Final Report Summary - ZISPLOC (Zinc and iron homeostasis in Arabidopsis thaliana: novel molecular factors and their influence on metal speciation and localization)
• Executive summary
The central objective of this project was to combine molecular genetics approaches with synchrotron-based elemental imaging techniques in the model plant Arabidopsis thaliana for advancing the mechanistic understanding of transition metal handling in plants. This innovative combination of approaches entailed the adaptation of physics techniques to molecular physiology scientific questions in a genetic model plant. Novel scientific insights have been gained that are very difficult to obtain through existing techniques, which make a significant contribution towards enabling the rational design of strategies for the molecular-assisted breeding of crop species with enhanced essential mineral content (bio-fortification), improved yields under minimal fertilizer input (sustainable agriculture), and for the development of technologies for the plant-based clean-up of heavy metal-contaminated soils.
There is a need to develop a comprehensive understanding of the functions of all central molecular components of the metal homeostasis network in plants, including their regulatory pathways, and of how these components function together in a network context. To date, the limited accessibility of techniques for the correct determination of metal localization and speciation are a major bottleneck for our understanding of plant metal homeostasis. Synchrotron-based imaging techniques have been used so far predominantly in a biogeochemical and ecological context using wild type plants. Their systematic application to model plants in a molecular genetics context with the aim to elucidate the functions of single genes or molecular pathways remains a challenge. The proposed project was aimed at making a decisive contribution to closing this gap, by pursuing the specific research goal to advance our understanding of the interface between zinc (Zn) and iron (Fe) homeostasis in the genetic model plant Arabidopsis thaliana.
This project was organized into three objectives. The first one was to take advantage of spectroscopic techniques to shed light on mechanisms involved in metal homeostasis using on-going projects in the host-laboratory. The second objective was to characterize a new mutant identified during a previous mutant screen (European project funded by a Marie Curie IIF fellowship: grant agreement number: 219457, Project acronym: CENTZIN) and understand the role of the mutated gene in Fe and Zn homeostasis. Finally, the last objective was to perform a different screen to identify new mutants involved in Fe homeostasis.
• A summary description of project context and objectives
A number of genes acting in iron and zinc homeostasis of Arabidopsis have been identified and functionally characterised at the molecular level. In previous work, a strong focus has been on membrane transport proteins acting in root metal uptake, vacuolar storage and re-mobilization, and short-and long-distance metal distribution in the plant. An understanding of the roles of the low-molecular-weight metal chelator nicotianamine and membrane transporters of nicotianamine and its metal complexes is emerging. Finally, the function and regulation of ferritin, a chloroplastic iron storage protein, has been elucidated in much detail. Recently published results have highlighted that metal homeostasis defects often manifest themselves in dramatically altered metal distribution between or within tissues or even at the sub-cellular level, which can explain major developmental defects such as male sterility. Furthermore, altered metal speciation is known to have dramatic effects on its intra- and inter-cellular as well as long-distance mobility of metals. However, in many cases methodological difficulties have either prevented or limited the resolution, precision or reliability of metal localization and in planta metal speciation in the past.
Although a number of metal homeostasis factors have been identified and characterized, the pathways involved in their regulation are less well understood and constitute a major current research topic. Only a couple of transcription factors have been identified that contribute to the transcriptional regulation of iron deficiency responses, and, apart from the well-characterized bHLH protein FIT (basic helix-loop-helix protein FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), our understanding of their individual roles and how they integrate into a regulatory network remain sketchy. The reason for this is probably that a number of genes of the regulatory pathways remain unidentified. Thus, the identification of additional genes contributing to iron-dependent regulation is critical for the construction of a comprehensive model of iron homeostasis in the future. Furthermore, it is now clear that iron-dependent regulation cannot be understood in isolation: the transcriptional response to iron deficiency includes increases in transcript levels of genes not acting in Fe acquisition, but in Zn detoxification instead. These Zn detoxification genes are transcriptionally responsive also to excess Zn. This common regulation could either be the result of overlapping or converging molecular regulatory pathways of responses to iron deficiency and excess zinc, or of converging physiological consequences of both nutrient regimes. Because of the lack of selectivity of IRT1 (Iron-Regulated Transporter 1), the major root iron uptake system of A. thaliana, Fe deficiency causes enhanced Zn influx into roots and thus the risk of Zn toxicity. Conversely, exposure to excess Zn causes iron deficiency by interfering with iron movement inside the plant and possibly also through competition during transport steps inside the plant and the formation of metalloproteins. Recent work suggests that all three of the described scenarios are relevant in vivo. To gain further insight into common and distinct regulatory pathways in iron and zinc homeostasis, it was proposed to follow a forward genetic approach combined with physiology and molecular biology, in a powerful combination with advanced imaging techniques to analyze more precisely metal localization and local speciation phenotypes.
