Metal metabolism in plant-galler interactions is largely unknown. We hypothesise that the mites manipulate metal distribution by sequestration of excessive levels and differential regulation of metalloproteins to support the main functions of gall-nutrition, protection and microenvironment. Using the Tilia cordata-eriophyid mites system, we aimed to reveal the role of metals in galls by investigating their distribution, speciation, gene expression and metabolome profiling. Complementary spectroscopy techniques (μXRF and μXANES tomographies, electron paramagnetic resonance), histochemical, metabolomic and transcriptomic analyses were employed. Mn was the most abundant micronutrient in the galls. Differential cell-specific Mn accumulation (idioblasts vs nutritive tissue) and speciation are essential for its homeostasis. Mn(II)-aquo complex, co-localised with Ca, sequestered in idioblasts, while Mn bound to stronger ligands including enzymes accumulated in the nutritive tissue. Zn, Cu and Fe predominately accumulated in the nutritive tissue to support intensive metabolic processes such as secondary and lipid metabolism, protein N-glycosylation and redox regulation. The slower rate of redox-sensitive spin probes' decay in the galls indicated a lower amount of antioxidants than in the leaf. We reveal essential functions of micronutrients in the galls, supporting the developmental and chemical changes in the host plant, and the nutrition of the galler.

• Klíčová slova
• Mn speciation, Tilia cordata galls, biotic stress, eriophyid mites, metal metabolism, micro‐XANES tomography, micro‐XRF tomography, nutritive tissue,
• MeSH
• kovy * metabolismus   MeSH
• listy rostlin * metabolismus   parazitologie   MeSH
• metabolom MeSH
• nádory rostlin * parazitologie   MeSH
• regulace genové exprese u rostlin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kovy * MeSH

Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type Arabidopsis and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.

• Klíčová slova
• Arabidopsis, chlorophyll biosynthesis, cytokinins, de-etiolation, ethylene, fluorescence, iReenCAM,
• Publikační typ
• časopisecké články MeSH

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 H2O2 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 H2O2/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.

• Klíčová slova
• OJIP, cell wall remodeling, cyclic electron transport, drought, germin-like proteins, late embryogenesis abundant proteins, pectin, polyphenol oxidase, resurrection plant, superoxide dismutase,
• Publikační typ
• časopisecké články MeSH

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O2. In green tissues, this putative effect is masked by photosynthetic O2 evolution. However, O2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

• Klíčová slova
• Arabidopsis thaliana, hypoxia, mitochondrial dysfunction stimulon, photosynthetic electron transfer, reactive oxygen species, retrograde signalling,
• MeSH
• anaerobióza MeSH
• Arabidopsis genetika   fyziologie   MeSH
• fotosyntéza * MeSH
• jaderné proteiny genetika   MeSH
• mitochondrie metabolismus   MeSH
• proteiny huseníčku genetika   MeSH
• signální transdukce * MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• proteiny huseníčku MeSH
• RCD1 protein, Arabidopsis MeSH Prohlížeč

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.

• Klíčová slova
• Arabidopsis halleri, Capsicum annuum, Glycine max, Hyperaccumulator, Leaf age, Micro X-ray fluorescence, Micronutrients, Noccaea caerulescens, Root, Zinc,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• TYMV infection, cadmium, chlorophyll fluorescence kinetics, metal transporters, micro X-ray fluorescence, non-hyperaccumulator, plant immunity, zinc,
• Publikační typ
• časopisecké články MeSH

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).

• Klíčová slova
• Cadmium, XANES, lipidomics, metabolomics, metal stress, metalloproteomics, soybean (Glycine max), sublethal toxicity,
• MeSH
• chlorofyl MeSH
• fotosyntéza MeSH
• Glycine max * MeSH
• kadmium * toxicita   MeSH
• listy rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• kadmium * MeSH