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Manganese (Mn), iron (Fe), and zinc (Zn) are essential for diverse processes in plants, but their availability is often limiting or excessive. Cation diffusion facilitator (CDF) proteins have been implicated in the allocation of those metals in plants, whereby most of our mechanistic understanding has been obtained in Arabidopsis. It is unclear to what extent this can be generalized to other dicots. We characterized all CDFs/metal tolerance proteins of sugar beet (Beta vulgaris spp. vulgaris), which is phylogenetically distant from Arabidopsis. Analysis of subcellular localization, substrate selectivities, and transcriptional regulation upon exposure to metal deficiencies and toxicities revealed unexpected deviations from their Arabidopsis counterparts. Localization and selectivity of some members were modulated by alternative splicing. Notably, unlike in Arabidopsis, Mn- and Zn-sequestrating members were not induced in Fe-deficient roots, pointing to differences in the Fe acquisition machinery. This was supported by low Zn and Mn accumulation under Fe deficiency and a strikingly increased Fe accumulation under Mn and Zn excess, coinciding with an induction of BvIRT1. High Zn load caused a massive upregulation of Zn-BvMTPs. The results suggest that the employment of the CDF toolbox is highly diverse amongst dicots, which questions the general applicability of metal homeostasis models derived from Arabidopsis.

Iron (Fe) deficiency causes reduced growth and yield in broccoli. This study elucidates how sodium nitroprusside (SNP), known as nitric oxide (NO) donor, mitigates the retardation caused by Fe deficiency in broccoli. The SNP caused substantial nitric oxide accumulation in the roots of Fe-deficient plants, which resulted in a significant improvement in chlorophyll levels, photosynthetic efficiency, and morphological growth parameters, showing that it has a favorable influence on recovering broccoli health. Ferric reductase activity and the expression of BoFRO1 (ferric chelate reductase) gene in roots were consistently increased by SNP under Fe deficiency, which likely resulted in increased Fe mobilization. Furthermore, proton (H

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca

The design of novel polymers bearing multiple ene-diynes in the main chain are reported, allowing to tune activity of DNA cleavage via the Bergman cyclization.

Manganese (Mn) is an essential microelement, but overaccumulation is harmful to many plant species. Most plants have similar minimal Mn requirements, but the tolerance to elevated Mn varies considerably. Mobilization of phosphate (P) by plant roots leads to increased Mn uptake, and shoot Mn levels have been reported to serve as an indicator for P mobilization efficiency in the presence of P deficiency. White lupin (Lupinus albus L.) mobilizes P and Mn with outstanding efficiency due to the formation of determinate cluster roots that release carboxylates. The high Mn tolerance of L. albus goes along with shoot Mn accumulation, but the molecular basis of this detoxification mechanism has been unknown. In this study, we identify LaMTP8.1 as the transporter mediating vacuolar sequestration of Mn in the shoot of white lupin. The function of Mn transport was demonstrated by yeast complementation analysis, in which LaMTP8.1 detoxified Mn in pmr1∆ mutant cells upon elevated Mn supply. In addition, LaMTP8.1 also functioned as an iron (Fe) transporter in yeast assays. The expression of LaMTP8.1 was particularly high in old leaves under high Mn stress. However, low P availability per se did not result in transcriptional upregulation of LaMTP8.1. Moreover, LaMTP8.1 expression was strongly upregulated under Fe deficiency, where it was accompanied by Mn accumulation, indicating a role in the interaction of these micronutrients in L. albus. In conclusion, the tonoplast-localized Mn transporter LaMTP8.1 mediates Mn detoxification in leaf vacuoles, providing a mechanistic explanation for the high Mn accumulation and Mn tolerance in this species.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca 2+ oscillations in Arabidopsis roots. These Ca 2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca 2+ -dependent protein kinases CPK21 and CPK23 as Ca 2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21 / 23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca 2+ -CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Manganese (Mn2+) is essential for a diversity of processes, including photosynthetic water splitting and the transfer of glycosyl moieties. Various Golgi-localized glycosyltransferases that mediate cell wall matrix polysaccharide biosynthesis are Mn2+ dependent, but the supply of these enzymes with Mn2+ is not well understood. Here, we show that the BIVALENT CATION TRANSPORTER 3 (BICAT3) localizes specifically to trans-cisternae of the Golgi. In agreement with a role in Mn2+ and Ca2+ homeostasis, BICAT3 rescued yeast (Saccharomyces cerevisiae) mutants defective in their translocation. Arabidopsis (Arabidopsis thaliana) knockout mutants of BICAT3 were sensitive to low Mn2+ and high Ca2+ availability and showed altered accumulation of these cations. Despite reduced cell expansion and leaf size in Mn2+-deficient bicat3 mutants, their photosynthesis was improved, accompanied by an increased Mn content of chloroplasts. Growth defects of bicat3 corresponded with an impaired glycosidic composition of matrix polysaccharides synthesized in the trans-Golgi. In addition to the vegetative growth defects, pollen tube growth of bicat3 was heterogeneously aberrant. This was associated with a severely reduced and similarly heterogeneous pectin deposition and caused diminished seed set and silique length. Double mutant analyses demonstrated that the physiological relevance of BICAT3 is distinct from that of ER-TYPE CA2+-ATPASE 3, a Golgi-localized Mn2+/Ca2+-ATPase. Collectively, BICAT3 is a principal Mn2+ transporter in the trans-Golgi whose activity is critical for specific glycosylation reactions in this organelle and for the allocation of Mn2+ between Golgi apparatus and chloroplasts.