One of the innovative aspects of the project was the use of cutting-edge imaging techniques to gain new insights of metal localization and speciation in Arabidopsis thaliana. Up to now, iron and zinc were mainly detected by histochemical techniques. Zinc can be visualized by adding fluorophores such as Zinpyr or Zinquin, and iron by the adjunction of Perls’stain. The resulting fluorescent metal-ligand complexes are then visualized by confocal laser scanning microscopy (CLSM). Quantification of metals in bulk tissues is commonly achieved employing digests of plant tissues (seed, root, stem or leaf) through the technique inductively coupled plasma mass spectrometry (ICP-MS) or atomic emission spectrometry (ICP-AES).
In this project, we went further than these well-known techniques and employed recently established physics techniques based on the interaction between radiation and matter to obtain complementary data on metal partitioning, speciation and local quantification in plants. For this purpose, experiments have been conducted on large instruments such as synchrotrons and nuclear microprobes. Their use in plant biology has been expanding as shown by several recent reviews.
Synchrotron radiation-based micro X-ray fluorescence (µXRF) in scanning mode permits to obtain two-dimensional (2D) elemental distribution maps. The main advantages of this technique are its sensitivity (in the sub-milligram per kilogram range) and the multi-elemental nature of the information collected. Moreover, the spatial resolution is about 1 µm x 1 µm, and new beamlines are under construction with spatial resolution in the nanometer range, much higher than that obtained by other techniques such as laser-ablation ICP-MS (LA-ICP-MS) that is about 10 µm x 10 µm. µXRF resolution criteria permit to study metal partitioning in plants at the cellular scale. For instance, µXRF has been used with success to determine nutrient distribution in the barley grain. Distribution maps show that Fe, Mn, Cu, Zn and K were mainly present in the envelope of the seed. Another study has demonstrated that TiO2 nanoparticles were taken up by wheat plantlets and transferred to the shoot. µXRF based on synchrotron radiation has thus proven its efficiency to image metallic elements in plants.
Performing µXRF on synchrotron beamlines often permits to couple a micro X-ray absorption spectroscopy (µXAS) analysis in situ (LUCIA, SOLEIL, France or ID21, ESRF, France for instance). µXAS is a spatially resolved technique, informative on metal speciation (oxidation state and binding environment of the element of interest). In addition to elemental distribution, the speciation of metals is of vital importance in understanding their metabolism in plants as well as the toxicity of contaminants and the availability of micronutrients. Moreover, with recent development of cryo-stages on synchrotron beamlines, artefacts such as speciation changes or metal remobilization can now be avoided. µXAS has mainly been used on bulk samples (freeze-dried plant tissues, mixed and pressed as a pellet), but a few research teams have already performed the analysis in situ on µXRF maps. The combination of synchrotron radiation-based µXRF and µXAS has thus been used in plant biology by only a few research groups in the world, but with promising results. For instance, in wheat plantlets exposed to TiO2, µXRF maps of roots have shown that Ti was internalized in roots and found inside the vascular cylinder. By using µXAS analysis on Ti hot spots located on µXRF maps, the authors demonstrated that Ti speciation inside roots, and later inside leaves, is not modified.
Synchrotron based-techniques are very powerful to image metals in plant tissue at a sub-micrometer spatial resolution, but they are not quantitative. To have access to this information, we analyzed plant sections by microparticle-induced X-ray emission (µPIXE) coupled to Rutherford backscattering spectroscopy (RBS) on nuclear microprobes. This technique permits local elemental quantification within plant organs and even at the sub-cellular scale: in the nucleus or in the call wall for instance. It is thus much more spatially resolved than classical ICP. For instance, a µPIXE/RBS study was performed on Hybanthus floribundus exposed to Ni. ICP-AES measurements show that leaves contain 7.8 g Ni kg-1 dry matter (DM). Ni concentration in the whole leaf section measured by µPIXE/RBS gave the same result: 7.8 g Ni kg-1 DM. Actually, this global concentration averages over vast differences ranging from 1.0 g Ni kg-1 DM in the adaxial epidermis to 8.6 g Ni kg-1 DM in the vascular tissue. The µPIXE/RBS technique is thus very well suited to measure local concentrations of elements. Moreover, during nuclear microprobe experiments, a spectrum of the whole periodic table (from sodium to uranium) is recorded, allowing the extraction of images for each element and thus co-localization analyses.
