Novel roles of dynamin-related protein in iron-deficiency in Arabidopsis thaliana
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Plants have developed sophisticated mechanisms to maintain optimal homeostasis for their survival in diverse environmental conditions. Among these mechanisms, the modulation of the plasma membrane (PM) proteome stands out as a crucial adaptation strategy. The PM serves as a dynamic interface essential for perceiving and adapting to extra- and intracellular stimuli, necessitating precise control over the composition of its proteome. One such control that is central to the fine-tuning of the PM protein composition is achieved through vesicular trafficking. Vesicular trafficking encompasses the sorting and trafficking of cargo proteins to and from the PM via the central intracellular sorting hub trans-Golgi network/early endosomes (TGN/EE). However, the roles of components involved in the post-Golgi trafficking of proteins required for critical cellular processes remain largely unknown. Here, we utilized the model plant Arabidopsis thaliana (Arabidopsis) as well as biochemical and transcriptomic approaches to uncover novel roles of the vesicular trafficking protein DYNAMIN-RELATED PROTEIN1A (DRP1A) in modulating the accumulation of proteins and cellular components essential for physiological processes including nutrient deficiency. In Arabidopsis, DRP1A is a plant-specific large GTPase that belongs to a family of sixteen dynamin-related proteins (DRPs). DRPs are divided into six subfamilies (DRP1-6) based on their domain structure and functions in fission of organelles and/or membrane vesicles. DRP1A is the best-studied of the five DRP1s (DRP1A-E) and studies have shown that loss-of-function drp1a mutants are defective in cytokinesis and constitutive bulk membrane endocytosis. Previous studies from our lab identified DRP1A in modulating the proper PM abundance of the leaf-specific immune receptor FLAGELLIN SENSING2 (FLS2) after elicitation with the bacterial pathogen-associated molecular pattern (PAMP) flg22. Underscoring its role as an endocytic accessory protein, drp1a mutants are impaired in the flg22-induced endocytosis of FLS2. DRP1A is also essential for the polar PM localization of root-specific hormone efflux carrier PINFORMED1 (PIN1) and the nutrient transporter BORON TRANSPORTER1 (BOR1) to specific subdomains of the PM. Given the significance of DRP1A in maintaining abundance and polar localization of PM proteins, we sought to identify other PMlocalized proteins that may be modulated by DRP1A. Vesicular trafficking regulates the PM abundance and polar localization of the root-specific iron (Fe) transporter IRON REGULATED TRANSPORTER1 (IRT1). While few vesicular trafficking components have been identified in this process, this study identified a novel role for DRP1A in modulating the abundance of IRT1 protein and mRNA under various growth conditions. We discovered that loss of DRP1A led to reduced PM accumulation of IRT1 which correlated with reduced Fe in root tissue of drp1a mutant seedlings as well as reduced chlorophyll content and protein accumulation of photosynthetic proteins that require Fe for their function and/or stability. Conversely, we uncovered that drp1a mutants showed increased protein accumulation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), potentially leading to increased intracellular reactive oxygen species (ROS). In turn, increased intracellular ROS may correlate with disrupting Fe-deficiency pathways, indicating a role for DRP1A in Fe homeostasis and stress responses. Under Fe-deficiency, IRT1 must be trafficked and reside in the soil-side of the PM, called outer domain, for uptake of Fe from the rhizosphere as needed; but IRT1 must be removed from the PM through endocytic internalization to prevent excess Fe uptake, which can be toxic. When exploring the physiological impact of loss of DRP1A during Fe-deficiency, we uncovered that under both continuous Fe-deficiency and induced Fedeficiency, drp1a mutants had reduced PM accumulation of IRT1 as well as reduced accumulation of PHOTOSYNTHETIC ELECTRON TRANSFER C, a photosynthetic protein that requires Fe as part of its Fe-S cluster to transport electrons from Photosystem II to Photosystem I. Additionally, we provide evidence that DRP1A played a role in modulating the PM abundance of RBOHD that appeared to be independent of induced Fe-deficiency. Taken these findings together, this study provides new insight on the role of DRP1A in modulating Fe-deficiency responses in plants, though much remains unknown about the underlying cellular and molecular mechanisms. Lastly, through qRT-PCR and RNA-seq analysis, we discovered that loss of DRP1A altered the transcriptional response of a subset of Fe-response genes, specifically the major Fe-deficiency transcription factor FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) and its downstream targets IRT1 and FERRIC REDUCTASE OXIDASE2 (FRO2). Our findings revealed DRP1A's specific role in potentially modulating the transcriptional response to Fe-deficiency in roots at the level of FIT during induced Fe-deficiency. These genes are root-specific Fe-response genes and leaf-specific Fe-response genes such as OLIGOPEPTIDE TRANSPORTER3 (OPT3) and POPEYE (PYE) were not significantly altered in the leaf, we potentially identified a role for DRP1A in propagating the yet unknown Fe-deficiency signal within the roots, leading to reduced FIT gene expression. These findings highlight an essential role of DRP1A in the modulating the abundance of membrane proteins and transcriptional regulation of a subset of genes many of which have roles in maintaining Fe-homeostasis. With increasing global food demands, understanding the role of vesicular trafficking in Fe uptake and utilization is critical for developing sustainable, nutrient-rich crops to combat malnutrition and food insecurity.
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Ph. D.