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.

Iron homeostasis and plant immune responses: Recent insights and translational implications.

Iron metabolism and the plant immune system are both critical for plant vigor in natural ecosystems and for reliable agricultural productivity. Mechanistic studies of plant iron home-ostasis and plant immunity have traditionally been carried out in isolation from each other; however, our growing understanding of both processes has uncovered significant connections. For example, iron plays a critical role in the generation of reactive oxygen intermediates during immunity and has been recently implicated as a critical factor for immune-initiated cell death via ferroptosis. Moreover, plant iron stress triggers immune activation, suggesting that sensing of iron depletion is a mechanism by which plants recognize a pathogen threat. The iron deficiency response engages hormone signaling sectors that are also utilized for plant immune signaling, providing a probable explanation for iron-immunity cross-talk. Finally, interference with iron acquisition by pathogens might be a critical component of the immune response. Efforts to address the global burden of iron deficiency-related anemia have focused on classical breeding and transgenic approaches to develop crops biofortified for iron content. However, our improved mechanistic understanding of plant iron metabolism suggests that such alterations could promote or impede plant immunity, depending on the nature of the alteration and the virulence strategy of the pathogen. Effects of iron biofortification on disease resistance should be evaluated while developing plants for iron biofortification.

Keep talking: crosstalk between iron and sulfur networks fine-tunes growth and development to promote survival under iron limitation.

Plants are capable of synthesizing all the molecules necessary to complete their life cycle from minerals, water, and light. This plasticity, however, comes at a high energetic cost and therefore plants need to regulate their economy and allocate resources accordingly. Iron-sulfur (Fe-S) clusters are at the center of photosynthesis, respiration, amino acid, and DNA metabolism. Fe-S clusters are extraordinary catalysts, but their main components (Fe2+ and S2-) are highly reactive and potentially toxic. To prevent toxicity, plants have evolved mechanisms to regulate the uptake, storage, and assimilation of Fe and S. Recent advances have been made in understanding the cellular economy of Fe and S metabolism individually, and growing evidence suggests that there is dynamic crosstalk between Fe and S networks. In this review, we summarize and discuss recent literature on Fe sensing, allocation, use efficiency, and, when pertinent, its relationship to S metabolism. Our future perspectives include a discussion about the open questions and challenges ahead and how the plant nutrition field can come together to approach these questions in a cohesive and more efficient way.

Defecatory dysfunction and other clinical variables are predictors of pessary discontinuation.

INTRODUCTION AND HYPOTHESIS: Pessaries provide first-line therapy for women with pelvic organ prolapse (POP) and stress urinary incontinence (SUI). The primary hypothesis was that defecatory dysfunction was associated with pessary discontinuation. METHODS: This was a retrospective cohort study of all women undergoing first pessary placement at one academic center from April 2014 to January 2017. Defecatory dysfunction was defined as the presence of constipation, rectal straining, rectal splinting, and/or incomplete defecation. Pessary discontinuation was defined as <1 year of pessary use and not using one at the most recent visit. Descriptive statistics; Person's chi-square, Fisher's exact, and Student's t test, and multivariate logistic regression analysis were used where appropriate. RESULTS: Charts of 1092 women were reviewed and 1071 were included. Mean age was 62 ± 15 years, mean body mass index (BMI) 28 ± 6 kg/m2, and mean parity 2 ± 1; 68% were Caucasian, 73% were menopausal, and 41% were sexually active. Reason for pessary use included POP (46%), SUI (24%), or both (30%). Overall pessary discontinuation rate was 77%; overall rate of defecatory dysfunction was 45%. In a logistic regression model, defecatory dysfunction in the form of incomplete defecation remained significantly associated with pessary discontinuation [odds ratio (OR) 3.29, 95% confidence interval (CI) 1.43-7.52]. Absence of bulge symptoms (OR 2.18, 95% CI 1.22-3.90), and younger age (OR 1.02, 95% CI 1.02-1.05) also remained significantly associated with pessary discontinuation. CONCLUSIONS: Pessary discontinuation was common, and defecatory dysfunction in the form of incomplete defecation had the strongest association with discontinuation. Understanding predictive factors of pessary discontinuation may help guide clinicians and patients when choosing treatment options for pelvic floor dysfunction.

The bHLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis.

