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Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography coupled online to sector-field ICP-MS. Our data identified known TM-binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10 to 15 kD containing Cu, Fe, S, and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu, and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Micronutrient manipulation can enhance crop resilience against pathogens, but the mechanisms are mostly unknown. We tested whether priming Capsicum annuum plants with zinc (5 μM Zn) or manganese (3 μM Mn) for six weeks increases their immunity against the generalist necrotroph Botrytis cinerea compared to deficient (0.1 μM Zn, 0.02 μM Mn) and control conditions (1 μM Zn, 0.6 μM Mn). Zinc priming reduced the pathogen biomass and lesion area and preserved CO 2 assimilation and stomatal conductance. Zinc mobilization at the infection site, visualized by micro-X-ray fluorescence, was accompanied by increased Zn protein binding obtained by size exclusion HPLC-ICP/MS. A common metabolic response to fungal infection in Zn- and Mn-primed plants was an accumulation of corchorifatty acid F, a signaling compound, and the antifungal compound acetophenone. In vitro tests showed that the binding of Zn 2+ increased, while Mn 2+ binding decreased acetophenone toxicity against B. cinerea at concentrations far below the toxicity thresholds of both metals in unbound (aquo complex) form. The metal-specific response to fungal infection included the accumulation of phenolics and amino acids (Mn), and the ligand isocitrate (Zn). The results highlight the importance of Zn for pepper immunity through direct involvement in immunity-related proteins and low molecular weight Zn-complexes, while Mn priming was inefficient., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

This study delves into the pH-dependent effects on the excited-state behavior of crocin, a hydrophilic carotenoid with diverse functions in biological systems. Steady-state spectroscopy demonstrates notable changes in absorption and fluorescence spectra, characterized by a pH-dependent blue shift and altered resolution of vibrational bands. Transient absorption spectra further elucidate these effects, highlighting a significant blue shift in the S 1 -S n peak with increasing pH. Detailed kinetic analysis shows the pH-dependent dynamics of crocin's excited states. At pH 11, a shortening of effective conjugation is observed, resulting in a prolonged S 1 /ICT lifetime. Conversely, at pH 9, our data suggest a more complex scenario, suggesting the presence of two distinct crocin species with different relaxation patterns. This implies structural alterations within the crocin molecule, potentially linked to the deprotonation of hydroxyl groups in crocin and/or saponification at high pH.

Cu + -chaperones are a diverse group of proteins that allocate Cu + ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu + -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu + with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu + -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu + -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