The continuing growth of the human population creates an inevitable necessity for higher crop yields, which are mandatory for the supply with adequate amounts of food. However, increasing grain yield may lead to a reduction of grain quality, such as a decline in protein and mineral nutrient concentrations causing the so-called hidden hunger. To assess the interdependence between quantity and quality and to evaluate the biofortification potential of wild barley, we conducted field studies, examining the interplay between plant development, yield, and nutrient concentrations, using HEB-YIELD, a subset of the wild barley nested association mapping population HEB-25. A huge variation of nutrient concentration in grains was obtained, since we identified lines with a more than 50% higher grain protein, iron, and zinc concentration in comparison to the recurrent parent ‘Barke’. We observed a negative relationship between grain yield and nutritional value in barley, indicated by predominantly negative correlations between yield and nutrient concentrations. Analyzing the genetic control of nutrient concentration in mature grains indicated that numerous genomic regions determine the final nutritional value of grains and wild alleles were frequently associated with higher nutrient concentrations. The targeted introgression of wild barley alleles may enable biofortification in future barley breeding.

Millions of people are anemic due to inadequate consumption of foods rich in iron and zinc. Plant-based foods provide most of our dietary nutrients but may also contain the toxic heavy metal cadmium (Cd). A low level of Cd silently enters the body through the diet. Once ingested, Cd may remain for decades. Hence, prolonged intake of Cd-containing foods endangers human health. Research that leads towards micronutrient enrichment and mitigation of Cd in foods has therefore dual significance for human health. The breeding of Cd-tolerant cultivars may enable them to grow on Cd-polluted soils; however, they may not yield Cd-free foods. Conversely, sequestration of Cd in roots can prevent its accumulation in grains, but this mechanism also retains nutrients, hence counteracting biofortification efforts. A specific restriction of the Cd absorption capacity of crops would prevent Cd entry into the plant system while maintaining micronutrient accumulation and may thus be a solution of the dilemma. After recapitulating existing strategies employed for the development of Cd-tolerant and biofortified cultivars, this viewpoint elaborates alternative approaches based on directed evolution and genome editing strategies for excluding Cd while enriching micronutrients in plant foods, which will concurrently help to eradicate malnutrition and prevent Cd intoxication.

Plant biology 24(1), 23 - 29 (2021). doi:10.1111/plb.13342, METAL TOLERANCE PROTEIN8 (MTP8) of Arabidopsis thaliana is a member of the CATION DIFFUSION FACILITATOR (CDF) family of proteins that transports primarily manganese (Mn), but also iron (Fe). MTP8 mediates Mn allocation to specific cell types in the developing embryo, and Fe re-allocation as well as Mn tolerance during imbibition. We analysed if an overexpression of MTP8 driven by the CaMV 35S promoter has an effect on Mn tolerance during imbibition and on Mn and Fe storage in seeds, which would render it a biofortification target. Fe, Mn and Zn concentrations in MTP8-overexpressing lines in wild type and vit1-1 backgrounds were analysed by ICP-MS. Distribution of metals in intact seeds was determined by synchrotron ��XRF tomography. MTP8 overexpression led to a strongly increased Mn tolerance of seeds during imbibition, supporting its effectiveness in loading excess Mn into the vacuole. In mature seeds, MTP8 overexpression did not cause a consistent increase in Mn and Fe accumulation, and it did not change the allocation pattern of these metals. Zn concentrations were consistently increased in bulk samples. The results demonstrate that Mn and Fe allocation is not determined primarily by the MTP8 expression pattern, suggesting either a cell type-specific provision of metals for vacuolar sequestration by upstream transport processes, or the determination of MTP8 activity by post-translational regulation., Published by Wiley- Blackwell, Oxford [u.a.]

