Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element–specific proteins reveal differentiation of the endomembrane system

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins (SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Several Yeast Species Induce Iron Deficiency Responses in Cucumber Plants (Cucumis sativus L.)

Iron (Fe) deficiency is a first-order agronomic problem that causes a significant decrease in crop yield and quality. Paradoxically, Fe is very abundant in most soils, mainly in its oxidized form, but is poorly soluble and with low availability for plants. In order to alleviate this situation, plants develop different morphological and physiological Fe-deficiency responses, mainly in their roots, to facilitate Fe mobilization and acquisition. Even so, Fe fertilizers, mainly Fe chelates, are widely used in modern agriculture, causing environmental problems and increasing the costs of production, due to the high prices of these products. One of the most sustainable and promising alternatives to the use of agrochemicals is the better management of the rhizosphere and the beneficial microbial communities presented there. The main objective of this research has been to evaluate the ability of several yeast species, such as Debaryomyces hansenii, Saccharomyces cerevisiae and Hansenula polymorpha, to induce Fe-deficiency responses in cucumber plants. To date, there are no studies on the roles played by yeasts on the Fe nutrition of plants. Experiments were carried out with cucumber plants grown in a hydroponic growth system. The effects of the three yeast species on some of the most important Fe-deficiency responses developed by dicot (Strategy I) plants, such as enhanced ferric reductase activity and Fe2+ transport, acidification of the rhizosphere, and proliferation of subapical root hairs, were evaluated. The results obtained show the inductive character of the three yeast species, mainly of Debaryomyces hansenii and Hansenula polymorpha, on the Fe-deficiency responses evaluated in this study. This opens a promising line of study on the use of these microorganisms as Fe biofertilizers in a more sustainable and environmentally friendly agriculture.

Growth and physiological impairments in Fe-starved alfalfa are associated with the downregulation of Fe and S transporters along with redox imbalance

Background Iron (Fe) is an essential plant nutrient. Its deficiency is a major constraint in crop production systems, affecting crop yield and quality. It is therefore important to elucidate the responses and adaptive mechanisms underlying Fe-deficiency symptoms in alfalfa. Materials and methods The experiment was carried out on 12-day-old alfalfa plants grown in hydroponics under Fe-sufficient and Fe-deficient conditions. Results The Fe-starved alfalfa showed decreased plant biomass, chlorophyll score, PSII efficiency, and photosynthesis performance index in young leaves under low Fe. Further, Fe shortage reduced the Fe, Zn, S and Ca concentration in root and shoot of alfalfa accompanied by the marked decrease of MsIRT1, MsZIP, MsSULTR1;1, MsSULTR1;2 and MsSULTR1;3 transcripts in root and shoot. It indicates that retardation caused by Fe-deficiency was also associated with the status of other elements, especially the reduced Fe and S may be coordinately attributed to the photosynthetic damages in Fe-deficient alfalfa. The ferric chelate reductase activity accompanied by the expression of MsFRO1 in roots showed no substantial changes, indicating the possible involvement of this Strategy I response in Fe-deficient alfalfa. However, the proton extrusion and expression of MsHAI1 were significantly induced following Fe-deficiency. In silico analysis further suggested their subcellular localization in the plasma membrane. Also, the interactome map suggested the partnership of MsFRO1 with plasma membrane H+-ATPase, transcription factor bHLH47, and nitrate reductase genes, while MsHAI1 partners include ferric reductase-like transmembrane component, plasma membrane ATPase, vacuolar-type H-pyrophosphatase, and general regulatory factor 2. In this study, SOD and APX enzymes showed a substantial increase in roots but unable to restore the oxidative damages in Fe-starved alfalfa. Conclusion These findings promote further studies for the improvement of Fe-starved alfalfa or legumes through breeding or transgenic approaches. Graphic Abstract

Influence of Ethylene Signaling in the Crosstalk Between Fe, S, and P Deficiency Responses in Arabidopsis thaliana

To cope with P, S, or Fe deficiency, dicot plants, like Arabidopsis, develop several responses (mainly in their roots) aimed to facilitate the mobilization and uptake of the deficient nutrient. Within these responses are the modification of root morphology, an increased number of transporters, augmented synthesis-release of nutrient solubilizing compounds and the enhancement of some enzymatic activities, like ferric reductase activity (FRA) or phosphatase activity (PA). Once a nutrient has been acquired in enough quantity, these responses should be switched off to minimize energy costs and toxicity. This implies that they are tightly regulated. Although the responses to each deficiency are induced in a rather specific manner, crosstalk between them is frequent and in such a way that P, S, or Fe deficiency can induce responses related to the other two nutrients. The regulation of the responses is not totally known but some hormones and signaling substances have been involved, either as activators [ethylene (ET), auxin, nitric oxide (NO)], or repressors [cytokinins (CKs)]. The plant hormone ET is involved in the regulation of responses to P, S, or Fe deficiency, and this could partly explain the crosstalk between them. In spite of these crosslinks, it can be hypothesized that, to confer the maximum specificity to the responses of each deficiency, ET should act in conjunction with other signals and/or through different transduction pathways. To study this latter possibility, several responses to P, S, or Fe deficiency have been studied in the Arabidopis wild-type cultivar (WT) Columbia and in some of its ethylene signaling mutants (ctr1, ein2-1, ein3eil1) subjected to the three deficiencies. Results show that key elements of the ET transduction pathway, like CTR1, EIN2, and EIN3/EIL1, can play a role in the crosstalk among nutrient deficiency responses.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

Physiological Responses to Fe Deficiency in Split-Root Tomato Plants: Possible Roles of Auxin and Ethylene?

Iron (Fe) bioavailability in soils is often limited and can be further exacerbated by a non- homogeneous distribution in the soil profile, which has been demonstrated to vary both in space and time. Consequently, plants respond with morphological and physiological modifications at the root level involving a complex local and systemic signaling machinery. The present work unravels the role of two phytohormones (i.e., ethylene and auxin) and their integrated signaling in plant response to Fe deficiency. Inhibitors of auxin polar transport and of ethylene biosynthesis (N-1-naphthylphthalamic acid - NPA and aminoethoxyvinylglycine - AVG, respectively) were applied on tomato (Solanum lycopersicum L.) plants grown by the split-root technique, which allows to simulate condition of Fe heterogeneous distribution. Results showed that plants, exposed to an uneven Fe supply, triggered a complex auxin-ethylene signaling. A systemic action of auxin on FERRIC REDUCTASE OXIDASE 1 (SlFRO1) expression was revealed, while ethylene signaling was effective both locally and systemically. In addition, the investigation of Fe concentration in tissues showed that when leaves overcame Fe deficiency a Fe “steady state” was maintained. Therefore, physiological adaptation to this heterogeneous Fe supply could be mediated by the integration of the complex signaling pathways prompted by both auxin and ethylene activities.

Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1)

Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2–STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer.