Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN 5.

Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system.

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.

Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters.

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON-REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.

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.

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure-function properties of PATELLIN2.

KEY MESSAGE: SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity. SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure-function relationships for biochemical-cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

The retromer, sorting nexins and the plant endomembrane protein trafficking.

Protein sorting in the endomembrane system is responsible for the coordination of cellular functions. Plant intracellular trafficking has its own unique features, which include specific regulatory aspects of endosomal sorting and recycling of cargo proteins, mediated by the retromer complex. Recent work has led to significant progress in understanding the role of Arabidopsis retromer subunits in recycling vacuolar sorting receptors and plasma membrane proteins. As a consequence, members of the sorting nexin (SNX) protein family and their interaction partners have emerged as critical protein trafficking regulators, in particular with regard to adaptation to environmental change, such as temperature fluctuations and nutrient deficiency. In this Review, we discuss the known and proposed functions of the comparatively small Arabidopsis SNX protein family. We review the available information on the role of the three Bin-Amphiphysin-Rvs (BAR)-domain-containing Arabidopsis thaliana (At)SNX proteins and discuss their function in the context of their potential participation in the plant retromer complex. We also summarize the role of AtSNX1-interacting proteins in different aspects of SNX-dependent protein trafficking and comment on the potential function of three novel, as yet unexplored, Arabidopsis SNX proteins.

Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR.

The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

Calcium-Promoted Interaction between the C2-Domain Protein EHB1 and Metal Transporter IRT1 Inhibits Arabidopsis Iron Acquisition.

Iron is a key transition element in the biosphere and is crucial for living organisms, although its cellular excess can be deleterious. Maintaining the balance of optimal iron availability in the model plant Arabidopsis (Arabidopsis thaliana) requires the precise operation of iron import through the principal iron transporter IRON-REGULATED TRANSPORTER1 (IRT1). Targeted inhibition of IRT1 can prevent oxidative stress, thus promoting plant survival. Here, we report the identification of an IRT1 inhibitor, namely the C2 domain-containing peripheral membrane protein ENHANCED BENDING1 (EHB1). EHB1 interacts with the cytoplasmically exposed variable region of IRT1, and we demonstrate that this interaction is greatly promoted by the presence of calcium. We found that EHB1 binds lipids characteristic of the plasma membrane, and the interaction between EHB1 and plant membranes is calcium-dependent. Molecular simulations showed that EHB1 membrane binding is a two-step process that precedes the interaction between EHB1 and IRT1. Genetic and physiological analyses indicated that EHB1 acts as a negative regulator of iron acquisition. The presence of EHB1 prevented the IRT1-mediated complementation of iron-deficient fet3fet4 yeast (Saccharomyces cerevisiae). Our data suggest that EHB1 acts as a direct inhibitor of IRT1-mediated iron import into the cell. These findings represent a major step in understanding plant iron acquisition, a process that underlies the primary production of bioavailable iron for land ecosystems.

COVID-19 in people with multiple sclerosis: A global data sharing initiative.

BACKGROUND: We need high-quality data to assess the determinants for COVID-19 severity in people with MS (PwMS). Several studies have recently emerged but there is great benefit in aligning data collection efforts at a global scale. OBJECTIVES: Our mission is to scale-up COVID-19 data collection efforts and provide the MS community with data-driven insights as soon as possible. METHODS: Numerous stakeholders were brought together. Small dedicated interdisciplinary task forces were created to speed-up the formulation of the study design and work plan. First step was to agree upon a COVID-19 MS core data set. Second, we worked on providing a user-friendly and rapid pipeline to share COVID-19 data at a global scale. RESULTS: The COVID-19 MS core data set was agreed within 48 hours. To date, 23 data collection partners are involved and the first data imports have been performed successfully. Data processing and analysis is an on-going process. CONCLUSIONS: We reached a consensus on a core data set and established data sharing processes with multiple partners to address an urgent need for information to guide clinical practice. First results show that partners are motivated to share data to attain the ultimate joint goal: better understand the effect of COVID-19 in PwMS.

Orphan crops at the food for future conference.