KEY MESSAGE: ILR3 and PYE function in a regulatory network that modulates GLS accumulation under iron deficiency. The molecular processes involved in the cross talk between iron (Fe) homeostasis and other metabolic processes in plants are poorly understood. In Arabidopsis thaliana the transcription factor IAA-LEUCINE RESISTANT3 (ILR3) regulates iron deficiency response, aliphatic glucosinolate (GLS) biosynthesis and pathogen response. ILR3 is also known to interact with its homolog, POPEYE (PYE), which also plays a role in Fe response. However, little is known about how ILR3 regulates such diverse processes, particularly, via its interaction with PYE. Since GLS are produced as part of a defense mechanism against wounding pathogens, we examined pILR3::ß-GLUCURONIDASE expression and found that Fe deficiency enhances the wound-induced expression of ILR3 in roots and that ILR3 is induced in response to the wounding pathogen, sugarbeet root cyst nematode (Heterodera schachtii). We also examined the expression pattern of genes involved in Fe homeostasis and aliphatic GLS biosynthesis in pye-1, ilr3-2 and pye-1xilr3-2 (pxi) mutants and found that ILR3 and PYE differentially regulate the expression of genes involved these processes under Fe deficiency. We measured GLS levels and sugarbeet root cyst nematode infection rates under varying Fe conditions, and found that long-chain GLS levels are elevated in ilr3-2 and pxi mutants. This increase in long-chain GLS accumulation is correlated with elevated nematode resistance in ilr3-2 and pxi mutants in the absence of Fe. Our findings suggest that ILR3 and PYE function in a regulatory network that controls wounding pathogen response in plant roots by modulating GLS accumulation under iron deficiency.

The E3 ligase BRUTUS facilitates degradation of VOZ1/2 transcription factors.

BRUTUS (BTS) is an iron binding E3 ligase that has been shown to bind to and influence the accumulation of target basic helix-loop-helix transcription factors through 26S proteasome-mediated degradation in Arabidopsis thaliana. Vascular Plant One-Zinc finger 1 (VOZ1) and Vascular plant One-Zinc finger 2 (VOZ2) are NAM, ATAF1/2 and CUC2 (NAC) domain transcription factors that negatively regulate drought and cold stress responses in plants and have previously been shown to be degraded via the 26S proteasome. However, the mechanism that initializes this degradation is unknown. Here, we show that BTS interacts with VOZ1 and VOZ2 and that the presence of the BTS RING domain is essential for these interactions. Through cell-free degradation and immunodetection analyses, we demonstrate that BTS facilitates the degradation of Vascular plant One-Zinc finger 1/2 (VOZ1/2) protein in the nucleus particularly under drought and cold stress conditions. In addition to its known role in controlling the iron-deficiency response in plants, here, we report that BTS may play a role in drought and possibly other abiotic stress responses by facilitating the degradation of transcription factors, VOZ1/2.

Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::ß-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

Chromate alters root system architecture and activates expression of genes involved in iron homeostasis and signaling in Arabidopsis thaliana.

Soil contamination by hexavalent chromium [Cr(VI) or chromate] due to anthropogenic activities has become an increasingly important environmental problem. To date few studies have been performed to elucidate the signaling networks involved on adaptive responses to (CrVI) toxicity in plants. In this work, we report that depending upon its concentration, Cr(VI) alters in different ways the architecture of the root system in Arabidopsis thaliana seedlings. Low concentrations of Cr (20-40 µM) promoted primary root growth, while concentrations higher than 60 µM Cr repressed growth and increased formation of root hairs, lateral root primordia and adventitious roots. We analyzed global gene expression changes in seedlings grown in media supplied with 20 or 140 µM Cr. The level of 731 transcripts was significantly modified in response to Cr treatment with only five genes common to both Cr concentrations. Interestingly, 23 genes related to iron (Fe) acquisition were up-regulated including IRT1, YSL2, FRO5, BHLH100, BHLH101 and BHLH039 and the master controllers of Fe deficiency responses PYE and BTS were specifically activated in pericycle cells. It was also found that increasing concentration of Cr in the plant correlated with a decrease in Fe content, but increased both acidification of the rhizosphere and activity of the ferric chelate reductase. Supply of Fe to Cr-treated Arabidopsis allowed primary root to resume growth and alleviated toxicity symptoms, indicating that Fe nutrition is a major target of Cr stress in plants. Our results show that low Cr levels are beneficial to plants and that toxic Cr concentrations activate a low-Fe rescue system.

A single amino acid alteration in PGR5 confers resistance to antimycin A in cyclic electron transport around PSI.

