Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

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 Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ 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 Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

The trans-Golgi-localized protein BICAT3 regulates manganese allocation and matrix polysaccharide biosynthesis.

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.

Cation diffusion facilitator proteins of Beta vulgaris reveal diversity of metal handling in dicotyledons.

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.

WHIRLY1 Acts Upstream of ABA-Related Reprogramming of Drought-Induced Gene Expression in Barley and Affects Stress-Related Histone Modifications.

WHIRLY1, a small plant-specific ssDNA-binding protein, dually located in chloroplasts and the nucleus, is discussed to act as a retrograde signal transmitting a stress signal from the chloroplast to the nucleus and triggering there a stress-related gene expression. In this work, we investigated the function of WHIRLY1 in the drought stress response of barley, employing two overexpression lines (oeW1-2 and oeW1-15). The overexpression of WHIRLY1 delayed the drought-stress-related onset of senescence in primary leaves. Two abscisic acid (ABA)-dependent marker genes of drought stress, HvNCED1 and HvS40, whose expression in the wild type was induced during drought treatment, were not induced in overexpression lines. In addition, a drought-related increase in ABA concentration in the leaves was suppressed in WHIRLY1 overexpression lines. To analyze the impact of the gain-of-function of WHIRLY1 on the drought-related reprogramming of nuclear gene expression, RNAseq was performed comparing the wild type and an overexpression line. Cluster analyses revealed a set of genes highly up-regulated in response to drought in the wild type but not in the WHIRLY1 overexpression lines. Among these genes were many stress- and abscisic acid (ABA)-related ones. Another cluster comprised genes up-regulated in the oeW1 lines compared to the wild type. These were related to primary metabolism, chloroplast function and growth. Our results indicate that WHIRLY1 acts as a hub, balancing trade-off between stress-related and developmental pathways. To test whether the gain-of-function of WHIRLY1 affects the epigenetic control of stress-related gene expression, we analyzed drought-related histone modifications in different regions of the promoter and at the transcriptional start sites of HvNCED1 and HvS40. Interestingly, the level of euchromatic marks (H3K4me3 and H3K9ac) was clearly decreased in both genes in a WHIRLY1 overexpression line. Our results indicate that WHIRLY1, which is discussed to act as a retrograde signal, affects the ABA-related reprogramming of nuclear gene expression during drought via differential histone modifications.

Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana.

BACKGROUND: Plants are continuously exposed to changing environmental conditions and biotic attacks that affect plant growth. In crops, the inability to respond appropriately to stress has strong detrimental effects on agricultural production and yield. Ca2+ signalling plays a fundamental role in the response of plants to most abiotic and biotic stresses. However, research on stimulus-specific Ca2+ signals has mostly been pursued in Arabidopsis thaliana, while in other species these events are little investigated . RESULTS: In this study, we introduced the Ca2+ reporter-encoding gene APOAEQUORIN into the crop species barley (Hordeum vulgare). Measurements of the dynamic changes in [Ca2+]cyt in response to various stimuli such as NaCl, mannitol, H2O2, and flagellin 22 (flg22) revealed the occurrence of dose- as well as tissue-dependent [Ca2+]cyt transients. Moreover, the Ca2+ signatures were unique for each stimulus, suggesting the involvement of different Ca2+ signalling components in the corresponding stress response. Alongside, the barley Ca2+ signatures were compared to those produced by the phylogenetically distant model plant Arabidopsis. Notable differences in temporal kinetics and dose responses were observed, implying species-specific differences in stress response mechanisms. The plasma membrane Ca2+ channel blocker La3+ strongly inhibited the [Ca2+]cyt response to all tested stimuli, indicating a critical role of extracellular Ca2+ in the induction of stress-associated Ca2+ signatures in barley. Moreover, by analysing spatio-temporal dynamics of the [Ca2+]cyt transients along the developmental gradient of the barley leaf blade we demonstrate that different parts of the barley leaf show quantitative differences in [Ca2+]cyt transients in response to NaCl and H2O2. There were only marginal differences in the response to flg22, indicative of developmental stage-dependent Ca2+ responses specifically to NaCl and H2O2. CONCLUSION: This study reveals tissue-specific Ca2+ signals with stimulus-specific kinetics in the crop species barley, as well as quantitative differences along the barley leaf blade. A number of notable differences to the model plants Arabidopsis may be linked to different stimulus sensitivity. These transgenic barley reporter lines thus present a valuable tool to further analyse mechanisms of Ca2+ signalling in this crop and to gain insights into the variation of Ca2+-dependent stress responses between stress-susceptible and -resistant species.