The aim of this study was to investigate how acclimation to medium-level, long-term, non-lethal iron limitation changes the electron flux around the Photosystem II of the oceanic diazotroph Trichodesmium erythraeum IMS101. Fe availability of about 5× and 100× lower than a replete level, i.e. conditions common in the natural environment of this cyanobacterium, were applied in chemostats. The response of the cells was studied not only in terms of growth, but also mechanistically, measuring the chlorophyll fluorescence of dark-adapted filaments via imaging fluorescence kinetic microscopy (FKM) with 0.3 ms time resolution. Combining these measurements with those of metal binding to proteins via online coupling of metal-free HPLC (size exclusion chromatography SEC) to sector-field ICP-MS allowed to track the fate of the photosystems, together with other metalloproteins. General increase of fluorescence has been observed, with the consequent decrease in the quantum yields φ of the PSII, while the efficiency ψ of the electron flux between PSII and the PSI remained surprisingly unchanged. This indicates the ability of Trichodesmium to cope with a situation that makes assembling the many iron clusters in Photosystem I a particular challenge, as shown by decreasing ratios of Fe to Mg in these proteins. The negative effect of Fe limitation on PSII may also be due to its fast turnover. A broader view was obtained from metalloproteomics via HPLC-ICP-MS, revealing a differential protein expression pattern under iron limitation with a drastic down-regulation especially of iron-containing proteins and some increase in low MW metal-binding complexes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hendrik Kuepper reports financial support was provided by Czech Academy of Sciences. Hendrik Kuepper reports financial support was provided by Ministry of Education Youth and Sports of the Czech Republic. Hendrik Kuepper reports financial support was provided by COST Association., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Low Zn availability in soils is a problem in many parts of the world, with tremendous consequences for food and feed production because Zn deficiency affects the yield and quality of plants. In this study we investigated the consequences of Zn-limitation in hydroponically cultivated soybean (Glycine max L.) plants. Parameters of photosynthesis biophysics were determined by spatially and spectrally resolved Kautsky and OJIP fluorescence kinetics and oxygen production at two time points (V4 stage, after five weeks, and pod development stage, R5-R6, after 8-10 weeks). Lower NPQ at 730 nm and lower quantum yield of electron transport flux until PSI acceptors were observed, indicating an inhibition of the PSI acceptor side. Metalloproteomics showed that down-regulation of Cu/Zn-superoxide dismutase (CuZnSOD) and Zn‑carbonic anhydrase (CA) were primary consequences of Zn-limitation. This explained the effects on photosynthesis in terms of decreased use of excitons, which consequently led to oxidative stress. Indeed, untargeted metabolomics revealed an accumulation of lipid oxidation products in the Zn-deficient leaves. Further response to Zn deficiency included up-regulation of gene expression of cell wall metabolism, response to (a)biotic stressors and antioxidant activity, which correlated with accumulation of antioxidants, Vit B6, (iso)flavonoids and phytoalexins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hendrik Kupper reports financial support was provided by Ministry of Education, Youth and Sports of the Czech Republic. Peter Konik reports financial support was provided by Ministry of Education Youth and Sports of the Czech Republic. Hendrik Kupper reports financial support was provided by European Cooperation in Science and Technology. Hendrik Kupper reports financial support was provided by Czech Academy of Sciences., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To understand the role of Zn and Cd in anti-viral defence, Zn/Cd hyperaccumulator Noccaea caerulescens plants grown with deficient (0.3 µM), replete (10 µM) and excess (100 µM) Zn 2+ and Cd (10 µM Zn 2+ + 1 µM Cd 2+ ) were infected with Turnip yellow mosaic virus (TYMV). Gas exchange and chlorophyll fluorescence kinetics analyses demonstrated direct TYMV effects on photosynthetic light reactions but N. caerulescens was more resistant against TYMV than the previously studied non-hyperaccumulator N. ochroleucum. Virus abundance and photosynthesis inhibition were the lowest in the high Zn and Cd treatments. RNAseq analysis of 10 µM Zn 2+ plants revealed TYMV-induced upregulation of Ca transporters, chloroplastic ZTP29 and defence genes, but none of those that are known to be strongly involved in hyperaccumulation. Synchrotron µ-XRF tomography, however, showed that Zn hyperaccumulation remained strongest in vacuoles of epidermal storage cells regardless of infection. This was in contrast to N. ochroleucum, where apoplastic Zn drastically increased in response to TYMV. These results suggest that the antiviral response of N. caerulescens is less induced by the onset of this biotic stress, but it is rather a permanent resistant state of the plant. Real-time qPCR revealed upregulation of ferritin in Zn10 infected plants, suggesting Fe deprivation as a virus defence strategy under suboptimal Zn supply., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pollution by potentially toxic trace metals, such as copper or zinc, is global. Both Cu and Zn are essential microelements, which in higher concentrations become toxic. The aquatic plant Pistia stratiotes(L. has great potential for phytoremediation. Also, it has an unusually large and easily detachable root cap, which makes it a suitable model for studying the potential role of the root cap in metal uptake. Plant response to environmentally relevant concentrations of Cu (0.1, 0.3, and 1 μM) and Zn (0.3, 1, and 3 μM) was investigated with the aim of studying their interaction and distribution at the root tissue level as well as revealing their tolerance mechanisms. Changes in the root anatomy and plant ionome were determined using light and fluorescence microscopy, ICP-MS, and μXRF imaging. Alterations in photosynthetic activity caused by Cu or Zn excesses were monitored by direct imaging of fast chlorophyll fluorescence kinetics (OJIP). Fe and Mn were preferentially localized in the root cap, while Ca, Cu, Ni, and Zn were mainly in the root tip regardless of the Cu/Zn treatment. Translocation of Cu and Zn to the leaves increased with higher doses, however the translocation factor was the lowest in the highest treatments. Measurements of photosynthetic parameters showed a higher susceptibility of electron transport flux from Q A  to Q B  under increasing Cu than Zn supply. This, along with our findings regarding the root anatomy and the differences in Ca accumulation and distribution, led to the conclusion that P. stratiotes is more effective for Zn remediation than Cu., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hendrik Kuepper reports financial support was provided by Ministry of Education Youth and Sports of the Czech Republic. Hendrik Kuepper reports financial support was provided by European Cooperation in Science and Technology. Hendrik Kuepper reports financial support was provided by Czech Academy of Sciences. Hendrik Kuepper and Alexander Lux reports financial support was provided by European Union., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Three basil plant varieties ( Ocimum basilicum var. Genovese, Ocimum × citriodorum , and Ocimum basilicum var. purpurascens ) were grown under moderate light (about 300 µmol photons m -2 s -1 ) in a glasshouse or growth chamber and then either transferred to an open field (average daily dose: 29.2 kJ m -2 d -1 ) or additionally exposed to UV-B irradiation in a growth chamber (29.16 kJ m -2 d -1 ), to reveal the variety-specific and light-specific acclimation responses. Total antioxidant capacity (TAC), phenolic profile, ascorbate content, and class III peroxidase (POD) activity were used to determine the antioxidant status of leaves under all four light regimes. Exposure to high solar irradiation at the open field resulted in an increase in TAC, total hydroxycinnamic acids (HCAs, especially caffeic acid), flavonoids, and epidermal UV-absorbing substances in all three varieties, as well as a two-fold increase in the leaf dry/fresh weight ratio. The supplemental UV-B irradiation induced preferential accumulation of HCAs (rosmarinic acid) over flavonoids, increased TAC and POD activity, but decreased the ascorbate content in the leaves, and inhibited the accumulation of epidermal flavonoids in all basil varieties. Furthermore, characteristic leaf curling and UV-B-induced inhibition of plant growth were observed in all basil varieties, while a pro-oxidant effect of UV-B was indicated with H 2 O 2 accumulation in the leaves and spotty leaf browning. The extent of these morphological changes, and oxidative damage depended on the basil cultivar, implies a genotype-specific tolerance mechanism to high doses of UV-B irradiation.

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (μXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component., (© The Author(s) 2023. Published by Oxford University Press.)