In summary, the proposed project entailed a multidisciplinary approach combining physics techniques with molecular biology and genetics for an insightful, innovative and original approach in plant biology which is powerful to understand molecular mechanisms of metal homeostasis.
Objective A
The central goal of the project was to implement comparative metal localization, speciation and local quantification in wild type and mutant Arabidopsis plants for the elucidation of the functions of individual genes. According to beamtime allocation specific questions related to the topic of this project have been addressed in a set of characterized genotypes (see results).
Recently developed techniques, namely µXRF, µXAS, µPIXE/RBS and µFTIR have been employed to obtain new and original data to complete first data obtained on studied mutants. For all these techniques, sample preparation is a crucial step and has been deeply optimized. Indeed, it can lead to artefacts such as metal remobilization or changes in metal speciation. The best way to preserve elemental partitioning and speciation is to keep the sample in cryo-conditions during the whole process. To determine metal speciation, reference compounds have been prepared as pellets in cryo-conditions and analysed (Zn-citrate, Zn-glutathione, Zn-His2, Fe-citrate, Fe-glutathione, Fe-His2 in the form of Fe(II) and Fe(III), Fe-ferritin). µXAS data have been fitted using Athena software by achieving linear combination fits with these reference compounds. µXRF data analyses have been achieved using PyMca software to obtain element maps. Absolute concentrations were obtained by processing the data through SIMNRA and Gupix softwares allowing comparing metal concentrations in different tissues and/or genotypes.
Objective B
To advance the number of known and characterized gene functions influencing the regulation of zinc and iron homeostasis, a mutant identified earlier, which fails to increase ZIF1promoter-GUS activity in response to growth in excess Zn, have been studied. This mutant has been preliminarily characterized to confirm the regulation defect and demonstrate that root and shoot Zn accumulation are altered. The phenotypic characterization of the mutant encompassed ZIF1promoter-GUS activity analyses of seedlings grown on different metal media, metal quantification in root and shoot tissues, growth assays (root length, fresh foliar and root biomasses) and photosynthesis study under a range of Zn and Fe metal supplies. Experiments have also been carried out to try to phenotypically rescue the plant phenotype using supplements on both soil and agar substrates.
Objective C
We decided to perform the screening on an EMS-mutagenized population of an iron homeostasis mutant to advance our understanding of Fe homeostasis. This mutant is being extensively studied in the host laboratory. The first step of this objective was to optimize the screening process by developing the most efficient medium to detect abnormal phenotypes. Several tests were performed both on differently composed agar media and on different type of soils with modified pH.
Then a batch of seeds of a common mutant genotype was further EMS-mutagenized and sown on soil. M2 seeds were pooled and M2 plants are presently being screened in the host laboratory. Putative mutant individuals will be back-crossed, and selfed to obtain the M3 generation.
• A description of the main S&T results/foregrounds
Objective A: Imaging of well-known mutants
The main goal of this part was to collaborate with other researchers and to take advantage of spectroscopic techniques to advance their knowledge on their research projects. This part of the project has been very beneficial in terms of networking between the plant biologist community and the spectroscopic community (Table 1). This work has also led to the adaptation of some instruments (cryo set-up on MicroXAS beamline, SLS, Switzerland & micro focused beam on RUBION nuclear microprobe, Germany) towards biological questions. It has also been very valuable for the fellow. The fellowship permitted her to deepen her knowledge of spectroscopic techniques and sample preparation as well as exploring different and complementary questions on the topic of metal homeostasis in plants. Additionally, it was also the opportunity of meeting new actors of both communities which represent potential collaborations for the future.
Finally, it is worth mentioning that this objective has been much further developed than initially foreseen. Indeed, getting beamtime is not an automatic process; however, all our proposals have been successful, leading to the granting of a high number of beamtime shifts, which required preparation, presence time and time for data evaluation. Thus, the amount of imaging data obtained in this project has been far larger than expected, thus enabling the completion of publishable imaging datasets for far more publications than we could have hoped for at the outset.
All the beamtime obtained through proposal acceptance or collaborations has been shared between my own project (Objective B) and collaborators’ projects. The order of priority between samples first presented in the ZISPLOC project has evolved according to new results obtained in the meantime. Several shifts of beamtime had also been allocated to sample preparation optimization, with Arabidopsis seedlings being particularly small and fragile.