Calcium (Ca2+) and manganese (Mn2+) are essential elements for plants and have similar ionic radii and binding coordination. They are assigned specific functions within organelles, but share many transport mechanisms to cross organellar membranes. Despite their points of interaction, those elements are usually investigated and reviewed separately. This review takes them out of this isolation. It highlights our current mechanistic understanding and points to open questions of their functions, their transport, and their interplay in the endoplasmic reticulum (ER), vesicular compartments (Golgi apparatus, trans-Golgi network, pre-vacuolar compartment), vacuoles, chloroplasts, mitochondria, and peroxisomes. Complex processes demanding these cations, such as Mn2+-dependent glycosylation or systemic Ca2+ signaling, are covered in some detail if they have not been reviewed recently or if recent findings add to current models. The function of Ca2+ as signaling agent released from organelles into the cytosol and within the organelles themselves is a recurrent theme of this review, again keeping the interference by Mn2+ in mind. The involvement of organellar channels [e.g. glutamate receptor-likes (GLR), cyclic nucleotide-gated channels (CNGC), mitochondrial conductivity units (MCU), and two-pore channel1 (TPC1)], transporters (e.g. natural resistance-associated macrophage proteins (NRAMP), Ca2+ exchangers (CAX), metal tolerance proteins (MTP), and bivalent cation transporters (BICAT)], and pumps [autoinhibited Ca2+-ATPases (ACA) and ER Ca2+-ATPases (ECA)] in the import and export of organellar Ca2+ and Mn2+ is scrutinized, whereby current controversial issues are pointed out. Mechanisms in animals and yeast are taken into account where they may provide a blueprint for processes in plants, in particular, with respect to tunable molecular mechanisms of Ca2+ versus Mn2+ selectivity.

Funder: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Id: http://dx.doi.org/10.13039/501100002322, Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects.

Modulation of gibberellic acid signaling through ScGAI regulates culm development including change in phytomer production and source–sink regulation in sugarcane., Sugarcane contributes more than 70% of sugar production and is the second largest feedstock for ethanol production globally. Since sugar accumulates in sugarcane culms, culm biomass and sucrose content are the most commercially important traits. Despite extensive breeding, progress in both cane yield and sugar content remains very slow in most countries. We hypothesize that manipulating the genetic elements controlling culm growth will alter source–sink regulation and help break down the yield barriers. In this study, we investigate the role of sugarcane ScGAI, an ortholog of SLR1/D8/RHT1/GAI, on culm development and source–sink regulation through a combination of molecular techniques and transgenic strategies. We show that ScGAI is a key molecular regulator of culm growth and development. Changing ScGAI activity created substantial culm growth and carbon allocation changes for structural molecules and storage. ScGAI regulates spatio-temporal growth of sugarcane culm and leaf by interacting with ScPIF3/PIF4 and ethylene signaling elements ScEIN3/ScEIL1, and its action appears to be regulated by SUMOylation in leaf but not in the culm. Collectively, the remarkable culm growth variation observed suggests that ScGAI could be used as an effective molecular breeding target for breaking the slow yield gain in sugarcane.

High-temperature (HT) stress is a major environmental stress that limits plant growth and development. MAPK cascades play key roles in plant growth and stress signaling, but their involvement in the HT stress response is poorly understood. Here, we describe a 47-kD MBP-phosphorylated protein (p47-MBPK) activated in tomato (Solanum lycopersicum) leaves under HT and identify it as SlMPK1 by tandem mass spectrometry analysis. Silencing of SlMPK1 in transgenic tomato plants resulted in enhanced tolerance to HT, while overexpression resulted in reduced tolerance. Proteomic analysis identified a set of proteins involved in antioxidant defense that are significantly more abundant in RNA interference-SlMPK1 plants than nontransgenic plants under HT stress. RNA interference-SlMPK1 plants also showed changes in membrane lipid peroxidation and antioxidant enzyme activities. Furthermore, using yeast two-hybrid screening, we identified a serine-proline-rich protein homolog, SlSPRH1, which interacts with SlMPK1 in yeast, in plant cells, and in vitro. We demonstrate that SlMPK1 can directly phosphorylate SlSPRH1. Furthermore, the serine residue serine-44 of SlSPRH1 is a crucial phosphorylation site in the SlMPK1-mediated antioxidant defense mechanism activated during HT stress. We also demonstrate that heterologous expression of SlSPRH1 in Arabidopsis (Arabidopsis thaliana) led to a decrease in thermotolerance and lower antioxidant capacity. Taken together, our results suggest that SlMPK1 is a negative regulator of thermotolerance in tomato plants. SlMPK1 acts by regulating antioxidant defense, and its substrate SlSPRH1 is involved in this pathway.