Soybean (Glycine max (L.) Merr.) plants were exposed to various Cd concentrations from background and low non-toxic (0.5-50 nM) via sublethally toxic (< 550 nM) to highly, ultimately lethally toxic (3 µM) concentrations. Plants were cultivated hydroponically for 10 weeks until pod development stage of the control plants. The threshold and mechanism of sublethal Cd toxicity was investigated by metabolomics and metalloproteomics (HPLC-ICP-MS) measuring metal binding to proteins in the harvested roots. Spatial distribution of Cd was revealed by µXRF-CT. Specific binding of Cd to proteins already at 50 nM Cd revealed the likely high-affinity protein binding targets in roots, identified by protein purification from natural abundance. This revealed allantoinase, aquaporins, peroxidases and protein disulfide isomerase as the most likely high-affinity targets of Cd binding. Cd was deposited in cortex cell vacuoles at sublethal and bound to the cell walls of the outer cortex and the vascular bundle at lethal Cd. Cd binding to proteins likely inhibits them, and possibly induces detoxification mechanisms, as verified by metabolomics: allantoic acid and allantoate increased due to sublethal Cd toxicity. Changes of the Cd binding pattern indicated a detoxification strategy at lower Cd, but saturated binding sites at higher Cd concentrations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The bacterium Gemmatimonas phototrophica AP64 isolated from a freshwater lake in the western Gobi Desert represents the first phototrophic member of the bacterial phylum Gemmatimonadota. This strain was originally cultured on agar plates because it did not grow in liquid medium. In contrast, the closely related species G. groenlandica TET16 grows both on solid and in liquid media. Here, we show that the growth of G. phototrophica in liquid medium can be induced by supplementing the medium with 20 mg CaCl 2 L -1 . When grown at a lower concentration of calcium (2 mg CaCl 2 L -1 ) in the liquid medium, the growth was significantly delayed, cells were elongated and lacked flagella. The elevated requirement for calcium is relatively specific as it can be partially substituted by strontium, but not by magnesium. The transcriptome analysis documented that several groups of genes involved in flagella biosynthesis and transport of transition metals were co-activated after amendment of 20 mg CaCl 2 L -1 to the medium. The presented results document that G. phototrophica requires a higher concentration of calcium for its metabolism and growth compared to other Gemmatimonas species.

Phytoextraction of rare earth elements (REE) from contaminated soils has gained importance during the last few decades. The Poços de Caldas municipality in Brazil is known for its mineral richness, including large reserves of REE. In this study, we report light REE (La, Ce, Sm, Pr, and Nd) in soils and plants collected in an area. Composite soil samples and plant individuals were collected, and total concentrations of LREE in soils were determined by wavelength dispersive X-ray fluorescence (WDXRF). The plant available LREE concentrations in soils were estimated upon the acetic acid method (F1 fractions) of the stepwise sequential extraction procedure, together with plant content that was analysed by inductively coupled plasma mass spectrometry (ICP-MS). The total sum concentrations of tested LREE in soils varied from 5.6 up to 37.9 g kg -1 , the bioavailable fraction was ca. 1%, and a linear relationship was found between them. The only exception was Sm, whose availability was lesser and did not show a linear relationship. The concentration of LREE in non-accumulator plants varied from 1.3-950 mg kg -1 for Ce, La 1.1-99 mg kg -1 , Sm 0.04-9.31 mg kg -1 , Pr 0.1-24.1 mg kg -1 , and Nd 0.55-81 mg kg -1 . The concentration of LREE among shoots did not show a linear relation either with the available fraction or total content. The screening also revealed Christella dentata (Forssk.) Brownsey & Jermy, Thelypteridaceae family, as a promising hyperaccumulator species. The concentrations of LREE among shoots of six individuals of this species were in the ranges from 115 to 1872 mg kg -1 for Ce, La 190-703 mg kg -1 , Sm 9-48 mg kg -1 , Pr 32-144 mg kg -1 , and Nd 105-478 mg kg -1 ., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Metal hyperaccumulation is an exclusive evolutionary trait contributing to efficient plant defence against biotic stress. The defence can be based on direct metal toxicity or the joint effects of accumulated metal and organic compounds, the latter being based on integrated signalling networks. While the role of metals in biotic stress defence of hyperaccumulators has been intensively studied, their role in the pathogen immunity of non-accumulator plants is far less understood. New findings show that in metal non-hyperaccumulating plants, localized hot spots of zinc, manganese, and iron increase plant immunity, while manipulation of nutrient availability may be used for priming against subsequent pathogen attack. Recent findings on the role of metals in plant-pathogen interactions are discussed considering the narrow line between deficiency and toxicity, host-pathogen nutrient competition and synergistic effects of simultaneous metal and biotic stress. We discuss the suitability of the direct-defence and joint-effects hypotheses in non-hyperaccumulating plants, and the involvement of metals as active centres of immunity-related enzymes. We also consider future challenges in revealing the mechanisms underlying metal-mediated plant immunity., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. This study aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica. We combined differential transcriptomics and proteomics, phenolic and sugar analysis, FTIR analysis of the cell wall polymers, and detailed analysis of the photosynthetic electron transport (PET) chain. The proteomic analysis allowed the relative quantification of 1192 different protein groups, of which 408 were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins related to photosynthetic processes were less abundant, while chlorophyll fluorescence measurements implied shifting from linear PET to cyclic electron transport (CET). The levels of H 2 O 2 scavenging enzymes, ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation provoked cell wall remodeling related to GLP-derived H 2 O 2 /HO ● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of the main metabolic pathways during desiccation aiming at crop drought tolerance improvement.

