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.

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.

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.

Hemerythrin E3 Ubiquitin Ligases as Negative Regulators of Iron Homeostasis in Plants.

Iron (Fe) is an essential nutrient for plants, but at the same time its redox properties can make it a dangerous toxin inside living cells. Homeostasis between uptake, use and storage of Fe must be maintained at all times. A small family of unique hemerythrin E3 ubiquitin ligases found in green algae and plants play an important role in avoiding toxic Fe overload, acting as negative regulators of Fe homeostasis. Protein interaction data showed that they target specific transcription factors for degradation by the 26S proteasome. It is thought that the activity of the E3 ubiquitin ligases is controlled by Fe binding to the N-terminal hemerythrin motifs. Here, we discuss what we have learned so far from studies on the HRZ (Hemerythrin RING Zinc finger) proteins in rice, the homologous BTS (BRUTUS) and root-specific BTSL (BRUTUS-LIKE) in Arabidopsis. A mechanistic model is proposed to help focus future research questions towards a full understanding of the regulatory role of these proteins in Fe homeostasis in plants.

Automated Imaging, Tracking, and Analytics Pipeline for Differentiating Environmental Effects on Root Meristematic Cell Division.

Exposure of plants to abiotic stresses, whether individually or in combination, triggers dynamic changes to gene regulation. These responses induce distinct changes in phenotypic characteristics, enabling the plant to adapt to changing environments. For example, iron deficiency and heat stress have been shown to alter root development by reducing primary root growth and reducing cell proliferation, respectively. Currently, identifying the dynamic temporal coordination of genetic responses to combined abiotic stresses remains a bottleneck. This is, in part, due to an inability to isolate specific intervals in developmental time where differential activity in plant stress responses plays a critical role. Here, we observed that iron deficiency, in combination with temporary heat stress, suppresses the expression of iron deficiency-responsive pPYE::LUC (POPEYE::luciferase) and pBTS::LUC (BRUTUS::luciferase) reporter genes. Moreover, root growth was suppressed less under combined iron deficiency and heat stress than under either single stress condition. To further explore the interaction between pathways, we also created a computer vision pipeline to extract, analyze, and compare high-dimensional dynamic spatial and temporal cellular data in response to heat and iron deficiency stress conditions at high temporal resolution. Specifically, we used fluorescence light sheet microscopy to image Arabidopsis thaliana roots expressing CYCB1;1:GFP, a marker for cell entry into mitosis, every 20 min for 24 h exposed to either iron sufficiency, iron deficiency, heat stress, or combined iron deficiency and heat stress. Our pipeline extracted spatiotemporal metrics from these time-course data. These metrics showed that the persistency and timing of CYCB1;1:GFP signal were uniquely different under combined iron deficiency and heat stress conditions versus the single stress conditions. These metrics also indicated that the spatiotemporal characteristics of the CYCB1;1:GFP signal under combined stress were more dissimilar to the control response than the response seen under iron deficiency for the majority of the 24-h experiment. Moreover, the combined stress response was less dissimilar to the control than the response seen under heat stress. This indicated that pathways activated when the plant is exposed to both iron deficiency and heat stress affected CYCB1;1:GFP spatiotemporal function antagonistically.

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.

Further insight into BRUTUS domain composition and functionality.

BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.

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.

Fixating on metals: new insights into the role of metals in nodulation and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation is one of the most promising and immediate alternatives to the overuse of polluting nitrogen fertilizers for improving plant nutrition. At the core of this process are a number of metalloproteins that catalyze and provide energy for the conversion of atmospheric nitrogen to ammonia, eliminate free radicals produced by this process, and create the microaerobic conditions required by these reactions. In legumes, metal cofactors are provided to endosymbiotic rhizobia within root nodule cortical cells. However, low metal bioavailability is prevalent in most soils types, resulting in widespread plant metal deficiency and decreased nitrogen fixation capabilities. As a result, renewed efforts have been undertaken to identify the mechanisms governing metal delivery from soil to the rhizobia, and to determine how metals are used in the nodule and how they are recycled once the nodule is no longer functional. This effort is being aided by improved legume molecular biology tools (genome projects, mutant collections, and transformation methods), in addition to state-of-the-art metal visualization systems.