In her 1929 essay A Room of One's Own, Virginia Wolf famously wrote, "One cannot think well, love well, sleep well, if one has not dined well." While this popular quote is perhaps not the most inspiring, it is an elegant reminder that food and the cultural practices surrounding food are paramount for our wellbeing. However, in our quest to feed a growing global population, we have become focused on increasing the production of a few staple crops and overlooked hundreds or thousands of locally and regionally important crops that may represent the future of agriculture. The growing interest in identifying and developing promising new crops and novel food sources prompted the 1st Cologne Conference on Food for Future, which took place between the 5 and 7th of September 2018 at the Rautenstrauch-Joest museum in Cologne, Germany. It offered a unique platform for researchers, journalists, politicians, and entrepreneurs to present and discuss their views, visions, and concerns on the topics of Food Security. This interdisciplinary meeting acted as a stage to cover diverse aspects of crop science, food research, and food production in the context of global food and nutrition security. Three sessions accommodated scientific contributions on the topics of "Orphan Crops", "Functional food", and "Innovative food sources and production systems", and two public events (a public lecture and a plenary discussion) engaged the citizens with informative discussions on relevant and mediatic topics. With delegates from Africa, Europe, and the United States of America, the conference aimed at building bridges between different communities through scientific exchange.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.

SORTING NEXIN1 is required for modulating the trafficking and stability of the Arabidopsis IRON-REGULATED TRANSPORTER1.

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.

Regularized quadratic cost-function for integrating wave-front gradient fields.

From the Bayesian regularization theory we derive a quadratic cost-function for integrating wave-front gradient fields. In the proposed cost-function, the term of conditional distribution uses a central-differences model to make the estimated function well consistent with the observed gradient field. As will be shown, the results obtained with the central-differences model are superior to the results obtained with the backward-differences model, commonly used in other integration techniques. As a regularization term we use an isotropic first-order differences Markov Random-Field model, which acts as a low-pass filter reducing the errors caused by the noise. We present simulated and real experiments of the proposal applied in the Foucault test, obtaining good results.

Foucault test: a quantitative evaluation method.

Reliable and accurate testing methods are essential to guiding the polishing process during the figuring of optical telescope mirrors. With the natural advancement of technology, the procedures and instruments used to carry out this delicate task have consistently increased in sensitivity, but also in complexity and cost. Fortunately, throughout history, the Foucault knife-edge test has shown the potential to measure transverse aberrations in the order of the wavelength, mainly when described in terms of physical theory, which allows a quantitative interpretation of its characteristic shadowmaps. Our previous publication on this topic derived a closed mathematical formulation that directly relates the knife-edge position with the observed irradiance pattern. The present work addresses the quite unexplored problem of the wavefront's gradient estimation from experimental captures of the test, which is achieved by means of an optimization algorithm featuring a proposed ad hoc cost function. The partial derivatives thereby calculated are then integrated by means of a Fourier-based algorithm to retrieve the mirror's actual surface profile. To date and to the best of our knowledge, this is the very first time that a complete mathematical-grounded treatment of this optical phenomenon is presented, complemented by an image-processing algorithm which allows a quantitative calculation of the corresponding slope at any given point of the mirror's surface, so that it becomes possible to accurately estimate the aberrations present in the analyzed concave device just through its associated foucaultgrams.

Foucault test: shadowgram modeling from the physical theory for quantitative evaluations.

The physical theory of the Foucault test has been investigated to represent the complex amplitude and irradiance of the shadowgram in terms of the wavefront error; however, most of the studies have limited the treatment for the particular case of nearly diffraction-limited optical devices (i.e., aberrations smaller than the wavelength). In this paper we discard this restriction, and in order to show a more precise interpretation from the physical theory we derive expressions for the complex amplitude and the irradiance over an optical device with larger aberrations. To the best of our knowledge, it is the first time an expression is obtained in closed form. As will be seen, the result of this derivation is obtained using some properties of the Hilbert transform that permit representing the irradiance in a simple form in terms of the partial derivatives of the wavefront error. Additionally, we briefly describe from this point of view a methodology for the quantitative analysis of the test.

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.

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.

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.

Determination of copper(II) ion concentration by lifetime measurements of green fluorescent protein.

The understanding of cellular processes and functions and the elucidation of their physiological mechanisms is an important aim in the life sciences. One important aspect is the uptake and the release of essential substances as well as their interactions with the cellular environment. As green fluorescent protein (GFP) can be genetically encoded in cells it can be used as an internal sensor giving a deeper insight into biochemical pathways. Here we report that the presence of copper(II) ions leads to a decrease of the fluorescence lifetime (τ(fl)) of GFP and provide evidence for Förster resonance energy transfer (FRET) as the responsible quenching mechanism. We identify the His(6)-tag as the responsible binding site for Cu(2+) with a dissociation constant K(d) = 9 ± 2 µM and a Förster radius R(0) = 2.1 ± 0.1 nm. The extent of the lifetime quenching depends on [Cu(2+)] which is comprehended by a mathematical titration model. We envision that Cu(2+) can be quantified noninvasively and in real-time by measuring τ(fl) of GFP.