Cellular clarity: a logistic regression approach to identify root epidermal regulators of iron deficiency response.

BACKGROUND: Plants respond to stress through highly tuned regulatory networks. While prior works identified master regulators of iron deficiency responses in A. thaliana from whole-root data, identifying regulators that act at the cellular level is critical to a more comprehensive understanding of iron homeostasis. Within the root epidermis complex molecular mechanisms that facilitate iron reduction and uptake from the rhizosphere are known to be regulated by bHLH transcriptional regulators. However, many questions remain about the regulatory mechanisms that control these responses, and how they may integrate with developmental processes within the epidermis. Here, we use transcriptional profiling to gain insight into root epidermis-specific regulatory processes. RESULTS: Set comparisons of differentially expressed genes (DEGs) between whole root and epidermis transcript measurements identified differences in magnitude and timing of organ-level vs. epidermis-specific responses. Utilizing a unique sampling method combined with a mutual information metric across time-lagged and non-time-lagged windows, we identified relationships between clusters of functionally relevant differentially expressed genes suggesting that developmental regulatory processes may act upstream of well-known Fe-specific responses. By integrating static data (DNA motif information) with time-series transcriptomic data and employing machine learning approaches, specifically logistic regression models with LASSO, we also identified putative motifs that served as crucial features for predicting differentially expressed genes. Twenty-eight transcription factors (TFs) known to bind to these motifs were not differentially expressed, indicating that these TFs may be regulated post-transcriptionally or post-translationally. Notably, many of these TFs also play a role in root development and general stress response. CONCLUSIONS: This work uncovered key differences in -Fe response identified using whole root data vs. cell-specific root epidermal data. Machine learning approaches combined with additional static data identified putative regulators of -Fe response that would not have been identified solely through transcriptomic profiles and reveal how developmental and general stress responses within the epidermis may act upstream of more specialized -Fe responses for Fe uptake.

POPEYE intercellular localization mediates cell-specific iron deficiency responses.

Plants must tightly regulate iron (Fe) sensing, acquisition, transport, mobilization, and storage to ensure sufficient levels of this essential micronutrient. POPEYE (PYE) is an iron responsive transcription factor that positively regulates the iron deficiency response, while also repressing genes essential for maintaining iron homeostasis. However, little is known about how PYE plays such contradictory roles. Under iron-deficient conditions, pPYE:GFP accumulates in the root pericycle while pPYE:PYE-GFP is localized to the nucleus in all Arabidopsis (Arabidopsis thaliana) root cells, suggesting that PYE may have cell-specific dynamics and functions. Using scanning fluorescence correlation spectroscopy and cell-specific promoters, we found that PYE-GFP moves between different cells and that the tendency for movement corresponds with transcript abundance. While localization to the cortex, endodermis, and vasculature is required to manage changes in iron availability, vasculature and endodermis localization of PYE-GFP protein exacerbated pye-1 defects and elicited a host of transcriptional changes that are detrimental to iron mobilization. Our findings indicate that PYE acts as a positive regulator of iron deficiency response by regulating iron bioavailability differentially across cells, which may trigger iron uptake from the surrounding rhizosphere and impact root energy metabolism.

The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development.

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.

Arogenate dehydratases: unique roles in light-directed development during the seed-to-seedling transition in Arabidopsis thaliana.