Many trace metals are essential micronutrients, but also potent toxins. Due to natural and anthropogenic causes, vastly different trace metal concentrations occur in various habitats, ranging from deficient to toxic levels. Therefore, one focus of plant research is on the response to trace metals in terms of uptake, transport, sequestration, speciation, physiological use, deficiency, toxicity, and detoxification. In this review, we cover most of these aspects for the essential micronutrients copper, iron, manganese, molybdenum, nickel, and zinc to provide a broader overview than found in other recent reviews, to cross-link aspects of knowledge in this very active research field that are often seen in a separated way. For example, individual processes of metal usage, deficiency, or toxicity often were not mechanistically interconnected. Therefore, this review also aims to stimulate the communication of researchers following different approaches, such as gene expression analysis, biochemistry, or biophysics of metalloproteins. Furthermore, we highlight recent insights, emphasizing data obtained under physiologically and environmentally relevant conditions.

Plant survival depends on the ability of roots to sense and acquire nutrients in soils, which harbor a rich diversity of microbes. A subset of this microcosm interacts with plant roots and collectively forms root-associated microbial communities, termed the root microbiota. Under phosphorus-limiting conditions, some plants can engage in mutualistic interactions, for example with arbuscular mycorrhizal fungi. Here, we describe how Arabidopsis thaliana, which lacks the genetic capacity for establishing the aforementioned symbiosis, interacts with soil-resident bacteria and fungi in soil from a long-term phosphorus fertilization trial. Long-term, contrasting fertilization regimes resulted in an ∼6-fold and ∼2.4-fold disparity in bioavailable and total phosphorous, respectively, which may explain differences in biomass of A. thaliana plants. Sequencing of marker genes enabled us to characterize bacterial and fungal communities present in the bulk soil, rhizosphere, and root compartments. Phosphorus had little effect on alpha- or beta-diversity indices, but more strongly influences bacterial and fungal community shifts in plant-associated compartments compared with bulk soil. The significant impact of soil P abundance could only be resolved at operational taxonomic unit level, and these subtle differences are more pronounced in the root compartment. We conclude that despite decades of different fertilization, both bacterial and fungal soil communities remained unexpectedly stable in soils tested, suggesting that the soil biota is resilient over time to nutrient supplementation. Conversely, low-abundance, root-associated microbes, which collectively represent 2 to 3% of the relative abundance of bacteria and fungi in the roots, exhibited a subtle, yet significant shift between the two soils. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Poly(ADP-ribose) polymerases (PARPs) have been implicated in responses of plants to DNA damage and numerous stresses, whereby the mechanistic basis of the interference is often unclear. Therefore, the identification of specific inhibitors and potential interactors of plant PARPs is desirable. For this purpose, we established an assay based on heterologous expression of PARP genes from the model plant Arabidopsis thaliana in yeast. Expression of AtPARPs caused an inhibition of yeast growth to different extent, which was alleviated by inhibitors targeted at human PARPs. This assay provides a fast and simple means to identify target proteins and pharmacological inhibitors of AtPARP1.

Metal accumulation in seeds is a prerequisite for germination and establishment of plants but also for micronutrient delivery to humans. To investigate metal transport processes and their interactions in seeds, we focused on METAL TOLERANCE PROTEIN8 (MTP8), a tonoplast transporter of the manganese (Mn) subclade of cation diffusion facilitators, which in Arabidopsis (Arabidopsis thaliana) is expressed in embryos of seeds. The x-ray fluorescence imaging showed that expression of MTP8 was responsible for Mn localization in subepidermal cells on the abaxial side of the cotyledons and in cortical cells of the hypocotyl. Accordingly, under low Mn availability, MTP8 increased seed stores of Mn, required for efficient seed germination. In mutant embryos lacking expression of VACUOLAR IRON TRANSPORTER1 (VIT1), MTP8 built up iron (Fe) hotspots in MTP8-expressing cells types, suggesting that MTP8 transports Fe in addition to Mn. In mtp8 vit1 double mutant seeds, Mn and Fe were distributed in all cell types of the embryo. An Fe transport function of MTP8 was confirmed by its ability to complement Fe hypersensitivity of a yeast mutant defective in vacuolar Fe transport. Imbibing mtp8-1 mutant seeds in the presence of Mn or subjecting seeds to wet-dry cycles showed that MTP8 conferred Mn tolerance. During germination, MTP8 promoted reallocation of Fe from the vasculature. These results indicate that cell type-specific accumulation of Mn and Fe in seeds depends on MTP8 and that this transporter plays an important role in the generation of seed metal stores as well as for metal homeostasis and germination efficiency under challenging environmental conditions.