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 µXRF 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., (© 2021 The Authors. Plant Biology published by John Wiley & Sons Ltd on behalf of German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

In almost all photosynthetic organisms the photosynthetic pigments chlorophyll and bacteriochlorophyll (BChl) are Mg 2+ containing complexes, but Mg 2+ may be exchanged against other metal ions when these are present in toxic concentrations, leading to inactivation of photosynthesis. In this report we studied mechanisms of copper toxicity to the photosynthetic apparatus of Acidiphilium rubrum, an acidophilic purple bacterium that uses Zn 2+ instead of Mg 2+ as the central metal in the BChl molecules ([Zn]-BChl) of its reaction centres (RCs) and light harvesting proteins (LH1). We used a combination of in vivo measurements of photosynthetic activity (fast fluorescence and absorption kinetics) together with analysis of metal binding to pigments and pigment-protein complexes by HPLC-ICP-sfMS to monitor the effect of Cu 2+ on photosynthesis of A. rubrum. Further, we found that its cytoplasmic pH is neutral. We compared these results with those obtained from Rhodospirillum rubrum, a purple bacterium for which we previously reported that the central Mg 2+ of BChl can be replaced in vivo in the RCs by Cu 2+ under environmentally realistic Cu 2+ concentrations, leading to a strong inhibition of photosynthesis. Thus, we observed that A. rubrum is much more resistant to copper toxicity than R. rubrum. Only slight changes of photosynthetic parameters were observed in A. rubrum at copper concentrations that were severely inhibitory in R. rubrum and in A. rubrum no copper complexes of BChl were found. Altogether, the data suggest that [Zn]-BChl protects the photosynthetic apparatus of A. rubrum from detrimental insertion of Cu 2+ (trans-metallation) into BChl molecules of its RCs., (Copyright © 2021. Published by Elsevier B.V.)

Toxicity of lanthanides is generally regarded as low, and they even have been suggested to be beneficial at low concentrations. This research was conducted to investigate effects of Lanthanum (La) on Desmodesmus quadricauda, a freshwater green microalga. The algal cultures were treated with nanomolar La concentrations under controlled environmentally relevant conditions. Intracellular localization of La was analyzed with μXRF tomography in frozen-hydrated samples. At sublethal concentration (128 nM) La was in hotspots inside the cells, while at lethal 1387 nM that led to release of other ions (K, Zn) from the cells, La filled most of the cells. La had no clear positive effects on growth or photosynthetic parameters, but increasing concentrations led to a dramatic decrease in cell counts. Chlorophyll fluorescence kinetic measurements showed that La led to the inhibition of photosynthesis. Maximal photochemical quantum yield of the PSII reaction center in dark-adapted state (F v /F m ) decreased at > 4.3 nM La during the 2nd week of treatment. Minimum dark-adapted fluorescence quantum yield (F 0 ) increased at > 13.5 nM La during the 2nd week of treatment except for control (0.2 nM La, baseline from chemicals) and 0.3 nM La. NPQ at the beginning of the actinic light phase showed significant increase for all the treatments. Metalloproteomics by HPLC-ICPMS showed that La binds to a >500 kDa soluble protein complex already in the sub-nM range of La treatments, in the low nM range to a small-sized (3 kDa) soluble peptide, and at >100 nM La additionally binds to a 1.5 kDa ligand., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Boron (B) and zinc (Zn) are essential micronutrients of plant nutrition programs in orchards for securing the crop quality and yield. Although orchard supplementation with B and Zn is a common practice to overcome deficiencies or maintain their optimal levels, the efficiency of combined B and Zn spraying in relation to European hazelnut ( Corylus avellana L.) phenological stage has not been investigated so far. Leaf and kernel mineral and functional traits were studied in cultivar Tonda di Giffoni after B and Zn spraying in four phenological stages. During the 2016/2017 season, 9-year-old trees were sprayed with B (0, 800, and 1,600 mg L -1 ) and Zn (0, 400, and 800 mg L -1 ) under three treatments: B 0 +Zn 0 , B 800 +Zn 400 , and B 1600 +Zn 800 implemented in three spring application programs scheduled from October to December (P1: four times, P2: early two times, and P3: late two times). B and Zn treatments in P1 and P3 led to higher Zn concentration both in leaves and in kernels compared with non-sprayed trees. Stabilized nut production increased 2.5-fold under B 800 +Zn 400 in all three programs. Kernel/nut ratio improved in both B+Zn treatments in P1 and P3, while the percentage of blank nuts was reduced compared with B 0 +Zn 0 . Increased radical scavenging activity in B+Zn-treated kernels and leaves was not attributed to the accumulation of phenolics in P3 compared with B 0 +Zn 0 , whereas B and Zn spraying reduced the level of lipid peroxidation in both studied organs. According to the results, combined B and Zn should be sprayed at the end of spring (P3) on hazelnut plantations in temperate areas such as Southern Chile, whereas early applications (P2) showed an irregularity in nut production and functional traits in nuts. Moderate and partialized rates of B and Zn and the time of implementation contribute to improving the quantitative and qualitative features crucial for future sustainable hazelnut production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Meriño-Gergichevich, Luengo-Escobar, Alarcón, Reyes-Díaz, Ondrasek, Morina and Ogass.)

