Laboratory of

Plant Biophysics & Biochemistry

IPMB

JCU

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Research fields and interests of our department

We are interested in the physiology, biophysics and biochemistry (incl. molecular biology) of photosynthetic organisms (green/brown/red algae, terrestrial and submerged higher plants, cyanobacteria). The main focus is on metal metabolism in terms of uptake, physiological use, sequestration/complexation, detoxification, toxicity and interaction with plant immunity against pathogens.
    Some of these mechanisms are plant-specific (e.g. inhibition of photosynthesis or detoxification by sequestration into vacuoles), but many of them are very similar to mechanisms found in animals (incl. humans). For example, all major families of metal transporting proteins that are important in plants are found in animals, and often in bacteria as well. In this way, analysing such mechanisms in a plant model is also directly relevant for understanding metal metabolism in other groups of organisms, incl. humans. As some examples, we investigate how metal transport proteins function and how expression of such metal transport proteins varies depending on physiological state and development of a tissue. We investigate which ligands bind to the metals inside various cells and cellular comparments, and with which kinetics and in which quantities metals are sequestered into storage sites. In terms of toxicity, we analyse in which sequence, in which causal relationship, in which metal concentration range and under which environmental conditions (e.g. irradiance) damage mechanisms occur that were proposed in previous studies. An investigation of the time sequences, concentration dependence and causal relationships is particularly important in the case of these potential damage mechanisms because, in principle, they can strongly influence each other. For example, a decreased efficiency of exciton usage for photosynthesis can increase the formation of reactive oxygen species. And the other way round, photosynthesis may become inhibited as a consequence of oxidative damage to the thylakoid membranes and the proteins involved in photosynthesis. As a new research field, we started working on the interactions between trace metal metabolism in plants and interactions of these plants with pathogens, because many proteins involved in plant immunity are metalloproteins, and non-protein metal complexes may play a role as well.
    Although our research primarily aims at analysing basic mechanisms of function, it is often closely related to practical applications. For example, even today heavy metal toxicity is still a major problem in many regions of the world - including middle Europe, where e.g. Cu- and Zn-toxicity is often caused by the agricultural use of Cu und Zn containing pesticides. In addition, metal refineries and abandoned hazardous sites in former and current industry cause such environmental problems. Further, hyperaccumulator plants, which naturally use an active accumulation of heavy metals in their above-ground tissues as a defence against herbivores and pathogens, are already successfully used for biotechnological detoxification ("phytoremediation", especially of Cd) and for commercial extraction of metals ("phytomining", important mainly for Ni). At the same time, agricultural plants in many regions of the world suffer from deficiency of essential trace metals, with Zn being a prominent example.
    In our projects, we do not only use various terrestrial and aquatic species of higher plants, but also microorganisms (algae, cyanobacteria, purple bacteria). First, we use such organisms as models for specific experiments that investigate general questions that are relevant for higher plants and possibly animals but that are difficult to analyse with such "higher" organisms. This applies e.g. to questions concerning the ecotoxicology of heavy metals..
    For these investigations, we also developed methods of our own, e.g. the "Fluorescence kinetic Microscopes" (FKM's), improved methods and instrumentation for ultra-trace HPLC-ICPMS, a method for quantification of pigments in complex mixtures by Gauss-Peak-Spectra (GPS), a method for quantitative mRNA in situ hybridization (QISH), and a chamber for measuring photosynthesis and oxygen evolution of filamentous algae.

More detailed descriptions of individual research fields, and links to major current projects/grants within them, are provided below.

Key methods applied in our projects involve the following (in our and collaborating labs); links provided below are to devices in our own labs unless specifically mentioned. You can download a short video presentation about key methods of our team here.

(1) Spectroscopy:
(a) UV/Vis/NIR- absorbance and fluorescence spectroscopy, with special emphasis on the analysis of chlorophyll fluorescence kinetics, microscopic (single-cell, time-resolved and in vivo) spectroscopy and pigment analysis in complex mixtures
(b) X-ray spectroscopy: X-ray absorption spectroscopy ((µ)XANES, EXAFS) at synchrotrons (e.g. DESY P64), X-ray fluorescence spectroscopy (µXRF) - see 2b below;
(c) elemental analysis (ICP-AES, ICP-MS, GF-AAS)
(2) Microscopy:
(a) light microscopy: spatially and spectrally resolved fluorescence kinetic microscopy from microseconds to hours (to study biophysics of photosynthetis and metal trafficking); confocal microscopy for quantitative mRNA in situ hybridisation; classical transmission and fluorescence microscopy
(b) X-ray fluorescence (µXRF) in 2D mode in vivo sing an in-house beamline (15µm resolution) and as tomography using synchrotron beamlines at DESY P06, Diamond i18 and ESRF ID16A
(3) Gas exchange measurements
(a) Infrared gas analyzer for measuring CO2 and water vapour exchange of leaves of terrestrial plants
(b) Clark-type electrodes in various chambers for algae and submerged water plants
(3) Quantitative macroscopic imaging
(a) Analysis of chlorophyll fluorescence kinetics of whole leaves and small plants
(b) Quantitative imaging of gels, blots and tissues in absorbance, fluorescence and chemiluminescence mode
(4) Biochemical and biophysical analysis of isolated proteins
(a) activity and metal binding assays of purified proteins
(b) spectroscopies listed under (1)
(c) metalloproteomics: analysis of metal binding to proteins via HPLC-ICPMS and mass spectrometric identification of novel proteins
(5) Molecular genetics:
(a) quantitative mRNA in situ hybridisation (QISH),
(b) quantitative PCR
(6) Preparative work:
(a) HPLC of low MW compounds and proteins: under anoxic conditions, medium-scale bioinert, small-scale strictly metal-free;
(b) gel electrophoresis of proteins and DNA/RNA;
(c) isolation and work with protoplasts