Tip growth of pollen tubes and root hairs of plants is oscillatory and orchestrated by tip-focussed variations of cytosolic free calcium ([Ca(2+)]cyt). Hyphae of filamentous fungi are also tubular tip-growing cells, and components of the Ca(2+) signalling machinery, such as Ca(2+) channels and Ca(2+) sensors, are known to be important for fungal growth. In this study, we addressed the questions if tip-focussed [Ca(2+)]cyt transients govern hyphal and whole-colony growth in the maize pathogen Colletotrichum graminicola, and whether colony-wide [Ca(2+)]cyt dynamics rely on external Ca(2+) or internal Ca(2+) stores. Ratiometric fluorescence microscopy of individual hyphae expressing the Ca(2+) reporter Yellow Cameleon 3.6 revealed that Ca(2+) spikes in hyphal tips precede the re-initiation of growth after wounding. Tip-focussed [Ca(2+)]cyt spikes were also observed in undisturbed growing hyphae. They occurred not regularly and at a higher rate in hyphae growing at a medium-glass interface than in those growing on an agar surface. Hyphal tip growth was non-pulsatile, and growth speed was not correlated with the rate of spike occurrence. A possible relationship of [Ca(2+)]cyt spike generation and growth of whole colonies was assessed by using a codon-optimized version of the luminescent Ca(2+) reporter Aequorin. Depletion of extracellular free Ca(2+) abolished [Ca(2+)]cyt spikes nearly completely, but had only a modest effect on colony growth. In a pharmacological survey, some inhibitors targeting Ca(2+) influx or release from internal stores repressed growth strongly. However, although some of those inhibitors also affected [Ca(2+)]cyt spike generation, the effects on both parameters were not correlated. Collectively, the results indicate that tip growth of C. graminicola is non-pulsatile and not mechanistically linked to tip-focused or global [Ca(2+)]cyt spikes, which are likely a response to micro-environmental parameters, such as the physical properties of the growth surface.

Saccharomyces cerevisiae Rch1 is structurally similar to both the vertebrate solute carrier SLC10A7 and Candida albicans Rch1. We show here that ScRCH1 is a functional homolog of CaRCH1. In S. cerevisiae, overexpression of ScRCH1 suppresses, but deletion of ScRCH1 does not affect, the lithium and rapamycin tolerance of pmr1 cells. Overexpression of ScRCH1 reduces expression of ENA1, prevents sustained accumulation of cytosolic calcium and reduces the activation level of calcium/calcineurin signaling in pmr1 cells. Therefore, similar to the situation in the pathogen C. albicans, ScRch1 negatively regulates the cytosolic homeostasis in response to high levels of extracellular calcium. ScRch1 proteins distribute as multiple foci in the plasma membrane prior to cell division, move toward and concentrate at the bud neck as the bud grows in size, and disperse again along the plasma membrane immediately prior to cytokinesis. Furthermore, our genetic and biochemical data also demonstrate that transcriptional expression of RCH1 is positively regulated by calcium/calcineurin signaling through the sole CDRE element in its promoter.

A long-term field experiment on a Haplic Phaeozem, established 1949 with four levels of potassium (K) supply (5, 69, 133, and 261 kg K ha−1), was analyzed for the interaction between K supply and yield loss of five crop species by water shortage. The crop species were cultivated simultaneously side-by-side in the following rotation: potato (Solanum tuberosum L.), silage maize (Zea mays L.), spring wheat (Triticum aestivum L.), beet (Beta vulgaris L.), and spring barley (Hordeum vulgare L.). The treatment with 133 kg K ha−1 supply had a nearly balanced K budget. In the treatments with lower supply, the soil delivered K from its mineral constituents. On the low-K plots (especially on those with only 5 kg K ha−1), crops suffered yield depressions of nearly all main harvest products (cereal grains, potato tubers, beet storage roots, silage maize) and by-products (straw, beet leaves) by up to 40.7% of dry matter. Only wheat grains were an exception. Potassium concentrations in the harvested plant parts decreased nearly in parallel to the reduction of their dry matter yields, with the exception of cereal grains, which kept stable concentrations even in the treatment with only 5 kg K ha−1. A comparison of four year-pairs with differing levels of precipitation in yield-relevant periods showed an average water shortage-induced depression of dry matter yields by 19.7% in the main harvest products. The severity of this yield depression was not mitigated by elevated K supply, with the exception of beet leaves, where the dry matter production was stabilized by high K supply. In this crop, the reduction of storage-root yield was associated with a decrease in harvest index and was therefore obviously caused by an inhibition of assimilate translocation from the leaves into these organs, in contrast to cereals, where water shortage primarily affected dry matter production in vegetative organs. It is concluded that the physiological causes of yield reduction by drought stress and the possibility of its amelioration by K supply differ between plant species and organs.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO2 fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown. By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma. BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca2+ ]stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.