Transport, functions, and interaction of calcium and manganese in plant organellar compartments.

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.

The vacuolar transporter LaMTP8.1 detoxifies manganese in leaves of Lupinus albus.

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.

A path toward concurrent biofortification and cadmium mitigation in plant-based foods.

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 to 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.

ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

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.

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

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.

The Tomato Mitogen-Activated Protein Kinase SlMPK1 Is as a Negative Regulator of the High-Temperature Stress Response.

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.

Poly(ADP-Ribose) Polymerases in Plants and Their Human Counterparts: Parallels and Peculiarities.

Poly(ADP-ribosyl)ation is a rapid and transient post-translational protein modification that was described first in mammalian cells. Activated by the sensing of DNA strand breaks, poly(ADP-ribose)polymerase1 (PARP1) transfers ADP-ribose units onto itself and other target proteins using NAD⁺ as a substrate. Subsequently, DNA damage responses and other cellular responses are initiated. In plants, poly(ADP-ribose) polymerases (PARPs) have also been implicated in responses to DNA damage. The Arabidopsis genome contains three canonical PARP genes, the nomenclature of which has been uncoordinated in the past. Albeit assumptions concerning the function and roles of PARP proteins in planta have often been inferred from homology and structural conservation between plant PARPs and their mammalian counterparts, plant-specific roles have become apparent. In particular, PARPs have been linked to stress responses of plants. A negative 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; in response to pathogen-associated molecular patterns, a positive role has been suggested. However, reports have been inconsistent, and the effects of PARP inhibitors appear to be more robust than the genetic abolition of PARP gene expression, indicating the presence of alternative targets of those drugs. Collectively, recent evidence suggests a conditionality of stress-related phenotypes of parp mutants and calls for a reconsideration of PARP inhibitor studies on plants. This review critically summarizes our current understanding of poly(ADP-ribosylation) and PARP proteins in plants, highlighting similarities and differences to human PARPs, areas of controversy, and requirements for future studies.

Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination.

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.

Trace metal metabolism in plants.

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.

ScGAI is a key regulator of culm development 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.

The Vacuolar Manganese Transporter MTP8 Determines Tolerance to Iron Deficiency-Induced Chlorosis in Arabidopsis.

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.

A yeast growth assay to characterize plant poly(ADP-ribose) polymerase (PARP) proteins and inhibitors.

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.

Quantity and distribution of arbuscular mycorrhizal fungal storage organs within dead roots.

The formation of storage organs, such as spores and vesicles, is a central part of the life cycle of an arbuscular mycorrhizal fungus (AMF), but the conditions under which this occurs in AMF are not well understood. Here, quantity and distribution of storage organs formed by the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae within dead (excised) roots were characterised. 'Trap roots' (TR), separated from the growth substrate by a 30-µm mesh, supported hyphal growth and formation of storage organs of the AMF. Hyphae developed both inside and on the outside of the TR and also within air gaps of surrounding nylon mesh compartments, but formation of vesicles and spores was confined to the interior and to the surface of the TR. Up to 20 % of the TR length harboured newly formed storage organs, resulting in a number of about 60 per mg TR dry weight. The portion of TR length containing storage organs was greater in coarse (diameter >300 µm) than in thin (<150 µm) TR, irrespective of whether the TR were sourced from an AMF host or non-host plant. We conclude that the AMF's extraradical mycelium produces its storage organs within dead roots in preference to air space in the substrate. Dead roots may indirectly supply nutrients to AMF (once they have been mineralised) or represent a protected space for the fungal structures to develop. The experimental technique described here allows for the preparation of AMF spores and vesicles of F. mosseae free of any mineral substrate.

Cytosolic free calcium dynamics as related to hyphal and colony growth in the filamentous fungal pathogen Colletotrichum graminicola.

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.

Newly characterized Golgi-localized family of proteins is involved in calcium and pH homeostasis in yeast and human cells.

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.