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

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

Computational approaches to identify regulators of plant stress response using high-throughput gene expression data

AbstractInsight into biological stress regulatory pathways can be derived from high-throughput transcriptomic data using computational algorithms. These algorithms can be integrated into a computational approach to provide specific testable predictions that answer biological questions of interest. This review conceptually organizes a wide variety of developed algorithms into a classification system based on desired type of output predictions. This classification is then used as a structure to describe completed approaches in the literature, with a focus on project goals, overall path of implemented algorithms, and biological insight gained. These algorithms and approaches are introduced mainly in the context of research on the model plant species Arabidopsis thaliana under stress conditions, though the nature of computational techniques makes these approaches easily applicable to a wide range of species, data types, and conditions

Host auxin machinery is important in mediating root architecture changes induced by <i>Pseudomonas fluorescens</i> GM30 and <i>Serendipita indica</i>

AbstractAuxin is a key phytohormone that is integral to plant developmental processes including those underlying root initiation, elongation, and branching. Beneficial microbes have been shown to have an impact on root development, potentially mediated through auxin. In this study, we explore the role of host auxin signaling and transport components in mediating the root growth promoting effects of beneficial microbes. Towards this end, we undertook co-culture studies of Arabidopsis thaliana plants with microbes previously reported to promote lateral root proliferation and produce auxin. Two types of beneficial microbes were included in the present study; a plant growth promoting bacterial species of interest, Pseudomonas fluorescens GM30, and a well-studied plant growth promoting fungal species, Serendipita indica (Piriformospora indica). Following co-culture, lateral root production was found restored in auxin transport inhibitor-treated plants, suggesting involvement of microbe and/or microbially-produced auxin in altering plant auxin levels. In order to clarify the role of host auxin signaling and transport pathways in mediating interactions with bacterial and fungal species, we employed a suite of auxin genetic mutants as hosts in co-culture screens. Our result show that the transport proteins PIN2, PIN3, and PIN7 and the signaling protein ARF19, are required for mediating root architecture effects by the bacterial and/or fungal species. Mutants corresponding to these proteins did not significantly respond to co-culture treatment and did not show increases in lateral root production and lateral root density. These results implicate the importance of host auxin signaling in both bacterial and fungal induced changes in root architecture and serve as a driver for future research on understanding the role of auxin-dependent and auxin-independent pathways in mediating plant-microbe interactions in economically important crop species.</jats:p

Table_1_Automated Imaging, Tracking, and Analytics Pipeline for Differentiating Environmental Effects on Root Meristematic Cell Division.docx

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</p

Experimentally validated regulatory relationships.

<p>Validated regulations between 4 early stage transcription factors and 7 known iron homeostasis transcription factors. Edges indicating positive regulations are green and edges indicating negative regulations are red. Edge indicating a direct connection validated by Y1H is darker.</p