Projects of the PBB department
- Trace metals in plants
- Regulation of photosynthesis
  - Light harvesting in aquatic microorganisms
  - Regulation of photosynthesis for nitrogen fixation in cyanobacteria
- Development of methods and scientific instruments
  - Ultratrace metal speciation analysis in proteins and pigments by metal-free HPLC coupled to sector-field ICP-MS (Küpper et al., 2019), 3D tour of the clean room
  - µXANES tomography (Mijovilovich et al., 2019)
  - benchtop µXRF - optimisation for in vivo imaging of trace elements (Mijovilovich et al., 2020), 3D tour of the µXRF lab
  - The Fluorescence kinetic Microscopes (FKM): with ms time resolution (Küpper et al., 2000), with additional spectral resolution (Küpper et al., 2007), and with µs time resolution (Küpper et al., 2019), 3D tour of the optics lab
  - Quantification of pigments in complex mixtures by Gauss-Peak-Spectra (GPS): 550-750nm for chlorophylls (Küpper et al., 2000), 330-750nm range for chlorophylls and carotenoids (Küpper et al., 2007)
  - Quantitative mRNA in situ hybridization (Küpper et al., 2007)
  - Chamber for measuring photosynthesis and oxygen evolution of filamentous algae (Küpper et al., 2004)