Calcium ion (Ca(2+) ) signalling triggered by insect herbivory is an intricate network with multiple components, involving positive and negative regulators. Real-time, noninvasive imaging of entire Arabidopsis thaliana rosettes was employed to monitor cytosolic free calcium ([Ca(2+) ]cyt ) elevations in local and systemic leaves in response to wounding and Spodoptera littoralis feeding. Luminescence emitted by the cytosol-localized Ca(2+) reporter aequorin was imaged using a high-resolution photon-counting camera system. Spodoptera littoralis feeding on Arabidopsis induced both local and systemic [Ca(2+) ]cyt elevations. Systemic [Ca(2+) ]cyt signals were found predominantly in adjacent leaves with direct vascular connections to the treated leaf and appeared with a delay of 1 to 2 min. Simulated herbivory by wounding always induced a local [Ca(2+) ]cyt response, but a systemic one only when the midrib was wounded. This systemic [Ca(2+) ]cyt response was suppressed by the presence of insect-derived oral secretions as well as in a mutant of the vacuolar cation channel, Two Pore Channel 1 (TPC1). Our results provide evidence that in Arabidopsis insect herbivory induces both local and systemic [Ca(2+) ]cyt signals that distribute within the vascular system. The systemic [Ca(2+) ]cyt signal could play an important signalling role in systemic plant defence.

Phosphorus (P) fertilizer recommendations in most European countries are based on plant-available soil P contents and long-term field experiments. Site-specific conditions are often neglected, resulting in excessive P fertilizer applications. P fertilization experiments including relevant site and soil parameters were evaluated in order to analyze the yield response. The database comprises about 2000 datasets from 30 field experiments from Germany and Austria. Statistical evaluations using a classification and regression tree approach, and multiple linear regression analysis indicate that besides plant-available soil P content, soil texture and soil organic matter content have a large influence on the effectiveness of P fertilization. This study methodology can be a basis for modification and specification of existing P fertilization recommendations and thus contribute to mitigate environmental impacts of P fertilization. Electronic supplementary material The online version of this article (10.1007/s13280-017-0971-1) contains supplementary material, which is available to authorized users.

Abiotic and biotic stress can have a detrimental impact on plant growth and productivity. Hence, there is a substantial demand for key factors of stress responses to improve yield stability of crops. Members of the poly(ADP-ribose)polymerase (PARP) protein family, which post-translationally modify (PARylate) nuclear proteins, have been suggested as such universal determinants of plant stress responses. A role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants. To further elucidate the role of PARP proteins under stress, T-DNA knockout mutants for the three Arabidopsis thaliana PARP genes were subjected to drought, osmotic, salt, and oxidative stress. To exclude a functional redundancy, which was indicated by a transcriptional upregulation of the remaining parp genes, a parp triple mutant was generated. Surprisingly, parp mutant plants did not differ from wild type plants in any of these stress experiments, independent from the number of PARP genes mutated. The parp triple mutant was also analyzed for callose formation in response to the pathogenassociated molecular pattern flg22. Unexpectedly, callose formation was unaltered in the mutant, albeit pharmacological PARP inhibition robustly blocked this immune response, confirming previous reports. Evidently, pharmacological inhibition appears to be more robust than the abolition of all PARP genes, indicating the presence of so-far undescribed proteins with PARP activity. This was supported by the finding that protein PARylation was not absent, but even increased in the parp triple mutant. Candidates for novel PARP-inhibitor targets may be found in the SRO protein family. These proteins harbor a catalytic PARP-like domain and are centrally involved in stress responses. Molecular modeling analyses, employing animal PARPs as templates, indeed indicated a capability of the SRO proteins RCD1 and SRO1 to bind nicotinamide-derived inhibitors. Collectively, the results of our study suggest that the stress-related phenotypes of parp mutants are highly conditional, and they call for a reconsideration of PARP inhibitor studies. In the context of this study, we also propose a unifying nomenclature of PARP genes and parp mutants, which is currently highly inconsistent and redundant.

To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.

Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca 2+ exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca 2+ homeostasis via an uncharacterized Ca 2+ transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca 2+ , and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca 2+ ion transport. Furthermore, defects in TMEM165 affected both Ca 2+ and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca 2+ /H + antiporters and that modification of the Golgi Ca 2+ and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.

