The small iron-deficiency-induced protein OLIVIA and its relation to the bHLH transcription factor POPEYE.

Iron (Fe) is a crucial micronutrient needed in many metabolic processes. To balance needs and potential toxicity, plants control the amount of Fe they take up and allocate to leaves and seeds during their development. One important regulator of this process is POPEYE (PYE). PYE is a Fe deficiency-induced key bHLH transcription factor (TF) for allocation of internal Fe in plants. In the absence of PYE, there is altered Fe translocation and plants develop a leaf chlorosis. NICOTIANAMINE SYNTHASE4 (NAS4), FERRIC-REDUCTION OXIDASE3 (FRO3), and ZINC-INDUCED FACILITATOR1 (ZIF1) genes are expressed at higher level in pye-1 indicating that PYE represses these genes. PYE activity is controlled in a yet unknown manner. Here, we show that a small Fe deficiency-induced protein OLIVIA (OLV) can interact with PYE. OLV has a conserved C-terminal motif, that we named TGIYY. Through deletion mapping, we pinpointed that OLV TGIYY and several regions of PYE can be involved in the protein interaction. An OLV overexpressing (OX) mutant line exhibited an enhanced NAS4 gene expression. This was a mild Fe deficiency response phenotype that was related to PYE function. Leaf rosettes of olv mutants remained smaller than those of wild type, indicating that OLV promotes plant growth. Taken together, our study identified a small protein OLV as a candidate that may connect aspects of Fe homeostasis with regulation of leaf growth.

FER-like iron deficiency-induced transcription factor (FIT) accumulates in nuclear condensates.

The functional importance of nuclear protein condensation remains often unclear. The bHLH FER-like iron deficiency-induced transcription factor (FIT) controls iron acquisition and growth in plants. Previously described C-terminal serine residues allow FIT to interact and form active transcription factor complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into FIT nuclear bodies (NBs). Using quantitative and qualitative microscopy-based approaches, we characterized FIT NBs as condensates that were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. FIT condensation may affect nuclear mobility and be relevant for integrating environmental and Fe nutrition signals.

Black sheep, dark horses, and colorful dogs: a review on the current state of the Gene Ontology with respect to iron homeostasis in Arabidopsis thaliana.

Cellular homeostasis of the micronutrient iron is highly regulated in plants and responsive to nutrition, stress, and developmental signals. Genes for iron management encode metal and other transporters, enzymes synthesizing chelators and reducing substances, transcription factors, and several types of regulators. In transcriptome or proteome datasets, such iron homeostasis-related genes are frequently found to be differentially regulated. A common method to detect whether a specific cellular pathway is affected in the transcriptome data set is to perform Gene Ontology (GO) enrichment analysis. Hence, the GO database is a widely used resource for annotating genes and identifying enriched biological pathways in Arabidopsis thaliana. However, iron homeostasis-related GO terms do not consistently reflect gene associations and levels of evidence in iron homeostasis. Some genes in the existing iron homeostasis GO terms lack direct evidence of involvement in iron homeostasis. In other aspects, the existing GO terms for iron homeostasis are incomplete and do not reflect the known biological functions associated with iron homeostasis. This can lead to potential errors in the automatic annotation and interpretation of GO term enrichment analyses. We suggest that applicable evidence codes be used to add missing genes and their respective ortholog/paralog groups to make the iron homeostasis-related GO terms more complete and reliable. There is a high likelihood of finding new iron homeostasis-relevant members in gene groups and families like the ZIP, ZIF, ZIFL, MTP, OPT, MATE, ABCG, PDR, HMA, and HMP. Hence, we compiled comprehensive lists of genes involved in iron homeostasis that can be used for custom enrichment analysis in transcriptomic or proteomic studies, including genes with direct experimental evidence, those regulated by central transcription factors, and missing members of small gene families or ortholog/paralog groups. As we provide gene annotation and literature alongside, the gene lists can serve multiple computational approaches. In summary, these gene lists provide a valuable resource for researchers studying iron homeostasis in A. thaliana, while they also emphasize the importance of improving the accuracy and comprehensiveness of the Gene Ontology.

Role of the plant-specific calcium-binding C2-DOMAIN ABSCISIC ACID-RELATED (CAR) protein family in environmental signaling.

Many signaling processes rely on information decoding at the plasma membrane, and membrane-associated proteins and their complexes are fundamental for regulating this process. Still many questions exist as to how protein complexes are assembled and function at membrane sites to change identity and dynamics of membrane systems. Peripheral membrane proteins containing a calcium and phospholipid-binding C2-domain can act in membrane-related signaling by providing a tethering function so that protein complexes form. C2 domain proteins termed C2-DOMAIN ABSCISIC ACID-RELATED (CAR) proteins are plant-specific, and the functional relevance of this C2 domain protein subgroup is just emerging. The ten Arabidopsis CAR proteins CAR1 to CAR10 have a single C2 domain with a plant-specific insertion, the so-called "CAR-extra-signature" or also termed "sig domain". Via this "sig domain" CAR proteins can bind signaling protein complexes of different kinds and act in biotic and abiotic stress, blue light and iron nutrition. Interestingly, CAR proteins can oligomerize in membrane microdomains, and their presence in the nucleus can be linked with nuclear protein regulation. This shows that CAR proteins may play unprecedented roles in coordinating environmental responses and assembling required protein complexes to transmit information cues between plasma membrane and nucleus. The aim of this review is to summarize structure-function characteristics of the CAR protein family and assemble findings from CAR protein interactions and physiological functions. From this comparative investigation we extract common principles about the molecular operations that CAR proteins may fulfill in the cell. We also deduce functional properties of the CAR protein family based on its evolution and gene expression profiles. We highlight open questions and suggest novel avenues to prove and understand the functional networks and roles played by this protein family in plants.

Role of SEC14-like phosphatidylinositol transfer proteins in membrane identity and dynamics.

Membrane identity and dynamic processes, that act at membrane sites, provide important cues for regulating transport, signal transduction and communication across membranes. There are still numerous open questions as to how membrane identity changes and the dynamic processes acting at the surface of membranes are regulated in diverse eukaryotes in particular plants and which roles are being played by protein interaction complexes composed of peripheral and integral membrane proteins. One class of peripheral membrane proteins conserved across eukaryotes comprises the SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs). These proteins share a SEC14 domain that contributes to membrane identity and fulfills regulatory functions in membrane trafficking by its ability to sense, bind, transport and exchange lipophilic substances between membranes, such as phosphoinositides and diverse other lipophilic substances. SEC14L-PITPs can occur as single-domain SEC14-only proteins in all investigated organisms or with a modular domain structure as multi-domain proteins in animals and streptophytes (comprising charales and land plants). Here, we present an overview on the functional roles of SEC14L-PITPs, with a special focus on the multi-domain SEC14L-PITPs of the SEC14-nodulin and SEC14-GOLD group (PATELLINs, PATLs in plants). This indicates that SEC14L-PITPs play diverse roles from membrane trafficking to organism fitness in plants. We concentrate on the structure of SEC14L-PITPs, their ability to not only bind phospholipids but also other lipophilic ligands, and their ability to regulate complex cellular responses through interacting with proteins at membrane sites.

High-Throughput Plant Gene Expression Analysis by 384-Format Reverse Transcription-Quantitative PCR for Investigating Plant Iron Homeostasis.

Analysis of plant gene expression is important in determining iron (Fe) homeostasis gene functions during plant development or in response to biotic and abiotic factors. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has many advantages. It is fast, inexpensive, accurate, and reproducible in any lab. Furthermore, RT-qPCR can be scaled up to study several genes of interest in many biological samples from any organism. We hereby provide a straightforward protocol on RT-qPCR analysis using a 384-well format for large-scale gene expression studies on Fe-regulated responses. The protocol highlights in detail, the steps ranging from choice and design of qPCR analysis, collection of plant material and RNA preparation, cDNA synthesis, set up of qPCR and run, thorough analysis of qPCR run data, and display of multiple gene expression data for convenient interpretation.

Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5.

Small GTPases comprise key proteins in signal transduction that function by conformational switching ability between GDP- and GTP-bound states. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking and cellular functions. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized functional properties and cellular localization of the essential small ARF-like GTPase TITAN5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. TTN5 showed rapid guanine nucleotide exchange capacity comparable to that of human counterparts, but a remarkably low GTP hydrolysis reaction. A TTN5Q70L mutant had enhanced nucleotide exchange activity, indicative of intracellular activation, while TTN5T30N with fast nucleotide dissociation can be considered a dominant-negative form. This suggests that TTN5 is present in GTP-loaded active form in the cells. YFP-tagged TTN5 and the two derived mutant variants were located at multiple sites of the endomembrane system in the epidermis of Arabidopsis seedlings and Nicotiana benthamiana leaves. While YFP-TTN5 and YFP-TTN5Q70L were highly mobile in the cells, mobility was reduced for TTN5T30N. Colocalization with endomembrane markers in combination with pharmacological treatments resolved localization at membrane sites and showed that YFP-TTN5 and YFP-TTN5Q70L were located in Golgi stacks, multivesicular bodies, while this was less the case for YFP-TTN5T30N. On the other hand, all three TTN5 forms were located at the plasma membrane. Hence, the unusual capacity of rapid nucleotide exchange activity of the small ARF-like GTPase TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system.

Effectiveness of an evidence-based care pathway to improve mobility and participation in older patients with vertigo and balance disorders in primary care (MobilE-PHY2): study protocol for a multicentre cluster-randomised controlled trial.

BACKGROUND: Vertigo, dizziness or balance disorders (VDB) are common leading symptoms in older people, which can have a negative impact on their mobility and participation in daily live, yet, diagnosis is challenging and specific treatment is often insufficient. An evidence-based, multidisciplinary care pathway (CPW) in primary care was developed and pilot tested in a previous study. The aim of the present study is to evaluate the effectiveness and safety of the CPW in terms of improving mobility and participation in community-dwelling older people with VDB in primary care. METHODS: For this multicentre cluster randomised controlled clinic trial, general practitioners (GP) will be recruited in two regions of Germany. A total of 120 patients over 60 years old with VDB will be included. The intervention is an algorithmized CPW. GPs receive a checklist for standardise clinical decision making regarding diagnostic screening and treatment of VDB. Physiotherapists (PT) receive a decision tree for evidence-based physiotherapeutic clinical reasoning and treatment of VDB. Implementation strategies comprises educational trainings as well as a workshop to give a platform for exchange for the GPs and PTs, an information meeting and a pocket card for home care nurses and informal caregivers and telephone peer counselling to give all participants the capability, opportunity and the motivation to apply the intervention. In order to ensure an optimised usual care in the control group, GPs get an information meeting addressing the national guideline. The primary outcome is the impact of VDB on participation and mobility of patients after 6 month follow-up, assessed using the Dizziness Handicap Inventory (DHI) questionnaire. Secondary outcomes are physical activity, static and dynamic balance, falls and fear of falling as well as quality of life. We will also evaluate safety and health economic aspects of the intervention. Behavioural changes of the participants as well as barriers, facilitating factors and mechanisms of impact of the implementation will be investigated with a comprehensive process evaluation in a mixed-methods design. DISCUSSION: With our results, we aim to improve evidence-based health care of community-dwelling older people with VDB in primary care. TRIAL REGISTRATION: DRKS, DRKS00028524 retrospectively registered on March 24, 2022.

Relationship between kinematic gait quality and caregiver-reported everyday mobility in children and youth with spastic Cerebral Palsy.

