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With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.

Plant xylem cells conduct water and mineral nutrients. Although most plant cells are totipotent, xylem cells are unusual and undergo terminal differentiation. Many genes regulating this process are well characterized, including the Vascular-related NAC Domain 7 (VND7), MYB46, and MYB83 transcription factors, which are proposed to act in interconnected feedforward loops (FFLs). Less is known regarding the molecular mechanisms underlying the terminal transition to xylem cell differentiation. Here, we generate whole-root and single-cell data, which demonstrate that VND7 initiates sharp switching of root cells to xylem cell identity. Based on these data, we identified 4 candidate VND7 downstream target genes capable of generating this switch. Although MYB46 responds to VND7 induction, it is not among these targets. This system provides an important model to study the emergent properties that may give rise to totipotency relative to terminal differentiation and reveals xylem cell subtypes.

Single-cell transcriptome profiling of heterogeneous tissues can provide high-resolution windows into developmental dynamics and environmental responses, but its application to plants has been limited. Here, we used the high-throughput Drop-seq approach to profile >12,000 cells from Arabidopsis roots. This identified numerous distinct cell types, covering all major root tissues and developmental stages, and illuminated specific marker genes for these populations. In addition, we demonstrate the utility of this approach to study the impact of environmental conditions on developmental processes. Analysis of roots grown with or without sucrose supplementation uncovers changes in the relative frequencies of cell types in response to sucrose. Finally, we characterize the transcriptome changes that occur across endodermis development and identify nearly 800 genes with dynamic expression as this tissue differentiates. Collectively, we demonstrate that single-cell RNA-seq can be used to profile developmental processes in plants and show how they can be altered by external stimuli.

There is a dynamic reciprocity between plants and their environment: soil physiochemical properties influence plant morphology and metabolism, and root morphology and exudates shape the environment surrounding roots. Here, we investigate the reproducibility of plant trait changes in response to three growth environments. We utilized fabricated ecosystem (EcoFAB) devices to grow the model grass Brachypodium distachyon in three distinct media across four laboratories: phosphate-sufficient and -deficient mineral media allowed assessment of the effects of phosphate starvation, and a complex, sterile soil extract represented a more natural environment with yet uncharacterized effects on plant growth and metabolism. Tissue weight and phosphate content, total root length, and root tissue and exudate metabolic profiles were consistent across laboratories and distinct between experimental treatments. Plants grown in soil extract were morphologically and metabolically distinct, with root hairs four times longer than with other growth conditions. Further, plants depleted half of the metabolites investigated from the soil extract. To interact with their environment, plants not only adapt morphology and release complex metabolite mixtures, but also selectively deplete a range of soil-derived metabolites. The EcoFABs utilized here generated high interlaboratory reproducibility, demonstrating their value in standardized investigations of plant traits.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguously identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.

Plant growth is commonly regulated by external cues such as light, temperature, water availability, and internal cues generated by the circadian clock. Changes in the rate of growth within the course of a day have been observed in the leaves, stems, and roots of numerous species. However, the relative impact of the circadian clock on the growth of grasses has not been thoroughly characterized. We examined the influence of diurnal temperature and light changes, and that of the circadian clock on leaf length growth patterns in Brachypodium distachyon using high-resolution time-lapse imaging. Pronounced changes in growth rate were observed under combined photocyles and thermocycles or with thermocycles alone. A considerably more rapid growth rate was observed at 28°C than 12°C, irrespective of the presence or absence of light. In spite of clear circadian clock regulated gene expression, plants exhibited no change in growth rate under conditions of constant light and temperature, and little or no effect under photocycles alone. Therefore, temperature appears to be the primary cue influencing observed oscillations in growth rate and not the circadian clock or photoreceptor activity. Furthermore, the size of the leaf meristem and final cell length did not change in response to changes in temperature. Therefore, the nearly five-fold difference in growth rate observed across thermocycles can be attributed to proportionate changes in the rate of cell division and expansion. A better understanding of the growth cues in B. distachyon will further our ability to model metabolism and biomass accumulation in grasses. [ABSTRACT FROM AUTHOR]

Plant growth is commonly regulated by external cues such as light, temperature, water availability, and internal cues generated by the circadian clock. Changes in the rate of growth within the course of a day have been observed in the leaves, stems, and roots of numerous species. However, the relative impact of the circadian clock on the growth of grasses has not been thoroughly characterized. We examined the influence of diurnal temperature and light changes, and that of the circadian clock on leaf length growth patterns in Brachypodium distachyon using high-resolution time-lapse imaging. Pronounced changes in growth rate were observed under combined photocyles and thermocycles or with thermocycles alone. A considerably more rapid growth rate was observed at 28°C than 12°C, irrespective of the presence or absence of light. In spite of clear circadian clock regulated gene expression, plants exhibited no change in growth rate under conditions of constant light and temperature, and little or no effect under photocycles alone. Therefore, temperature appears to be the primary cue influencing observed oscillations in growth rate and not the circadian clock or photoreceptor activity. Furthermore, the size of the leaf meristem and final cell length did not change in response to changes in temperature. Therefore, the nearly five-fold difference in growth rate observed across thermocycles can be attributed to proportionate changes in the rate of cell division and expansion. A better understanding of the growth cues in B. distachyon will further our ability to model metabolism and biomass accumulation in grasses. [ABSTRACT FROM AUTHOR]

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response. [ABSTRACT FROM AUTHOR]

Time-resolved hypocotyl length measurements in seedlings have the potential to greatly aid genetic studies looking at light, hormone, and circadian regulation of cell expansion. Recently, several computer-based tools have been developed to quantify hypocotyl length during photomorphogenesis and early seedling development. Here we detail a method for quantifying Arabidopsis seedling hypocotyls in an image-based assay, focusing on light-grown seedlings responding to shade conditions.