Abiotic and biotic stress can have a detrimental impact on plant growth and productivity. Hence, there is a substantial demand for key factors of stress responses to improve yield stability of crops. Members of the poly(ADP-ribose)polymerase (PARP) protein family, which post-translationally modify (PARylate) nuclear proteins, have been suggested as such universal determinants of plant stress responses. A role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants. To further elucidate the role of PARP proteins under stress, T-DNA knockout mutants for the three Arabidopsis thaliana PARP genes were subjected to drought, osmotic, salt, and oxidative stress. To exclude a functional redundancy, which was indicated by a transcriptional upregulation of the remaining parp genes, a parp triple mutant was generated. Surprisingly, parp mutant plants did not differ from wild type plants in any of these stress experiments, independent from the number of PARP genes mutated. The parp triple mutant was also analyzed for callose formation in response to the pathogenassociated molecular pattern flg22. Unexpectedly, callose formation was unaltered in the mutant, albeit pharmacological PARP inhibition robustly blocked this immune response, confirming previous reports. Evidently, pharmacological inhibition appears to be more robust than the abolition of all PARP genes, indicating the presence of so-far undescribed proteins with PARP activity. This was supported by the finding that protein PARylation was not absent, but even increased in the parp triple mutant. Candidates for novel PARP-inhibitor targets may be found in the SRO protein family. These proteins harbor a catalytic PARP-like domain and are centrally involved in stress responses. Molecular modeling analyses, employing animal PARPs as templates, indeed indicated a capability of the SRO proteins RCD1 and SRO1 to bind nicotinamide-derived inhibitors. Collectively, the results of our study suggest that the stress-related phenotypes of parp mutants are highly conditional, and they call for a reconsideration of PARP inhibitor studies. In the context of this study, we also propose a unifying nomenclature of PARP genes and parp mutants, which is currently highly inconsistent and redundant.

Iron (Fe) deficiency is a widespread nutritional disorder on calcareous soils. To identify genes involved in the Fe deficiency response, Arabidopsis (Arabidopsis thaliana) transfer DNA insertion lines were screened on a high-pH medium with low Fe availability. This approach identified METAL TOLERANCE PROTEIN8 (MTP8), a member of the Cation Diffusion Facilitator family, as a critical determinant for the tolerance to Fe deficiency-induced chlorosis, also on soil substrate. Subcellular localization to the tonoplast, complementation of a manganese (Mn)-sensitive Saccharomyces cerevisiae yeast strain, and Mn sensitivity of mtp8 knockout mutants characterized the protein as a vacuolar Mn transporter suitable to prevent plant cells from Mn toxicity. MTP8 expression was strongly induced on low-Fe as well as high-Mn medium, which were both strictly dependent on the transcription factor FIT, indicating that high-Mn stress induces Fe deficiency. mtp8 mutants were only hypersensitive to Fe deficiency when Mn was present in the medium, which further suggested an Mn-specific role of MTP8 during Fe limitation. Under those conditions, mtp8 mutants not only translocated more Mn to the shoot than did wild-type plants but suffered in particular from critically low Fe concentrations and, hence, Fe chlorosis, although the transcriptional Fe deficiency response was up-regulated more strongly in mtp8. The diminished uptake of Fe from Mn-containing low-Fe medium by mtp8 mutants was caused by an impaired ability to boost the ferric chelate reductase activity, which is an essential process in Fe acquisition. These findings provide a mechanistic explanation for the long-known interference of Mn in Fe nutrition and define the molecular processes by which plants alleviate this antagonism.

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.

In addition to establishing symbiotic relationships with arbuscular mycorrhizal fungi, legumes also enter into a nitrogen-fixing symbiosis with rhizobial bacteria that results in the formation of root nodules. Several genes involved in the development of both arbuscular mycorrhiza and legume nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K+) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast (Saccharomyces cerevisiae) mutant toward those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant-negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants.

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.

In root nodules of leguminous plants, such as Vicia faba L., N 2 is fixed by rhizobial bacteroids within infected cells. These cells are located in the centre of the nodule, whereas the vascular system serving import and export of solutes is located in the periphery. Within the infected central tissue, metabolites may travel symplastically by using bands of interconnected uninfected cells. Structural evidence, however, speaks against symplastic movement between infected cells themselves. The present work examined the possibility of an apoplastic step in amino acid export from infected cells. Incubation experiments with dissected central tissue demonstrated the release of amino compounds by infected cells. The predominant compound released was asparagine, which is also the major amino acid in xylem sap of legumes forming indeterminate nodules. During incubation of central infected tissue, medium acidification by plasma membrane H + -ATPase quickly turned into slight alkalization, probably caused by the released amino acids. In vivo, this process would lead to an increased apoplastic pH with consequences for processes relying on the proton gradient across the plasma membrane. Uptake of 14 C-labelled amino acids by uninfected and infected cells was studied using protoplasts isolated from the central nodule tissue. Uninfected protoplasts accumulated amino acids with low specificity in a ΔpH-dependent, bi-phasic manner, whereas infected protoplasts did not absorb amino acids from the medium. This indicates that uninfected protoplasts not only function in metabolite transport, but also in collection of amino acids from the apoplast. Taken together, both experimental approaches demonstrate the possibility of an apoplastic export step for amino acids in the central tissue of indeterminate legume nodules.