In Arabidopsis thaliana, the main route of cyclic electron transport around PSI is sensitive to antimycin A, but the site of inhibition has not been clarified. We discovered that ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts was less sensitive to antimycin A in Arabidopsis that overaccumulated PGR5 (PROTON GRADIENT REGULATION 5) originating from Pinus taeda (PtPGR5) than that in the wild type. Consistent with this in vitro observation, infiltration of antimycin A reduced PSII yields and the non-photochemical quenching (NPQ) of Chl fluorescence in wild-type leaves but not in leaves accumulating PtPGR5. There are eight amino acid differences between PGR5 of Arabidopsis (AtPGR5) and PtPGR5 in their mature forms. To determine the site conferring antimycin A resistance, a series of AtPGR5 and PtPGR5 variants was introduced into the Arabidopsis pgr5 mutant. We determined that the presence of lysine rather than valine at the third amino acid position was necessary and sufficient for resistance to antimycin A. High levels of resistance to antimycin A required overaccumulation of PtPGR5 in ruptured chloroplasts, suggesting that PtPGR5 is partly resistant to antimycin A. In contrast, PSII yield was almost fully resistant to antimycin A in leaves accumulating endogenous levels of PtPGR5 or AtPGR5 V3K that had lysine instead of valine at the third position. NPQ was also dramatically recovered in leaves of these lines. These results imply that partial recovery of PSI cyclic electron transport is sufficient for maintaining redox homeostasis in photosynthesis. Our discovery suggests that antimycin A inhibits the function of PGR5 or proteins localized close to PGR5.

The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots.

Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. Using cell type-specific transcriptional profiling, we identified a pericycle-specific iron deficiency response and a bHLH transcription factor, POPEYE (PYE), that may play an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron-deficient conditions. Chromatin immunoprecipitation-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by regulating the expression of known iron homeostasis genes and other genes involved in transcription, development, and stress response. PYE interacts with PYE homologs, including IAA-Leu Resistant3 (ILR3), another bHLH transcription factor that is involved in metal ion homeostasis. Moreover, ILR3 interacts with a third protein, BRUTUS (BTS), a putative E3 ligase protein, with metal ion binding and DNA binding domains, which negatively regulates the response to iron deficiency. PYE and BTS expression is also tightly coregulated. We propose that interactions among PYE, PYE homologs, and BTS are important for maintaining iron homeostasis under low iron conditions.

Transcription factors and hormones: new insights into plant cell differentiation.

Plant development is a continuous process, mainly due to the presence of stem cell niches within the root and shoot. The interplay between a host of transcription factors determines whether the cells within the meristem maintain their stem cell state, differentiate into leaves or form secondary meristems, which develop into shoots and flowers. Several recent studies provide new insight into how transcription factors and phytohormones interact within meristems to control cell proliferation and differentiation.

E3 ligase BRUTUS Is a Negative Regulator for the Cellular Energy Level and the Expression of Energy Metabolism-Related Genes Encoded by Two Organellar Genomes in Leaf Tissues.

E3 ligase BRUTUS (BTS), a putative iron sensor, is expressed in both root and shoot tissues in seedlings of Arabidopsis thaliana. The role of BTS in root tissues has been well established. However, its role in shoot tissues has been scarcely studied. Comparative transcriptome analysis with shoot and root tissues revealed that BTS is involved in regulating energy metabolism by modulating expression of mitochondrial and chloroplast genes in shoot tissues. Moreover, in shoot tissues of bts-1 plants, levels of ADP and ATP and the ratio of ADP/ATP were greatly increased with a concomitant decrease in levels of soluble sugar and starch. The decreased starch level in bts-1 shoot tissues was restored to the level of shoot tissues of wild-type plants upon vanadate treatment. Through this study, we expand the role of BTS to regulation of energy metabolism in the shoot in addition to its role of iron deficiency response in roots.

Solving the puzzle of Fe homeostasis by integrating molecular, mathematical, and societal models.

To ensure optimal utilization and bioavailability, iron uptake, transport, subcellular localization, and assimilation are tightly regulated in plants. Herein, we examine recent advances in our understanding of cellular responses to Fe deficiency. We then use intracellular mechanisms of Fe homeostasis to discuss how formalizing cell biology knowledge via a mathematical model can advance discovery even when quantitative data is limited. Using simulation-based inference to identify plausible systems mechanisms that conform to known emergent phenotypes can yield novel, testable hypotheses to guide targeted experiments. However, this approach relies on the accurate encoding of domain-expert knowledge in exploratory mathematical models. We argue that this would be facilitated by fostering more "systems thinking" life scientists and that diversifying your research team may be a practical path to achieve that goal.