BACKGROUND: 3D gait analysis (3DGA) is a common assessment in Cerebral Palsy (CP) to quantify the extent of movement abnormalities. Yet, 3DGA is performed in laboratories and may thus be of debatable significance to everyday life. AIM: The aim was to assess the relationship between kinematic gait abnormality and everyday mobility in ambulatory children and youth with spastic CP. METHODS: 73 paediatric and juvenile patients with uni- or bilateral spastic CP (N = 21 USCP, N = 52, BSCP, age: 4-20 y, GMFCS I-III) underwent a 3DGA, while the MobQues47 Questionnaire quantified caregiver-reported mobility. We calculated the Gait Profile Score (GPS), a metric that summarizes how far the lower limb joint angles during walking deviate from those of matched controls. RESULTS: The GPS correlated well with indoor and outdoor mobility (rho = -0.69 and -0.70, both p < 0.001) and the relationships were not significantly different for USCP and BSCP. Still, mobility was lower in BSCP (p < 0.001) and more compromised outdoors (p = 0.002). Indoor mobility could be predicted by walking speed, GPS and age (adj. R2 = 0.62). Outdoor mobility was best predicted by walking speed and GPS (adj. R2 = 0.60). The additive explained variance by the GPS was even higher outdoors than indoors (17.1% vs. 11.4%). CONCLUSIONS: Measuring movement deviations with 3DGA seems equally meaningful in uni- and bilaterally affected children and has considerable relevance for real-life ambulation, particurlarly outdoors, where children with spastic CP typically face greater difficulties. Therapeutic strategies that achieve faster walking and reduction of kinematic deviations may increase outdoor mobility.

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

The Iron Deficiency-Regulated Small Protein Effector FEP3/IRON MAN1 Modulates Interaction of BRUTUS-LIKE1 With bHLH Subgroup IVc and POPEYE Transcription Factors.

In light of climate change and human population growth one of the most challenging tasks is to generate plants that are Fe-efficient, resilient to low Fe supply and Fe-biofortified. For such endeavors, it is crucial to understand the regulation of Fe acquisition and allocation in plants. One open question is how identified Fe-regulatory proteins comprising positive and negative regulators act together to steer Fe homeostasis. bHLH transcription factors (TFs) belonging to the subgroups IVb and IVc can initiate a bHLH cascade controlling the -Fe response in roots. In Arabidopsis thaliana, the -Fe-induced genes are sub-divided into several gene co-expression clusters controlled by different sets of TFs. Some of the co-expressed genes encode regulatory E3 ligase proteins BRUTUS (BTS)/BTS-LIKE (BTSL) and small proteins belonging to the group of FE UPTAKE-INDUCING PEPTIDE/IRON MAN (FEP/IMA). Recently, it was described that FEP1/IMA3 and FEP3/IMA1 proteins inhibit the repression of bHLH factors by BTS. We had postulated that -Fe-regulated co-expression clusters provide new information about regulatory protein interaction complexes. Here, we report a targeted yeast two-hybrid screen among 23 proteins of the -Fe response. This identified a novel protein interactome involving another E3 ligase, namely BTSL1, basic helix-loop-helix (bHLH) protein POPEYE (PYE) and transcription factors of the subgroup IVc as well as FEP3/IMA1. Because of the difficulty in stable BTSL1 protein expression in plant cells, we used a yeast two hybrid-based deletion mapping, homology modeling and molecular docking, to pinpoint interaction sites in BTSL1 and FEP3/IMA1. bHLH IVc TFs have similar residues at their C-terminus as FEP3/IMA1 interacting sites. FEP3/IMA1 attenuated interaction of BTSL1 and bHLH proteins in a yeast three-hybrid assay, in line with physiological data pointing to enhanced Fe acquisition and allocation in FEP3/IMA1 overexpression and btsl1 btsl2 mutant plants. Hence, exploiting -Fe-induced gene co-expression networks identified FEP3/IMA1 as a small effector protein that binds and inhibits the BTSL1 complex with PYE and bHLH subgroup IVc proteins. Structural analysis resolved interaction sites. This information helps improving models of Fe regulation and identifying novel targets for breeding of Fe-efficient crops.

Strategies and Bottlenecks in Hexaploid Wheat to Mobilize Soil Iron to Grains.