Phomopsis. longicolla is a hemibiotrophic fungus causing significant soybean yield loss worldwide. To reveal the role of zinc in plant-pathogen interactions, soybean seedlings were grown hydroponically with a range of Zn concentrations, 0.06 µM (deficient, Zn0), 0.4 µM (optimal growth), 1.5 µM, 4 µM, 12 µM, and toxic 38 μM, and were subsequently inoculated with P. longicolla via the roots. In vivo analysis of metal distribution in tissues by micro-X-ray fluorescence showed local Zn mobilization in the root maturation zone in all treatments. Decreased root and pod biomass, and photosynthetic performance in infected plants treated with 0.4 µM Zn were accompanied with accumulation of Zn, jasmonoyl-L-isoleucine (JA-Ile), jasmonic acid, and cell wall-bound syringic acid (cwSyA) in roots. Zn concentration in roots of infected plants treated with 1.5 µM Zn was seven-fold higher than in the 0.4 µM Zn treatment, which together with accumulation of JA-Ile, cwSyA, cell wall-bound vanilic acid and leaf jasmonates contributed to maintaining photosynthesis and pod biomass. Host-pathogen nutrient competition and phenolics accumulation limited the infection in Zn-deficient plants. The low infection rate in Zn 4 µM-treated roots correlated with salicylic and 4-hydroxybenzoic acid, and cell wall-bound p-coumaric acid accumulation. Zn toxicity promoted pathogen invasion and depleted cell wall-bound phenolics. The results show that manipulation of Zn availability improves soybean resistance to P. longicolla by stimulating phenolics biosynthesis and stress-inducible phytohormones., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity&#95;Sample&#95;RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

This work reveals, by imaging in vivo measurements in the Cd/Zn hyperaccumulator Arabidopsis halleri, in how far Cd stress affects macronutrient (Ca, K) and micronutrient (Fe, Zn) distribution in the leaves. We directly correlate these changes with biophysics of the photosynthetic light reactions. Plants were grown for 2 months at 10 μM Zn (=control), and supplemented with 10, 15, 50 or 75 μM Cd. Direct imaging of OJIP transients revealed that bundle sheath cells were more sensitive to Cd toxicity than mesophyll cells further from the vein. Progressive inhibition of photosystem (PS) II reaction centres and decrease in quantum yield of electron transport between Q A and Q B and further to PSI acceptors was observed. This was correlated with the decreased dynamics of Q A re-oxidation and lower operating efficiency of PSII. Analysis by a benchtop micro X-ray fluorescence device showed that Cd mostly accumulated in the veins, and restricted Fe and Zn distribution from the veins, especially in the 75 μM Cd, while K concentration increased in the whole leaf. Calcium distribution was apparently not affected by Cd, but Cd excess inhibited trichome formation and thereby diminished total Ca concentration in the leaves. The results point to differential tissue sensitivity to Cd, evident by heterogeneous inhibition of photosynthesis. Part of this may be a result of selective disturbances in the leaf nutrient homeostasis. The better photosynthetic performance away from the veins compared to the bundle sheath cells, however, indicates that direct inhibition of photosynthesis by Cd dominates over inhibition caused by micronutrient deficiency., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Combining high-carbohydrate food with polyphenol-rich food is a possible way of producing slowly digestible starch with beneficial health properties. In Japan, non-glutinous and glutinous rice are cooked with adzuki bean and the colour of the cooked rice is pale red. In this article, we show that (1) the red colour of rice could be attributed to the oxidation of adzuki bean procyanidins, (2) pancreatin-induced starch digestion of the red-coloured non-glutinous rice was slower than white rice and (3) the digestion of amylose and potato starch but not amylopectin became slower by heating with procyanidin B2. Furthermore, the rate of starch digestion of red-coloured rice was not affected by nitrite treatment under simulated gastric conditions. The above results show that procyanidins could bind to amylose independent of the starch source by heating and could suppress starch digestion by α-amylase in the intestine.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H 2 O 2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

In the previous work, sheath-flow probe electrospray ionization (sfPESI) equipped with a touch sensor was developed for conducting samples. In this work, a capacitiance-sensitive touch sensor that can be applicable to samples prepared in a nonconducting plastic multiwell plate was developed. The radiofrequency with 5 kHz and 4.5 Vpp was applied to the metal substrate on which the plastic plate was placed. The probe tip stopped at the position where it touched the surface of the liquid solution prepared in the plastic multiwell plate by detecting the displacement current flowing through the capacitance of the circuit. By coupling a nondisposable sfPESI probe with a table-top 3-axis robot, consecutive analysis of peptides, proteins, drugs, and real samples was performed. The carry-over by the consecutive analyses was suppressed to minimal by cleansing the probe tip using the solvent of water/methanol/acetonitrile (1/1/1).