Results obtained here are confidential (only described in the last periodic report in section 3.2.2. work progress and achievements during the period) and will lead to the publication of 5 articles in high impact factor journals that will associate both communities (researchers from large instruments and plant biologists). This objective of the project will lead to the publication of 5 papers as co-authors in high impact factor journals (one is under review in Nature Plants, and one will be soon submitted to PNAS).
Objective B: Characterization of a new mutant
A batch of ZIF1pGUS seeds (background Columbia) was EMS mutagenized. A screening was performed on 4800 M2 seeds by germinating them onto 1xHoaglands (1xHG) with 30 µM ZnSO4. 14 d old seedlings were assessed for chlorosis (i.e. Zn sensitivity) and other phenotypes (such as root growth or leaf/cotyledon morphology). A single cotyledon was GUS-stained. Mutants were selected for chlorosis and lack of the appropriate transcriptional response (i.e. induction of ZIF1 and thus blue staining). From those mutants, one was selected and called roz1 (regulator of ZIF1) and three alleles have been discovered so far and confirmed by crossing:
- roz1-1: A1.116
- roz1-2: D3.115 and possibly D3.333
- roz1-3: E1.417
- Elemental distribution and metal speciation
Elemental distribution and speciation were studied on the roz1-2 mutant by µXRF and µXANES. Local concentration was determined by µPIXE on the roz1-3 mutant.
An experiment was performed at the Zn-edge at MicroXAS beamline (Swiss Light Source) but the data set is not complete due to time spent to optimize the cryogenic set-up. Nevertheless, partial results obtained show that Zn in roots was mainly detected in the vasculature and in the cell walls. In leaves, Zn was seen in trichomes as well as in hot spots throughout leaf cross-sections and especially in the epidermis for the mutant exposed to high concentrations of Zn. These results were also confirmed by µPIXE analysis on the Slovenian nuclear microprobe (in collaboration with Katharina Vogel-Mikus). No major differences were detected between WT and mutants (Figure 1).
Zn was not enough concentrated in tissues cultivated under basal conditions to be detected by this technique (with the actual setup) except in the tips of the leaves of the WT. In high Zn conditions, Zn was detected in the base of the trichomes (adaxial side) and as small dots on the abaxial side (stomata?). In the cross-sections it was observed as small dots in the epidermis (stomatal cells?). Same distribution patterns were shown on cross-sections in low Fe condition.
Seedlings were also analyzed at the Fe-K edge on ID21 beamline (European Synchrotron Radiation Facility). On this beamline, Zn is not reachable but Fe and lighter elements such as P, S, Cl (and Ca, K) that were not analyzable on MicroXAS (analysis chamber not under vacuum loss of light element signal) were accessible.
In whole roots, the main difference between WT and mutant is the accumulation of Mn in the quiescent center for the mutant whereas it is more concentrated in the root cap for the WT. When analyzing root cross-sections, differences were detected in Fe distribution as well; it was adsorbed on root epidermis for both genotypes but in the mutant Fe was also detected in the cell walls of parenchymal cells.
In whole leaves, Fe, Mn and P distributions seem to be altered in the mutant: these elements are more homogeneously distributed in the leaf whereas they are more concentrated in the trichomes in the WT. Analysis of leaf cross-sections demonstrated that Fe was mainly localized in cell walls and vasculature. It was also detected in the base of the trichome for the WT but not for the mutant (as already shown by analysis of whole leaves).
Fe speciation was analyzed in situ for roots and leaves. Several spectra were acquired per point of interest to increase the signal/noise ratio. Several points were analyzed per tissue and averaged in Table 2. Differences in Fe distribution between WT and mutant were detected mainly in the trichome and in cell walls in roots. However this difference in distribution does not seem to be linked with a different speciation. Fe(III) seems to be more predominant in the mutant (especially in leaf surface and root epidermis) than in the WT.
- Phenotypic characterization
Mutant seeds were grown on soil together with the WT. It appears that all alleles of roz1 seedlings experienced a delayed development (Figure 2), with mutants having rosette of few leaves when the WT was giving seeds.
The three alleles of roz1 were also germinated on Hoagland plates for 7 days and then transferred on plates on different media for 7 more days: Hoagland (1 µM Zn, 5 µM Fe), +Zn (30 µM ZnSO4) and -Fe (washed agar, no Fe). After 15 days, GUS staining wad performed on the first true leaves, fresh biomass was recorded, photosynthetic pigment concentrations were determined by spectrophotometry and elemental concentrations were assessed by ICP-AES.