A hybrid model connecting regulatory interactions with stem cell divisions in the root.

Stem cells give rise to the entirety of cells within an organ. Maintaining stem cell identity and coordinately regulating stem cell divisions is crucial for proper development. In plants, mobile proteins, such as WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and SHORTROOT (SHR), regulate divisions in the root stem cell niche. However, how these proteins coordinately function to establish systemic behaviour is not well understood. We propose a non-cell autonomous role for WOX5 in the cortex endodermis initial (CEI) and identify a regulator, ANGUSTIFOLIA (AN3)/GRF-INTERACTING FACTOR 1, that coordinates CEI divisions. Here, we show with a multi-scale hybrid model integrating ordinary differential equations (ODEs) and agent-based modeling that quiescent center (QC) and CEI divisions have different dynamics. Specifically, by combining continuous models to describe regulatory networks and agent-based rules, we model systemic behaviour, which led us to predict cell-type-specific expression dynamics of SHR, SCARECROW, WOX5, AN3 and CYCLIND6;1, and experimentally validate CEI cell divisions. Conclusively, our results show an interdependency between CEI and QC divisions.

Broadening the impact of plant science through innovative, integrative, and inclusive outreach.

Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re-envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.

Computational solutions for modeling and controlling plant response to abiotic stresses: a review with focus on iron deficiency.

Computational solutions enable plant scientists to model protein-mediated stress responses and characterize novel gene functions that coordinate responses to a variety of abiotic stress conditions. Recently, density functional theory was used to study proteins active sites and elucidate enzyme conversion mechanisms involved in iron deficiency responsive signaling pathways. Computational approaches for protein homology modeling and the kinetic modeling of signaling pathways have also resolved the identity and function in proteins involved in iron deficiency signaling pathways. Significant changes in gene relationships under other stress conditions, such as heat or drought stress, have been recently identified using differential network analysis, suggesting that stress tolerance is achieved through asynchronous control. Moreover, the increasing development and use of statistical modeling and systematic modeling of transcriptomic data have provided significant insight into the gene regulatory mechanisms associated with abiotic stress responses. These types of in silico approaches have facilitated the plant science community's future goals of developing multi-scale models of responses to iron deficiency stress and other abiotic stress conditions.

MAGIC: Live imaging of cellular division in plant seedlings using lightsheet microscopy.

Imaging technologies have been used to understand plant genetic and developmental processes, from the dynamics of gene expression to tissue and organ morphogenesis. Although the field has advanced incredibly in recent years, gaps remain in identifying fine and dynamic spatiotemporal intervals of target processes, such as changes to gene expression in response to abiotic stresses. Lightsheet microscopy is a valuable tool for such studies due to its ability to perform long-term imaging at fine intervals of time and at low photo-toxicity of live vertically oriented seedlings. In this chapter, we describe a detailed method for preparing and imaging Arabidopsis thaliana seedlings for lightsheet microscopy via a Multi-Sample Imaging Growth Chamber (MAGIC), which allows simultaneous imaging of at least four samples. This method opens new avenues for acquiring imaging data at a high temporal resolution, which can be eventually probed to identify key regulatory time points and any spatial dependencies of target developmental processes.

BioVision Tracker: A semi-automated image analysis software for spatiotemporal gene expression tracking in Arabidopsis thaliana.

Fluorescence microscopy can produce large quantities of data that reveal the spatiotemporal behavior of gene expression at the cellular level in plants. Automated or semi-automated image analysis methods are required to extract data from these images. These data are helpful in revealing spatial and/or temporal-dependent processes that influence development in the meristematic region of plant roots. Tracking spatiotemporal gene expression in the meristem requires the processing of multiple microscopy imaging channels (one channel used to image root geometry which serves as a reference for relating locations within the root, and one or more channels used to image fluorescent gene expression signals). Many automated image analysis methods rely on the staining of cell walls with fluorescent dyes to capture cellular geometry and overall root geometry. However, in long time-course imaging experiments, dyes may fade which hinders spatial assessment in image analysis. Here, we describe a procedure for analyzing 3D microscopy images to track spatiotemporal gene expression signals using the MATLAB-based BioVision Tracker software. This software requires either a fluorescence image or a brightfield image to analyze root geometry and a fluorescence image to capture and track temporal changes in gene expression.