Background: Many metals are essential for plants and humans. Knowledge of metal distribution in plant tissues in vivo contributes to the understanding of physiological mechanisms of metal uptake, accumulation and sequestration. For those studies, X-rays are a non-destructive tool, especially suited to study metals in plants., Results: We present microfluorescence imaging of trace elements in living plants using a customized benchtop X-ray fluorescence machine. The system was optimized by additional detector shielding to minimize stray counts, and by a custom-made measuring chamber to ensure sample integrity. Protocols of data recording and analysis were optimised to minimise artefacts. We show that Zn distribution maps of whole leaves in high resolution are easily attainable in the hyperaccumulator Noccaea caerulescens . The sensitivity of the method was further shown by analysis of micro- (Cu, Ni, Fe, Zn) and macronutrients (Ca, K) in non-hyperaccumulating crop plants (soybean roots and pepper leaves), which could be obtained in high resolution for scan areas of several millimetres. This allows to study trace metal distribution in shoots and roots with a wide overview of the object, and thus avoids making conclusions based on singular features of tiny spots. The custom-made measuring chamber with continuous humidity and air supply coupled to devices for imaging chlorophyll fluorescence kinetic measurements enabled direct correlation of element distribution with photosynthesis. Leaf samples remained vital even after 20 h of X-ray measurements. Subtle changes in some of photosynthetic parameters in response to the X-ray radiation are discussed., Conclusions: We show that using an optimized benchtop machine, with protocols for measurement and quantification tailored for plant analyses, trace metal distribution can be investigated in a reliable manner in intact, living plant leaves and roots. Zinc distribution maps showed higher accumulation in the tips and the veins of young leaves compared to the mesophyll tissue, while in the older leaves the distribution was more homogeneous., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Zinc is essential for the functioning of numerous proteins in plants. To investigate how Zn homeostasis interacts with virus infection, Zn-tolerant Noccaea ochroleucum plants exposed to deficient (Zn'0'), optimal (Zn10), and excess Zn (Zn100) concentrations, as well as Cd amendment, were infected with Turnip yellow mosaic virus (TYMV). Imaging analysis of fluorescence kinetics from the μs (OJIP) to the minutes (Kautsky effect, quenching analysis) time domain revealed strong patchiness of systemic virus-induced photosystem II (PSII) inhibition. That was more pronounced in Zn-deficient plants, while Zn excess acted synergistically with TYMV, in both cases resulting in reduced PSII reaction centers. Infected Cd-treated plants, already severely stressed, showed inhibited non-photochemical quenching and PSII activity. Quantitative in situ hybridization at the cellular level showed increased gene expression of ZNT5 and downregulation of HMA4 in infected Zn-deficient leaves. In Zn10 and Zn100 infected leaves, vacuolar sequestration of Zn increased by activation of HMA3 (mesophyll) and MTP1 (epidermis). This correlated with Zn accumulation in the mesophyll and formation of biomineralization dots in the cell wall (Zn100) visible by micro X-ray fluorescence tomography. The study reveals the importance of adequate Zn supply and distribution in the maintenance of photosynthesis under TYMV infection, achieved by tissue-targeted activation of metal transporter gene expression., (Copyright © 2020 Morina, Mishra, Mijovilovich, Matoušková, Brückner, Špak and Küpper.)

Solving the global environmental and agricultural problem of chronic low-level cadmium (Cd) exposure requires better mechanistic understanding. Here, soybean (Glycine max) plants were exposed to Cd concentrations ranging from 0.5 nM (background concentration, control) to 3 µM. Plants were cultivated hydroponically under non-nodulating conditions for 10 weeks. Toxicity symptoms, net photosynthetic oxygen production and photosynthesis biophysics (chlorophyll fluorescence: Kautsky and OJIP) were measured in young mature leaves. Cd binding to proteins [metalloproteomics by HPLC-inductively coupled plasma (ICP)-MS] and Cd ligands in light-harvesting complex II (LHCII) [X-ray absorption near edge structure (XANES)], and accumulation of elements, chloropyll, and metabolites were determined in leaves after harvest. A distinct threshold concentration of toxicity onset (140 nM) was apparent in strongly decreased growth, the switch-like pattern for nutrient uptake and metal accumulation, and photosynthetic fluorescence parameters such as Φ RE10 (OJIP) and saturation of the net photosynthetic oxygen release rate. XANES analyses of isolated LHCII revealed that Cd was bound to nitrogen or oxygen (and not sulfur) atoms. Nutrient deficiencies caused by inhibited uptake could be due to transporter blockage by Cd ions. The changes in specific fluorescence kinetic parameters indicate electrons not being transferred from PSII to PSI. Inhibition of photosynthesis combined with inhibition of root function could explain why amino acid and carbohydrate metabolism decreased in favour of molecules involved in Cd stress tolerance (e.g. antioxidative system and detoxifying ligands)., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

For quick, noninvasive, and high-sensitivity surface analysis of foods and agricultural products, a touch sensor was developed and applied to sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS). Upon making contact with the sample, the probe stopped by detecting the current flowing through the circuit and analytes on the sample surface were extracted in the solvent preloaded in the plastic capillary. By lifting up the probe to the default position, an electrospray ionization mass spectrum of the sample was obtained. By scanning the sample stage using a programming tool, a point analysis of targeted positions of biological samples with a spot diameter of ≤0.3 mm was achieved. It took less than 10 s for one sample spot. This method was applied to various plants and animal tissues.

In 2007, probe electrospray ionization/mass spectrometry (PESI/MS) was developed. In this technique, the needle is moved down along a vertical axis and the tip of the needle touched to the sample. After capturing the sample at the needle tip, the needle is then moved up and a high voltage is applied to the needle at the highest position to generate electrospray. Due to the discontinuous sampling followed by the generation of spontaneous electrospray, sequential and exhaustive electrospray takes place depending on the surface activity of the analytes. As modified versions of PESI, dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI) have been developed. These methods are complementary to each other and they can be applicable to surface and bulk analysis of various biological samples. In this article, the characteristics of these methods and their applications to real samples will be reviewed., (Copyright © 2020 Kenzo Hiraoka, Osamu Ariyada, Dilshadbek T. Usmanov, Lee C. Chen, Satoshi Ninomiya, Kentaro Yoshimura, Sen Takeda, Zhang Yu, Mridul K. Mandal, Hiroshi Wada, Stephanie Rankin-Turner, and Hiroshi Nonami.)