Our knowledge of iron (Fe) uptake and mobilization in plants is mainly based on Arabidopsis and rice. Although multiple players of Fe homeostasis have been elucidated, there is a significant gap in our understanding of crop species, such as wheat. It is, therefore, imperative not only to understand the different hurdles for Fe enrichment in tissues but also to address specifically the knowns/unknowns involved in the plausible mechanism of Fe sensing, signaling, transport, and subsequent storage in plants. In the present review, a unique perspective has been described in light of recent knowledge generated in wheat, an economically important crop. The strategies to boost efficient Fe uptake, transcriptional regulation, and long-distance mobilization in grains have been discussed, emphasizing recent biotechnological routes to load Fe in grains. This article also highlights the new elements of physiological and molecular genetics that underpin the mechanistic insight for the identified Fe-related genes and discusses the bottlenecks in unloading the Fe in grains. The information presented here will provide much-needed resources and directions to overcome challenges and design efficient strategies to enhance the Fe density in wheat grains.

German translation and pre-testing of Consolidated Framework for Implementation Research (CFIR) and Expert Recommendations for Implementing Change (ERIC).

BACKGROUND: Implementation frameworks may support local implementation strategies with a sound theoretical foundation. The Consolidated Framework for Implementation Research (CFIR) facilitates identification of contextual barriers and facilitators, and the Expert Recommendations for Implementing Change (ERIC) allows identifying adequate implementation strategies. Both instruments are already used in German-speaking countries; however, no standardised and validated translation is available thus far. The aim of this study was to translate the CFIR and ERIC framework into German, in order to increase the use of these frameworks and the adherence to evidence-based implementation efforts in German-speaking countries. METHODS: The translation of the original versions of the CFIR and ERIC framework was guided by the World Health Organisation's recommendations for the process of translating and adapting both conceptual frameworks. Accordingly, a four-step process was employed: first, forward translation from English into German was conducted by a research team of German native speakers with fluent knowledge of the English language. Second, a bilingual expert panel comprising one researcher with German as his mother tongue and expert command of the English language and one English language expert and university teacher reviewed the translation and discussed inconsistencies with the initial translators. Third, back-translation into English was conducted by an English native speaking researcher. The final version was pre-tested with 12 German researchers and clinicians who were involved in implementation projects using cognitive interviews. RESULTS: The translation and review process revealed some inconsistencies between the original version and the German translations. All issues could be solved by discussion. Central aspects of the items were confirmed in 60 to 70% of the items, and modifications were proposed in 30% of the items. Finally, we revised one CFIR-item heading after pre-testing. The final version was given consent by all involved parties. CONCLUSIONS: Now, two validated and tested implementation frameworks to guide implementation efforts are available in the German language and can be used to increase the application of agreed-on implementation strategies into practice.

Development of a complex intervention to improve mobility and participation of older people with vertigo, dizziness and balance disorders in primary care: a mixed methods study.

BACKGROUND: Vertigo, dizziness and balance disorders (VDB) are common in older people and cause restrictions in mobility and social participation. Due to a multifactorial aetiology, health care is often overutilised, but many patients are also treated insufficiently in primary care. The purpose of this study was to develop a care pathway as a complex intervention to improve mobility and participation in older people with VDB in primary care. METHODS: The development process followed the UK Medical Research Council guidance using a mixed-methods design with individual and group interviews carried out with patients, physical therapists (PTs), general practitioners (GPs), nurses working in community care and a multi-professional expert panel to create a first draft of a care pathway (CPW) and implementation strategy using the Consolidated Framework of Implementation Research and the Expert recommendations for Implementing Change. Subsequently, small expert group modelling of specific components of the CPW was carried out, with GPs, medical specialists and PTs. The Behaviour Change Wheel was applied to design the intervention´s approach to behaviour change. To derive theoretical assumptions, we adopted Kellogg´s Logic Model to consolidate the hypothesized chain of causes leading to patient-relevant outcomes. RESULTS: Individual interviews with patients showed that VDB symptoms need to be taken more seriously by GPs. Patients demanded age-specific treatment offers, group sessions or a continuous mentoring by a PT. GPs required a specific guideline for diagnostics and treatment options including psychosocial interventions. Specific assignment to and a standardized approach during physical therapy were desired by PTs. Nurses favoured a multi-professional documentation system. The structured three-day expert workshop resulted in a first draft of CPW and potential implementation strategies. Subsequent modelling resulted in a CPW with components and appropriate training materials for involved health professionals. A specific implementation strategy is now available. CONCLUSION: A mixed-methods design was suggested to be a suitable approach to develop a complex intervention and its implementation strategy. We will subsequently test the intervention for its acceptability and feasibility in a feasibility study accompanied by a comprehensive process evaluation to inform a subsequent effectiveness trial. TRIAL REGISTRATION: The research project is registered in "Projektdatenbank Versorgungsforschung Deutschland" (Project-ID: VfD_MobilE-PHY_17_003910; date of registration: 30.11.2017).