It has long been known that plants facing a localized stress or attack may launch responses in unaffected distant tissues, hence preparing the entire plant for the potential spread of the challenge. Traditionally, this plant-wide “systemic” coordination of responses has been assigned largely to

The central tissue of Vicia faba L. root nodules is composed of cells infected with Rhizobium bacteroids and uninfected cells. For the study of various processes, such as plasma membrane transport, it is essential to separate both cell types. Initial attempts to isolate protoplasts according to protocols described in the literature resulted in non-spherical and osmotically inactive material, which is in agreement with previous descriptions. In the study reported herein, it was shown that the plasma membrane of non-spherical infected protoplasts is not intact. A new isolation and separation protocol was developed, based on dissection of the nodule prior to cell wall digestion, non-shaking digestion in hypertonic medium, and a combined procedure for release of protoplasts into slightly hypotonic medium and separation of protoplast fractions by isopycnic density gradient centrifugation. Infected and uninfected protoplasts that were isolated according to this protocol were spherical, osmotically active and excluded propidium iodide, confirming the intactness of their plasma membrane. The common fluorescein diacetate test was shown to be artefactual in infected cells, since viable bacteroids also stain in defective cells. Light and electron microscopic examination of infected protoplasts showed that protoplasts still contained starch after isolation and bacteroids in intact protoplasts had unusually high amounts of polyhydroxybutyrate. The vacuoles of infected protoplasts contained protein and membrane-enclosed structures, and were of non-acid pH; traits that are typical of protein storage vacuoles.

Symbiotic dinitrogen fixation of legume nodules is fuelled by phloem-imported carbohydrates. These have to pass several cell layers to reach cells infected with Rhizobium bacteroids. It is unclear whether apoplastic steps are involved in carbohyd-rate translocation within the nodule. Protoplasts were isolated from the infected and uninfected cells of the central tissue of Vicia faba nodules using a recently developed protocol. These protoplasts were used to elucidate pathways for sugar transport in this tissue. Both types of protoplasts released protons into the medium. Acidification was inhibited by vanadate and erythrosin B. However, it was stimulated by fusicoccin only in uninfected cells. A symport of sugars with protons can therefore be energized in both cell types. Uptake of 14C-labelled sugars was determined using a phthalate centrifugation technique. Uninfected protoplasts accumulated glucose through high-affinity H+/glucose-symport that was not competitively inhibited by fructose or sucrose. Uninfected protoplasts also absorbed sucrose with biphasic kinetics. At 0.1, 1, and 10 mM sucrose, uptake was inhibited by CCCP. Fusicoccin did not stimulate the linear phase of sucrose uptake. Glucose inhibited sucrose uptake nearly completely. This was not related to sucrose cleavage in the medium because sucrose was absorbed at a much higher rate than glucose, and glucose concentration did not increase in sucrose-containing protoplast suspensions. By contrast with uninfected protoplasts, infected cells did not show transporter-mediated glucose or sucrose uptake. The findings underline a role of uninfected cells in sugar translocation. Infected cells are not apoplastically supplied with sugars and possibly depend on uninfected cells for carbon supply.

Changes in cytosolic free calcium ([Ca(2+)]cyt) are an early and essential element of signalling networks activated by the perception of pathogen-associated molecular patterns (PAMPs), such as flg22. The flg22-induced calcium signal has been described on whole-plant, but not on single-cell scale so far. Also, the Ca(2+) sources and channels contributing to its generation are still obscure. Ratiometric fluorescence imaging employing the calcium reporter Yellow Cameleon 3.6 was performed to analyse the flg22-induced calcium signature in single guard cells of Arabidopsis thaliana. Calcium stores and channel types involved in its generation were determined by a pharmacological approach. In contrast to the calcium signal determined on whole-plant level, the signature on single-cell level is not characterized by one sustained response, but by oscillations in [Ca(2+)]cyt. These oscillations were abolished by EGTA and lanthanum, as well as by U73122, neomycin and TMB-8, but only partially or not at all affected by inhibitors of glutamate receptor-like channels and cyclic nucleotide-gated channels. Our analyses suggest that the response observed on whole-plant level is the summary of oscillations occurring in single cells. Parallel to external calcium, influx via channels located at internal stores contributes to the signal.