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Magnesium (Mg 2+ ) is the ubiquitous metal ion present in chlorophyll and bacteriochlorophyll (BChl), involved in photosystems in photosynthetic organisms. In the present study we investigated targets of toxic copper binding to the photosynthetic apparatus of the anoxygenic purple bacterium Rhodospirillum rubrum. This was done by a combination of in vivo measurements of flash photolysis and fast fluorescence kinetics combined with the analysis of metal binding to pigments and pigment-protein complexes isolated from Cu-stressed cells by HPLC-ICPMS (ICP-sfMS). This work concludes that R. rubrum is highly sensitive to Cu 2+ , with a strong inhibition of the photosynthetic reaction centres (RCs) already at 2 μM Cu 2+ . The inhibition of growth and of RC activity was related to the formation of Cu-containing BChl degradation products that occurred much more in the RC than in LH1. These results suggest that the shift of metal centres in BChl from Mg 2+ to Cu 2+ can occur in vivo in the RCs of R. rubrum under environmentally realistic Cu 2+ concentrations, leading to a strong inhibition of the function of these RCs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1 , qsk1 , and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins., (© 2019 Wu et al.)

In previous work, probe electrospray ionization/mass spectrometry (PESI/MS) and sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS) were reported for the rapid and minimally invasive analysis of food. In this work, a modified version of sfPESI will be reported. The sample surface was pricked with an acupuncture needle inserted in the sfPESI probe that protruded from the terminus of the tip by 5 mm. The invasion depth of the needle into the sample was ∼1 mm. After sampling, the needle was retracted into the solvent-preloaded capillary with a protrusion length of 0.1-0.2 mm from the tip. A mass spectrum of the sample captured on the needle was obtained by applying a high voltage to the needle. This method could be applicable to profiling analyses of plants with the epicuticular wax covering on the surfaces that are difficult to analyze by sf-PESI. The on-site mass spectrometric analysis for a growing apricot in the field was performed to monitor the developing stage of the fruit.

Chlorophyll fluorescence kinetic analysis has become an important tool in basic and applied research on plant physiology and agronomy. While early systems recorded the integrated kinetics of a selected spot or plant, later systems enabled imaging of at least the slower parts of the kinetics (20-ms time resolution). For faster events, such as the rise from the basic dark-adapted fluorescence yield to the maximum (OJIP transient), or the fluorescence yield decrease during reoxidation of plastoquinone A after a saturating flash, integrative systems are used because of limiting speed of the available imaging systems. In our new macroscopic and microscopic systems, the OJIP or plastonique A reoxidation fluorescence transients are directly imaged using an ultrafast camera. The advantage of such systems compared to nonimaging measurements is the analysis of heterogeneity of measured parameters, for example between the photosynthetic tissue near the veins and the tissue further away from the veins. Further, in contrast to the pump-and-probe measurement, direct imaging allows for measuring the transition of the plant from the dark-acclimated to a light-acclimated state via a quenching analysis protocol in which every supersaturating flash is coupled to a measurement of the fast fluorescence rise. We show that pump-and-probe measurement of OJIP is prone to artifacts, which are eliminated with the direct measurement. The examples of applications shown here, zinc deficiency and cadmium toxicity, demonstrate that this novel imaging platform can be used for detection and analysis of a range of alterations of the electron flow around PSII., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

In electrospray, excess charges are supplied to a sample solution by the occurrence of electrochemical reactions. Recently, different versions of electrospray, e.g., dielectric barrier electrospray ionization, inductive desorption electrospray ionization, and electrostatic-ionization driven by dielectric polarization, have been reported in which the sample solution was not in direct contact with the metal electrode but separated by dielectric materials. The objective of the current work is to elucidate the mechanism of dielectric barrier electrospray. A sealed borosilicate glass capillary inserted with a fine acupuncture needle was used as a probe. A sample solution (~ 400 nL) was captured on the glass capillary tip and a positive high voltage (HV) pulse (+ 4.5 kV) was applied to the internal metal electrode. Mass spectra were measured as a function of the HV pulse width from μs to 10 s. Ions started to be detected with the pulse width of ~ 5 ms. The ion intensities increased slowly with time and reached a plateau in a few seconds. The charge distribution of cytochrome c [M + nH] n+ shifted to higher n values from a few ms to seconds. In addition to cone-jet mode normal electrospray that lasted until all the liquid sample was depleted from the glass tip, the polarization-induced electrospray ionization was observed at the early stage of the HV application. Graphical Abstract ᅟ.