Improving mobility and participation of older people with vertigo, dizziness and balance disorders in primary care using a care pathway: feasibility study and process evaluation.

BACKGROUND: Community-dwelling older people are frequently affected by vertigo, dizziness and balance disorders (VDB). We previously developed a care pathway (CPW) to improve their mobility and participation by offering standardized approaches for general practitioners (GPs) and physical therapists (PTs). We aimed to assess the feasibility of the intervention, its implementation strategy and the study procedures in preparation for the subsequent main trial. METHODS: This 12-week prospective cohort feasibility study was accompanied by a process evaluation designed according to the UK Medical Research Council's Guidance for developing and evaluating complex interventions. Patients with VDB (≥65 years), GPs and PTs in primary care were included. The intervention consisted of a diagnostic screening checklist for GPs and a guide for PTs. The implementation strategy included specific educational trainings and a telephone helpline. Data for mixed-method process evaluation were collected via standardized questionnaires, field notes and qualitative interviews. Quantitative data were analysed using descriptive statistics, qualitative data using content analysis. RESULTS: A total of five GP practices (seven single GPs), 10 PT practices and 22 patients were included in the study. The recruitment of GPs and patients was challenging (response rates: GP practices: 28%, PT practices: 39%). Ninety-one percent of the patients and all health professionals completed the study. The health professionals responded well to the educational trainings; the utilization of the telephone helpline was low (one call each from GPs and PTs). Familiarisation with the routine of application of the intervention and positive attitudes were emphasized as facilitators of the implementation of the intervention, whereas a lack of time was mentioned as a barrier. Despite difficulties in the GPs' adherence to the intervention protocol, the GPs, PTs and patients saw benefit in the intervention. The patients' treatment adherence to physical therapy was good. There were minor issues in data collection, but no unintended consequences. CONCLUSION: Although the process evaluation provided good support for the feasibility of study procedures, the intervention and its implementation strategy, we identified a need for improvement in recruitment of participants, the GP intervention part and the data collection procedures. The findings will inform the main trial to test the interventions effectiveness in a cluster RCT. TRIAL REGISTRATION: Projektdatenbank Versorgungsforschung Deutschland (German registry Health Services Research) VfD_MobilE-PHY_17_003910, date of registration: 30.11.2017; Deutsches Register Klinischer Studien (German Clinical Trials Register) DRKS00022918, date of registration: 03.09.2020 (retrospectively registered).

Comparative Transcriptomics of Lowland Rice Varieties Uncovers Novel Candidate Genes for Adaptive Iron Excess Tolerance.

Iron (Fe) toxicity is a major challenge for plant cultivation in acidic waterlogged soil environments, where lowland rice is a major staple food crop. Only few studies have addressed the molecular characterization of excess Fe tolerance in rice, and these highlight different mechanisms for Fe tolerance. Out of 16 lowland rice varieties, we identified a pair of contrasting lines, Fe-tolerant Lachit and -susceptible Hacha. The two lines differed in their physiological and morphological responses to excess Fe, including leaf growth, leaf rolling, reactive oxygen species generation and Fe and metal contents. These responses were likely due to genetic origin as they were mirrored by differential gene expression patterns, obtained through RNA sequencing, and corresponding gene ontology term enrichment in tolerant vs. susceptible lines. Thirty-five genes of the metal homeostasis category, mainly root expressed, showed differential transcriptomic profiles suggestive of an induced tolerance mechanism. Twenty-two out of these 35 metal homeostasis genes were present in selection sweep genomic regions, in breeding signatures, and/or differentiated during rice domestication. These findings suggest that Fe excess tolerance is an important trait in the domestication of lowland rice, and the identified genes may further serve to design the targeted Fe tolerance breeding of rice crops.