As seen on Figure 3, roz1 mutants seemed to grow smaller and paler than the WT in all conditions even if the difference is less evident in –Fe condition.
The lack of induction of ZIF1pGUS was checked after exposure for 7 days in high Zn condition (Figure 4). Results were in agreement with the previously obtained ones: in high Zn in the WT the staining expanded over the vasculature but not in the mutant.
Determination of the fresh root and shoot biomasses confirmed those observations.
Likewise, chlorophyll a, chlorophyll b and carotenoid pigment concentrations in the leaves of the three (potentially four) alleles of roz1 mutants were decreased as compared with the WT. Again the –Fe condition resulted in comparatively sick seedlings for the WT and the mutants.
Elemental composition showed that roz mutants had higher P concentration in leaves, lower Mg in roots and lower K in both root and shoot. Again, differences between wild type and mutants were less pronounced upon cultivation under –Fe conditions. Impaired P metabolism has already been evidenced by the previous µXRF analysis. In basal condition, ICP results also confirmed the altered Mn partitioning seen by µXRF as well as Fe behaviour in roots. Moreover, in all alleles of roz1 mutant over-accumulated Zn in both root and shoot.
Nucleic DNA was extracted to identify the exact location of the mutation in the roz1-1 mutant. Results are not yet available.
Different phenotypic rescue experiments were pursued (adding Fe or Zn-rich solution or on high pH soil) but none of these were conclusive.
Objective C: New screening
Towards objective C, screening conditions have been optimized to permit to faster the screening process afterwards. Several conditions have been tested comparing wild-type and a previously characterized iron-deficient mutant to obtain an efficient Fe-depleted medium: on plates (by addition of different concentrations of ferrozine) or on soil (by increasing the soil pH).
Plate conditions were too stringent for the irt1 mutant. A protocol was established to screen for Fe deficiency symptoms on soil (Figure 5). The intermediate pH level was chosen since it permitted to maximize differences between the WT and the irt1 phenotype with no dead plants. Higher pH was obtained through the addition of 10 g CaCO3, 5 g CaO and 5 g K2HPO4 for 500 g of soil.
Seeds of irt1 were EMS-mutagenized and sown on normal minitray soil for seed production and watered with sequestrene to avoid an overly dramatic Fe deficiency. M2 seeds were collected and are being cultivated on modified minitray soil for a first screen. Mutagenesis and screening were performed together with Dr Maria Bernal, who will pursue the project in the future.
• The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
First of all, this fellowship has been a unique opportunity for the fellow to broaden her knowledge and increase her employability. Indeed, the fellow terminated the contract two months before its expiration because she got a permanent position as scientist at the French centre for scientific research (CNRS). She is now developing a new activity and starting her own group.
This fellowship also contributes to networking inside and outside Europe. The fellow is involved in drafting a proposal for a European project (ETN) with the host institute that will be submitted next year. She will also continue to work with previous colleagues who are moving to Belgium and Brasil.
Another goal of this fellowship was to bridge the gap between the plant biologist and the spectroscopist communities. This goal was achieved with great success. In collaboration with scientists at synchrotrons and nuclear microprobes, developments have been made on beamlines to make them suitable for biological samples (cryo-stage at microXAS beamline in the Swiss Light Synchrotron, micro-focused beam at the nuclear microprobe in the Ruhr Univeristät Bochum, important novel software developments), and people working there have been sensitized to biological issues (fragile, inhomogeneous and diluted samples). In particular, this has been highlighted by the RUBION day (annual day of the nuclear microprobe in the Ruhr University Bochum) which was entitled this year “material science meets life science” where Prof. Ute Kraemer was invited to give the keynote lecture (1h30) to the user’s community based on the fellow’s work.
Results obtained during beamtime sessions will permit to publish research articles in journal with high impact factor (one article is now under review in the Nature Plants journal) and will give a large visibility to this work and the combination of both molecular biology and spectroscopy as a powerful tool to answer biological questions and address the role of genes in metal homeostasis. This will contribute to giving Europe a leading position in this field worldwide. Some other new collaborations were started thanks to this fellowship and will be pursued after the end of the contract.