The seed-to-seedling transition is impacted by changes in nutrient availability and light profiles, but is still poorly understood. Phenylalanine affects early seedling development; thus, the roles of arogenate dehydratases (ADTs), which catalyze phenylalanine formation, were studied in germination and during the seed-to-seedling transition by exploring the impact of light conditions and specific hormone responses in adt mutants of Arabidopsis thaliana. ADT gene expression was assessed in distinct tissues and for light-quality dependence in seedlings for each of the six-member ADT gene family. Mutant adt seedlings were evaluated relative to wild type for germination, photomorphogenesis (blue, red, far red, white light, and dark conditions), anthocyanin accumulation, and plastid development-related phenotypes. ADT proteins are expressed in a light- and tissue-specific manner in transgenic seedlings. Among the analyzed adt mutants, adt3, adt5, and adt6 exhibit significant defects in germination, hypocotyl elongation, and root development responses during the seed-to-seedling transition. Interestingly, adt5 exhibits a light-dependent disruption in plastid development, similar to a phyA mutant. These data indicate interactions between photoreceptors, hormones, and regulation of phenylalanine pools in the process of seedling establishment. ADT5 and ADT6 may play important roles in coordinating hormone and light signals for normal early seedling development.

Plant peroxisome proteostasis-establishing, renovating, and dismantling the peroxisomal proteome.

Plant peroxisomes host critical metabolic reactions and insulate the rest of the cell from reactive byproducts. The specialization of peroxisomal reactions is rooted in how the organelle modulates its proteome to be suitable for the tissue, environment, and developmental stage of the organism. The story of plant peroxisomal proteostasis begins with transcriptional regulation of peroxisomal protein genes and the synthesis, trafficking, import, and folding of peroxisomal proteins. The saga continues with assembly and disaggregation by chaperones and degradation via proteases or the proteasome. The story concludes with organelle recycling via autophagy. Some of these processes as well as the proteins that facilitate them are peroxisome-specific, while others are shared among organelles. Our understanding of translational regulation of plant peroxisomal protein transcripts and proteins necessary for pexophagy remain based in findings from other models. Recent strides to elucidate transcriptional control, membrane dynamics, protein trafficking, and conditions that induce peroxisome turnover have expanded our knowledge of plant peroxisomal proteostasis. Here we review our current understanding of the processes and proteins necessary for plant peroxisome proteostasis-the emergence, maintenance, and clearance of the peroxisomal proteome.

More than meets the eye: Emergent properties of transcription factors networks in Arabidopsis.

Uncovering and mathematically modeling Transcription Factor Networks (TFNs) are the first steps in engineering plants with traits that are better equipped to respond to changing environments. Although several plant TFNs are well known, the framework for systematically modeling complex characteristics such as switch-like behavior, oscillations, and homeostasis that emerge from them remain elusive. This review highlights literature that provides, in part, experimental and computational techniques for characterizing TFNs. This review also outlines methodologies that have been used to mathematically model the dynamic characteristics of TFNs. We present several examples of TFNs in plants that are involved in developmental and stress response. In several cases, advanced algorithms capture or quantify emergent properties that serve as the basis for robustness and adaptability in plant responses. Increasing the use of mathematical approaches will shed new light on these regulatory properties that control plant growth and development, leading to mathematical models that predict plant behavior. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Clustering and Differential Alignment Algorithm: Identification of Early Stage Regulators in the Arabidopsis thaliana Iron Deficiency Response.

Time course transcriptome datasets are commonly used to predict key gene regulators associated with stress responses and to explore gene functionality. Techniques developed to extract causal relationships between genes from high throughput time course expression data are limited by low signal levels coupled with noise and sparseness in time points. We deal with these limitations by proposing the Cluster and Differential Alignment Algorithm (CDAA). This algorithm was designed to process transcriptome data by first grouping genes based on stages of activity and then using similarities in gene expression to predict influential connections between individual genes. Regulatory relationships are assigned based on pairwise alignment scores generated using the expression patterns of two genes and some inferred delay between the regulator and the observed activity of the target. We applied the CDAA to an iron deficiency time course microarray dataset to identify regulators that influence 7 target transcription factors known to participate in the Arabidopsis thaliana iron deficiency response. The algorithm predicted that 7 regulators previously unlinked to iron homeostasis influence the expression of these known transcription factors. We validated over half of predicted influential relationships using qRT-PCR expression analysis in mutant backgrounds. One predicted regulator-target relationship was shown to be a direct binding interaction according to yeast one-hybrid (Y1H) analysis. These results serve as a proof of concept emphasizing the utility of the CDAA for identifying unknown or missing nodes in regulatory cascades, providing the fundamental knowledge needed for constructing predictive gene regulatory networks. We propose that this tool can be used successfully for similar time course datasets to extract additional information and infer reliable regulatory connections for individual genes.

