeit by unknown mechanisms. Here we report the gall-forming aphid-like parasite phylloxera, Daktulosphaira vitifoliae, induces stomata on the adaxial surface of grape leaves where stomata typically do not occur. We characterized the function of the phylloxera-induced stomata by tracing transport of assimilated carbon. Because induction of stomata suggests a significant manipulation of primary metabolism, we also characterized the gall transcriptome to infer the level of global reconfiguration of primary metabolism and the subsequent changes in downstream secondary metabolism. Phylloxera feeding induced stomata formation in proximity to the insect and promoted the assimilation and importation of carbon into the gall. Gene expression related to water, nutrient, and mineral transport; glycolysis; and fermentation increased in leaf-gall tissues. This shift from an autotrophic to a heterotrophic profile occurred concurrently with decreased gene expression for nonmevalonate and terpenoid synthesis and increased gene expression in shikimate and phenylpropanoid biosynthesis, secondary metabolite systems that alter defense status in grapes. These functional insect-induced stomata thus comprise part of an extended phenotype, whereby D. vitifoliae globally reprograms grape leaf development to alter patterns of primary metabolism, nutrient mobilization, and defense investment in favor of the galling habit.

Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.).

BACKGROUND: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. RESULTS: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. CONCLUSIONS: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.