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

Rapid, direct, on-site and noninvasive food analysis is strongly needed for quality control of food. To satisfy this demand, the technique of dipping probe electrospray ionization/mass spectrometry (dPESI/MS) was developed. The sample surface was pricked with a fine acupuncture needle and a sample of ∼200 pL was captured at the needle tip. After drying the sample, the needle tip was dipped into the solvent for ∼50 ms and was moved upward. A high-voltage was applied to the needle to generate electrospray when the needle reached the highest position, and mass spectra were measured with a time-of-flight mass spectrometer. For evaluation of the method, the technique was used to analyze foods such as vegetables, salmon flesh, cow's milk, yogurt, and soy-bean milk. The detected major ions for cow's milk and yogurt were [(Lac) n  + Ca] 2+ with n = 1-6 (where (Lac) is lactose), indicating that Ca 2+ is tightly bound by Lac molecules., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Probe electrospray ionization (PESI) is only applicable to liquid or wet samples. In this study, a sheath-flow PESI method for remote sampling mass spectrometry that can be applied to dry samples was developed., Methods: An acupuncture needle (0.12 mm outer diameter, 700 nm tip diameter) was inserted into a gel-loading tip with a 0.1 mm protrusion out of the tip. Analytes were extracted by filling the latter tip with solvent and softly touching the sample surface for a short time (<1 s). A high voltage was applied to the acupuncture needle, and mass spectra of analytes were obtained by self-aspirating electrospray., Results: Dry samples, such as lines of ballpoint pen ink on paper, pharmaceutical tablets, instant coffee, brown rice, and narcotics, gave strong ion signals. The sample carryover was negligible. The sequential electrospray was observed to be similar to conventional PESI. The limits of detection (LODs) for morphine and rhodamine B were found to be of the order of picograms., Conclusions: Because of its simplicity and versatility, sheath-flow PESI is a promising technique for on-site and nondestructive profile analysis of dry samples with bulky and complicated shapes, with a spatial resolution of ~0.3 mm., (Copyright © 2017 John Wiley & Sons, Ltd.)

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.

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Direct and rapid trace-level gas analysis is highly needed in various fields such as safety and security, quality control, food analysis, and forensic medicine. In many cases, the real samples are bulky and are not accessible to the space-limited ion source of the mass spectrometer. In order to circumvent this problem, we developed an airtight atmospheric-pressure chemical ionization (APCI) ion source equipped with a flexible 1-m-long, 2-mm-i.d. PTFE sniffing tube. The ambient air bearing sample gas was sucked into the heated PTFE tube (130 °C) and was transported to the air-tight ion source without using any extra pumping system or a Venturi device. Analytes were ionized by an ac corona discharge located at 1.5 mm from the inlet of the mass spectrometer. By using the airtight ion source, all the ionized gas in the ion source was introduced into the vacuum of the mass spectrometer via only the evacuation of the mass spectrometer (1.6 l min -1 ). Sub-pg limits of detection were obtained for carbaryl and trinitrotoluene. Owing to its flexibility and high sensitivity, the sniffing tube coupled with a mass spectrometer can be used as the stethoscope for the high-sensitive gas analysis. The experimental results obtained for drugs, hydrogen peroxide and small alkanes were discussed by DFT calculations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Commercial strawberry, Fragaria x ananassa Duch., is a species sensitive to salinity. Under saline conditions, Na + uptake by the plant is increased, while K + uptake is significantly reduced. Maintaining an adequate K + /Na + cytosolic ratio determines the ability of the plant to survive in saline environments. The goal of the present work was to clone and functionally characterize the genes AKT1 and HKT1 involved in K + and Na + transport in strawberry and to determine the relationship of these genes with the responses of three Fragaria spp. genotypes having different ecological adaptations to salt stress. FaHKT1 and FcHKT1 proteins from F. x ananassa and F. chiloensis have 98.1% of identity, while FaAKT1 and FcAKT1 identity is 99.7%. FaHKT1 and FaAKT1 from F. x ananassa, were functionally characterized in Xenopus oocytes. FaHKT1, belongs to the group I of HKT transporters and is selective for Na + . Expression of FaAKT1 in oocytes showed that the protein is a typical inward-rectifying and highly K + -selective channel. The relative expression of Fragaria HKT1 and AKT1 genes was studied in roots of F. x ananassa cv. Camarosa and of F. chiloensis (accessions Bau and Cucao) grown under salt stress. The expression of AKT1 was transiently increased in 'Camarosa', decreased in 'Cucao' and was not affected in 'Bau' upon salt stress. HKT1 expression was significantly increased in roots of 'Cucao' and was not affected in the other two genotypes. The increased relative expression of HKT1 and decreased expression of AKT1 in 'Cucao' roots correlates with the higher tolerance to salinity of this genotype in comparison with 'Camarosa' and 'Bau'., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e. , ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

Experimental and theoretical studies on the oxidation of saturated hydrocarbons (n-hexane, cyclohexane, n-heptane, n-octane and isooctane) and ethanol in 28 Torr O 2 or air plasma generated by a hollow cathode discharge ion source were made. Ions corresponding to [M + 15] + and [M + 13] + in addition to [M - H] + and [M - 3H] + were detected as major ions where M is the sample molecule. The ions [M + 15] + and [M + 13] + were assigned as oxidation products, [M - H + O] + and [M - 3H + O] + , respectively. By the tandem mass spectrometry analysis of [M - H + O] + and [M - 3H + O] + , H 2 O, olefins (and/or cycloalkanes) and oxygen-containing compounds were eliminated from these ions. Ozone as one of the terminal products in the O 2 plasma was postulated as the oxidizing reagent. As an example, the reactions of C 6 H 14 +• with O 2 and of C 6 H 13 + (CH 3 CH 2 CH + CH 2 CH 2 CH 3 ) with ozone were examined by density functional theory calculations. Nucleophilic interaction of ozone with C 6 H 13 + leads to the formation of protonated ketone, CH 3 CH 2 C(=OH + )CH 2 CH 2 CH 3 . In air plasma, [M - H + O] + became predominant over carbocations, [M - H] + and [M - 3H] + . For ethanol, the protonated acetic acid CH 3 C(OH) 2 + (m/z 61.03) was formed as the oxidation product. The peaks at m/z 75.04 and 75.08 are assigned as protonated ethyl formate and protonated diethyl ether, respectively, and that at m/z 89.06 as protonated ethyl acetate. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)