CRISPR/Cas9 Editing of the Wheat Iron Sensor <i>TaHRZ1</i> Confirms Its Conserved Role in Iron Homeostasis and Allocation in Grains

Plant Cell & Environment · 2026-04-03

Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum L. (wheat) HRZ homoeologs referred to as TaHRZ1 and TaHRZ2, were identified by BLAST searches using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode for proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain of TaHRZ1, coupled with devlopmental regulators GRF4-GIF1 chimeric protein, achieved 6.4%-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved Fe partitioning compared to the non-edited plants. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.

John Matthew McDowell: A Visionary Leader in Molecular Oomycete-Plant Interactions and a Wonderful Mentor and Friend to Many

Molecular Plant-Microbe Interactions · 2025-01-01

John M. McDowell, a leader in the field of molecular plant-microbe interactions, friend and colleague of many scientists in the molecular plant-microbe community, and a dedicated member of the International Society of Molecular Plant-Microbe Interactions, passed away in December 2024. John was known to many, not just because of his own seminal scientific discoveries, but because of his ability to synthesize the progress made in the field of molecular plant-microbe interactions, and in particular, the interplay between oomycete pathogens and the plant immune system, which led to many outstanding reviews. His ability to zoom out from minute details to the big picture also made him an effective mentor, teacher, editor in chief of the MPMI journal, NSF program director, and a great colleague, who would always be ready to ask the illuminating questions that helped you in deciding where to direct future efforts or provide sensitive hypotheses to test next. And in whatever he did, in whatever situation he was, he was always supportive, encouraging, lifting people up, and making them feel good about themselves and getting everybody even more excited about the research they were doing. He was the most even-keeled, kind-hearted person, an example to all of how to be better - not just as a scientist but as a human. Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Iron deficiency changes regulatory mechanisms governing sieve element cell differentiation

Nature Communications · 2025-11-20

Plant cell differentiation incorporates environmental cues over time to optimize overall growth. Iron deficiency influences development, such as root hair, cortical, and endodermal cell differentiation. However, the mechanisms by which iron deficiency regulates cell differentiation are not well characterized. Root sieve elements serve as an excellent model for cell differentiation since all cells, from undifferentiated to differentiated, are present in a distinct cell file. Here, we use semi-automated image analysis to show that iron deficiency delays sieve element differentiation, particularly enucleation and cell wall thickening, and consequently delays phloem sap unloading to roots. Using Dynamic Bayesian modeling we also characterize how iron deficiency changes the fundamental structure of the gene regulatory network associated with sieve element differentiation. We identify DOF1.5 as a positive regulator of sieve element enucleation and, consequently, of root sap translocation. These results clarify how abiotic stress can influence overall plant growth as a consequence of negatively influencing vascular differentiation. Plant growth and development respond to environmental cues. Here the authors infer gene regulatory networks controlling sieve element differentiation and show that under iron deficiency, enucleation and, consequently, phloem sap translocation are delayed.

Integrative spatial transcriptomic analysis pinpoints the role of the ferroxidase, <scp>TaMCO3</scp> , in wheat root tip iron mobilization

The Plant Journal · 2025-04-01 · 3 citations

Roots play a critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about the molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences result in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (-Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilization. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple multicopper oxidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Further characterization of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the -Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to -Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in -Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

Integrative spatial transcriptomic analysis pinpoints the role of the ferroxidase, TaMCO3, in wheat root tip iron mobilization

bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-24

SUMMARY Roots play an critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences results in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (–Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilisation. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple m ulti- c opper o xidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Detailed characterisation of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the –Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to –Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in –Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

CRISPR/Cas9 editing of the wheat iron sensor <i>TaHRZ1</i> confirms its conserved role in iron homeostasis and allocation in grains

bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-29 · 1 citations

Abstract Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum (wheat) HRZ homoeologs belonging to TaHRZ1 and TaHRZ2, were identified by BLAST search using rice ( Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain in all the TaHRZ1 homoeologs, coupled with GRF4-GIF1 chimeric protein, achieved 6.4-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved iron partitioning compared to the wild type. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3 , and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.

The Black American experience: Answering the global challenge of broadening participation in STEM/agriculture

The Plant Cell · 2024-01-29

From the stars to your table - plants as complex conduits for iron nutrition

2024-06-08

Iron is a ubiquitous micronutrient that plays critical roles in central metabolic processes for all living organisms. The mechanisms by which plants extract iron from soil and maintain iron homeostasis are particularly intriguing. While it is relatively abundant, in most soils iron is insoluble and therefore of limited bioavailability, however excess iron accumulation in plants can lead to cellular damage. Thus, plants must extract sufficient quantities of iron from recalcitrant soil environments, while also ensuring that iron content does not exceed a specific range. Arabidopsis and other dicots have evolved mechanisms to sense iron deficiency in the shoot, which triggers roots to solubilize, reduce and uptake iron across multiple root cell types before transport to the shoot. Using a combination of molecular and confocal microscopy analysis, cell-type specific transcriptional profiling, and mathematical modeling we have uncovered several molecular mechanisms that control how plants recognize and respond to iron deficiency stress in a root cell-specific manner. Our findings provide new evidence for how distinct alternations in the root cortex control carbon metabolism in response to iron deprivation, and how iron deficiency causes specific developmental alterations in the root vasculature and epidermis. Together, these mechanisms operate to fine-tune root growth and physiology in the face of suboptimal growth conditions, while also providing new avenues for exploring inter- and intracellular nutrient stress response in plants.

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

BMC Genomics · 2023-10-18 · 1 citations

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

PLANT PHYSIOLOGY · 2022-08-03 · 15 citations

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.