Trace metals in plants
Research direction	Current lead scientists of the field in our department	Current and recent grants related to this research direction led by our department
Mechanisms of trace metal toxicity and deficiency stress	Prof. Hendrik Küpper Dr. Elisa Andresen Dr. Filis Morina	Project KOROLID (CZ.02.1.01/0.0/0.0/15_003/0000336, Ministry of Education, Youth and Sports, co-financed by the European Union) Project updates on ResearchGate Goals of this part of KOROLID: a) Environmental risk assessment. While many potential mechanisms of metal toxicity have been proposed, most of them were investigated only under rather artificial laboratory conditions. Therefore, using aquatic and terrestrial model organisms we will evaluate the relevance of different proposed mechanisms of metal-induced inhibition under environmentally relevant conditions and compare the results with those obtained under conditions that were used in identifying the putative toxicity mechanisms. (b) Improved agriculture through more targeted fertilization, plus breeding crops that tolerate varying metal concentrations or that are able to grow in adverse conditions where current crops either suffer from deficiency or toxicity and/or contain an elemental composition that is suboptimal for human health. We want how in plants metal (Cr, Cu, La, Mn, Nd, Zn) deficiency and onset of metal(loid) (As, Cd, Cr, Cu, La, Mn, Nd, Zn) toxicity lead to a change in metal distribution between different tissues, different cellular compartments and different metal-binding proteins, rather than a general concentration change in many tissues. (c) The analysis of differences in photosynthesis biophysics and metal/acid resistance of Zn-BChl containing bacteria compared to their Mg-BChl containing relatives will help in understanding why almost all organisms utilize the Mg-complexes, and under which exact conditions it becomes advantageous to use alternatives. Project "Trace Metal Metabolism in Plants - PLANTMETALS" (COST Association grant CA19116) Project homepage Objectives of the COST Action PLANTMETALS to which our department will contribute within this field of our research (a) Our work will aid environmental risk assessment. While many potential mechanisms of metal toxicity have been proposed, most of them were investigated only under rather artificial laboratory conditions. Therefore, using aquatic and terrestrial model organisms we will evaluate the relevance of different proposed mechanisms of metal-induced inhibition under environmentally relevant conditions and compare the results with those obtained under conditions that were used in identifying the putative toxicity mechanisms. We have chosen Cu, Cr and Cd as model heavy metals, plus arsenic as a toxic metalloid, because they most frequently reach toxic concentrations in contaminated environments. But this also applies to "rare earth" elements (e.g. La, Nd) in their mining regions. (b) Our work on mechanisms of trace metal metabolism, although being basic research by itself, will help to improve agriculture. This will be through more targeted fertilization as thresholds and mechanisms of beneficial and toxic effects of trace metals will be better known. This will furthermore help other groups in the network in breeding crops that tolerate varying metal concentrations or that are able to grow in adverse conditions where current crops either suffer from deficiency or toxicity and/or contain an elemental composition that is suboptimal for human health. Project "Relationship between naturally zinc containing bacteriochlorophyll (Zn-BChl) and ecological adaptation in Acidiphilium bacteria" (Czech Science Foundation grant 16-03211S) Project updates on ResearchGate Goal: The purpose of the project is to investigate mechanisms of sublethal copper toxicity in two contrasting groups of purple bacteria: Acidiphilium and Rhodospirillum. We want to find out and in how far differences in copper resistance and damage caused by copper toxicity in these organisms are related to the different natural central ion in their BChl (Mg2+ in Rhodospirillum vs. Zn2+ in Rhodospirillum).
Role of trace metals in plant immunity against biotic stress	Dr. Filis Morina Prof. Hendrik Küpper	Project KOROLID (CZ.02.1.01/0.0/0.0/15_003/0000336, Ministry of Education, Youth and Sports, co-financed by the European Union) Project updates on ResearchGate Goal of this part of KOROLID: Improved agriculture via better knowledge about the role of metals in plant immunity against pathogens. This is an exciting emerging field, since it turned out that many proteins that are essential for plant immunity need metal binding for their function. Project "Trace Metal Metabolism in Plants - PLANTMETALS" (COST Association grant CA19116) Project homepage Objectives of the COST Action PLANTMETALS to which our department will contribute within this field of our research. Our research should contribute to better understanding of the mechanisms of metal-induced plant immunity and the crosstalk between abiotic and biotic stress, both in hyperaccumulators and in crop plants. This is an exciting emerging field, since it turned out that many proteins that are essential for plant immunity need metal binding for their function. By exploring the role of essential micronutrients (Zn, Mn, Ni) in plant response to pathogenic fungi (generalist and specialist) we will corelate micronutrient distribution and accumulation with the induction of organic defence pathways (such as phytohormone signalling), reactive oxygen species accumulation, accumulation of secondary metabolites and changes in the cell wall.
Mechanisms of trace metal resistance and hyperaccumulation	Prof. Hendrik Küpper Dr. Filis Morina	Project KOROLID (CZ.02.1.01/0.0/0.0/15_003/0000336, Ministry of Education, Youth and Sports, co-financed by the European Union) Project updates on ResearchGate Goal of this part of KOROLID: Phytoremediation of polluted soils and aquifers. Heavy metal hyperaccumulators are of great interest because of their use in phytoremediation of contaminated soils and in phytomining, but also as models for fundamental mechanisms of metal metabolism. Our project will contribute to the understanding of physiological and biochemical mechanisms of hyperaccumulation by revealing mechanisms of metal uptake, transport and detoxification. Project "Trace Metal Metabolism in Plants - PLANTMETALS" (COST Association grant CA19116) Project homepage Objectives of the COST Action PLANTMETALS (citation from the MoU) to which our department will contribute within this field of our research: Our work will contribute to better phytoremediation of polluted soils and aquifers. Heavy metal hyperaccumulators are of great interest because of their use in phytoremediation of contaminated soils and in phytomining, but also as models for fundamental mechanisms of metal metabolism. Our project will contribute to the understanding of physiological and biochemical mechanisms of hyperaccumulation by revealing mechanisms of metal uptake, transport and detoxification. Project "Roles of apoplastic and symplastic transport in cadmium and zinc uptake in the Cd/Zn hyperaccumulator Sedum alfredii" (Czech Academy of Sciences - National Natural Sciences Foundation of China (collaboration grant NSFC-21-05 with Dr. Qi Tao as PI in PR China) Project updates on ResearchGate Goal: Although most studies have reported that trace metal ions in hyperaccumulators reach the xylem mostly through the symplastic pathway, our latest research indicated a significance of the apoplastic pathway. This project aims at determining the importance of the apoplastic compared to the symplastic pathway of metal loading into the xylem and metal distribution inside the plant.
Regulation of photosynthesis
Light harvesting in aquatic microorganisms	Dr. Radek Litvin Dr. David Bina	Czech Science Foundation project 20-01159S Interactions between pigments for efficient light harvesting and photoprotection in photosynthesis. (with Jakub Psencik, Charles universiy in Prague) The goal of the project is to elucidate the role of two types of interactions between pigments for efficient light harvesting and photoprotection in photosynthetic light-harvesting complexes, and to use the results for optimization of artificial antenna complexes based on self-assembly of pigments.
Regulation of photosynthesis for nitrogen fixation in cyanobacteria	Prof. Hendrik Küpper	Project KOROLID (CZ.02.1.01/0.0/0.0/15_003/0000336, Ministry of Education, Youth and Sports, co-financed by the European Union) Goal of this part of KOROLID: More realistic estimation of the productivity of ecosystems, especially the oceans. This will be tackled by a project on regulation of photosynthesis and nitrogen fixation under iron limitation stress, involving the expression of alternative protein isoforms which we first observed as a phenomenon in an earlier study. This project will furthermore study how the oxygen-sensitive iron centers in the nitrogenase are protected from damage, where still controversial theories exist.