Glycerolipid profile differences between perennial and annual stem zones in the perennial model plant Arabis alpina.

The perennial life style is a successful ecological strategy, and Arabis alpina is a recently developed model Brassicaceae species for studying it. One aspect, poorly investigated until today, concerns the differing patterns of allocation, storage, and metabolism of nutrients between perennials and annuals and the yet unknown signals that regulate this process. A. alpina has a complex lateral stem architecture with a proximal vegetative perennial (PZ) and a distal annual flowering zone (AZ) inside the same stems. Lipid bodies (LBs) with triacylglycerols (TAGs) accumulate in the PZ. To identify potential processes of lipid metabolism linked with the perennial lifestyle, we analyzed lipid species in the PZ versus AZ. Glycerolipid fractions, including neutral lipids with mainly TAGs, phospholipids, and glycolipids, were present at higher levels in the PZ as compared to AZ or roots. Concomitantly, contents of specific long-chain and very long-chain fatty acids increased during formation of the PZ. Corresponding gene expression data, gene ontology term enrichment, and correlation analysis with lipid species pinpoint glycerolipid-related genes to be active during the development of the PZ. Possibilities that lipid metabolism genes may be targets of regulatory mechanisms specifying PZ differentiation in A. alpina are discussed.

Cytokinin-promoted secondary growth and nutrient storage in the perennial stem zone of Arabis alpina.

Perennial plants maintain their lifespan through several growth seasons. Arabis alpina serves as a model Brassicaceae species to study perennial traits. Lateral stems of A. alpina have a proximal vegetative zone with a dormant bud zone and a distal senescing seed-producing inflorescence zone. We addressed how this zonation is distinguished at the anatomical level, whether it is related to nutrient storage and which signals affect the zonation. We found that the vegetative zone exhibits secondary growth, which we termed the perennial growth zone (PZ). High-molecular-weight carbon compounds accumulate there in cambium and cambium derivatives. Neither vernalization nor flowering were requirements for secondary growth and the sequestration of storage compounds. The inflorescence zone with only primary growth, termed the annual growth zone (AZ), or roots exhibited different storage characteristics. Following cytokinin application cambium activity was enhanced and secondary phloem parenchyma was formed in the PZ and also in the AZ. In transcriptome analysis, cytokinin-related genes represented enriched gene ontology terms and were expressed at a higher level in the PZ than in the AZ. Thus, A. alpina primarily uses the vegetative PZ for nutrient storage, coupled to cytokinin-promoted secondary growth. This finding lays a foundation for future studies addressing signals for perennial growth.

Reactive oxygen species coordinate the transcriptional responses to iron availability in Arabidopsis.

Reactive oxygen species play a central role in the regulation of plant responses to environmental stress. Under prolonged iron (Fe) deficiency, increased levels of hydrogen peroxide (H2O2) initiate signaling events, resulting in the attenuation of Fe acquisition through the inhibition of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). As this H2O2 increase occurs in a FIT-dependent manner, our aim was to understand the processes involved in maintaining H2O2 levels under prolonged Fe deficiency and the role of FIT. We identified the CAT2 gene, encoding one of the three Arabidopsis catalase isoforms, as regulated by FIT. CAT2 loss-of-function plants displayed severe susceptibility to Fe deficiency and greatly increased H2O2 levels in roots. Analysis of the Fe homeostasis transcription cascade revealed that H2O2 influences the gene expression of downstream regulators FIT, BHLH genes of group Ib, and POPEYE (PYE); however, H2O2 did not affect their upstream regulators, such as BHLH104 and ILR3. Our data shows that FIT and CAT2 participate in a regulatory loop between H2O2 and prolonged Fe deficiency.