Phenylalanine is required to promote specific developmental responses and prevents cellular damage in response to ultraviolet light in soybean (Glycine max) during the seed-to-seedling transition.

UV-radiation elicits a suite of developmental (photomorphogenic) and protective responses in plants, but responses early post-germination have received little attention, particularly in intensively bred plants of economic importance. We examined germination, hypocotyl elongation, leaf pubescence and subcellular responses of germinating and/or etiolated soybean (Glycine max (L.) Merr.) seedlings in response to treatment with discrete wavelengths of UV-A or UV-B radiation. We demonstrate differential responses of germinating/young soybean seedlings to a range of UV wavelengths that indicate unique signal transduction mechanisms regulate UV-initiated responses. We have investigated how phenylalanine, a key substrate in the phenylpropanoid pathway, may be involved in these responses. Pubescence may be a key location for phenylalanine-derived protective compounds, as UV-B irradiation increased pubescence and accumulation of UV-absorbing compounds within primary leaf pubescence, visualized by microscopy and absorbance spectra. Mass spectrometry analysis of pubescence indicated that sinapic esters accumulate in the UV-irradiated hairs compared to unirradiated primary leaf tissue. Deleterious effects of some UV-B wavelengths on germination and seedling responses were reduced or entirely prevented by inclusion of phenylalanine in the growth media. Key effects of phenylalanine were not duplicated by tyrosine or tryptophan or sucrose, nor is the specificity of response due to the absorbance of phenylalanine itself. These results suggest that in the seed-to-seedling transition, phenylalanine may be a limiting factor in the development of initial mechanisms of UV protection in the developing leaf.

Pirin1 (PRN1) is a multifunctional protein that regulates quercetin, and impacts specific light and UV responses in the seed-to-seedling transition of Arabidopsis thaliana.

Pirins are cupin-fold proteins, implicated in apoptosis and cellular stress in eukaryotic organisms. Pirin1 (PRN1) plays a role in seed germination and transcription of a light- and ABA-regulated gene under specific conditions in the model plant system Arabidopsis thaliana. Herein, we describe that PRN1 possesses previously unreported functions that can profoundly affect early growth, development, and stress responses. In vitro-translated PRN1 possesses quercetinase activity. When PRN1 was incubated with G-protein-α subunit (GPA1) in the inactive conformation (GDP-bound), quercetinase activity was observed. Quercetinase activity was not observed when PRN1 was incubated with GPA1 in the active form (GTP-bound). Dark-grown prn1 mutant seedlings produced more quercetin after UV (317 nm) induction, compared to levels observed in wild type (WT) seedlings. prn1 mutant seedlings survived a dose of high-energy UV (254 nm) radiation that killed WT seedlings. prn1 mutant seedlings grown for 3 days in continuous white light display disoriented hypocotyl growth compared to WT, but hypocotyls of dark-grown prn1 seedlings appeared like WT. prn1 mutant seedlings transformed with GFP constructs containing the native PRN1 promoter and full ORF (PRN1::PRN1-GFP) were restored to WT responses, in that they did not survive UV (254 nm), and there was no significant hypocotyl disorientation in response to white light. prn1 mutants transformed with PRN1::PRN1-GFP were observed by confocal microscopy, where expression in the cotyledon epidermis was largely localized to the nucleus, adjacent to the nucleus, and diffuse and punctate expression occurred within some cells. WT seedlings transformed with the 35S::PRN1-GFP construct exhibited widespread expression in the epidermis of the cotyledon, also with localization in the nucleus. PRN1 may play a critical role in cellular quercetin levels and influence light- or hormonal-directed early development.