From a scientific point of view, the fellowship permitted to gain a deeper understanding on the role of some genes in metal homeostasis in plants. In particular one of those genes was implied in Pb homeostasis in the hyperaccumulating species A. halleri. This new piece of information will help going toward more efficient phytoremediation technologies to depollute metal contaminated sites. Moreover, the lab is also very interested in the phytomining concept. Phytomining consists in growing hyperaccumulator plants on contaminated sites and being able to recycle the accumulated metal to re-use it for industrial applications. Indeed, starting from June 2015, the patent protecting this technology will fall into the public domain and research will have an important role to improve the efficiency of phytomining. This application is very promising and raises a lot of public attention. Prof. Kraemer’s lab is a key player in that area as proven by the journal, TV and radio teams received in the lab (Süddeutsche Zeitung Magazin, Motherboard online magazine, radio eins rbb Wissenschaft, DRadio Wissen Grünstreifen, Die große Show der Naturwunder on ARD national television; all accessible through http://www.ruhr-uni-bochum.de/pflaphy/Seiten_en/index_e.html(opens in new window)) .
Progress has also been made on the implication of a specific gene in Zn loading into developing seeds. Understanding the mechanisms of metal accumulation in plants will help for the rational design of strategies for the molecular-assisted breeding of crop species with enhanced essential mineral content (biofortification). This issue is of crucial importance since the World Health Organization estimates that two billion people suffer from Zn deficiency, making Zn deficiency one of the leading human nutritional disorder.
Communication was also developed for the public at large with the publication of postcard in the special program “die Welt unter der Lupe” of the Ruhr University Bochum. This initiative led to the publication of a postcard per month presenting a microscopic image and distributed inside the University but also in the centre of the city. Likewise, some images from experiments performed at ESRF were published on the ESRF website under the rubric “Beauty of Science” (http://www.esrf.eu/home/news/beauty-of-science/content-news/beauty-of-science/beauty-of-trichomes.html(opens in new window)) and were also showcased on the lab website (http://www.ruhr-uni-bochum.de/pflaphy/Seiten_en/index_e.html(opens in new window)). The work performed by the fellow was mentioned on an article published in the ESRF News (number 67, July 2014, p 9) and presented to the imaging communities of the ESRF as an invited talk during the ESRF User’s meeting in 2014.
Prof. Ute Kraemer’s lab in general is very active in the communication towards the general public around the topics that represent a question of general interest and has even developed a Twitter account in English to make available latest information (@PlantAdaptation) to a large population.
Finally, every year the lab organises an Open day at the Lehrstuhl für Pflanzenphysiologie to promote science in general and plant science in particular to students at the Ruhr Universität Bochum. It is particularly interesting since people from the lab come from different scientific communities (bioinformatics, spectroscopy, ecology, molecular biology) and different cultures/countries (Brasil, India, Spain, Poland, Egypt, France, Australia,…). Indeed, a survey carried out in the lab showed that this highly international and multidisciplinary team is an asset to attract students.
Very importantly, the fellow has participated in/presented at the FESPB/EPSO conference in Dublin (22-26 June 2014). This allowed her to present her results to a large community of researchers and engage in scientific exchange and networking.
• The address of the project public website, if applicable as well as relevant contact details.
http://www.ecolab.omp.eu/profils/Larue_Camille(opens in new window)
The central objective of this project was to combine molecular genetics approaches with synchrotron-based elemental imaging techniques in the model plant Arabidopsis thaliana for advancing the mechanistic understanding of transition metal handling in plants. This innovative combination of approaches entailed the adaptation of physics techniques to molecular physiology scientific questions in a genetic model plant. Novel scientific insights have been gained that are very difficult to obtain through existing techniques, which make a significant contribution towards enabling the rational design of strategies for the molecular-assisted breeding of crop species with enhanced essential mineral content (bio-fortification), improved yields under minimal fertilizer input (sustainable agriculture), and for the development of technologies for the plant-based clean-up of heavy metal-contaminated soils.
There is a need to develop a comprehensive understanding of the functions of all central molecular components of the metal homeostasis network in plants, including their regulatory pathways, and of how these components function together in a network context. To date, the limited accessibility of techniques for the correct determination of metal localization and speciation are a major bottleneck for our understanding of plant metal homeostasis. Synchrotron-based imaging techniques have been used so far predominantly in a biogeochemical and ecological context using wild type plants. Their systematic application to model plants in a molecular genetics context with the aim to elucidate the functions of single genes or molecular pathways remains a challenge. The proposed project was aimed at making a decisive contribution to closing this gap, by pursuing the specific research goal to advance our understanding of the interface between zinc (Zn) and iron (Fe) homeostasis in the genetic model plant Arabidopsis thaliana.
This project was organized into three objectives. The first one was to take advantage of spectroscopic techniques to shed light on mechanisms involved in metal homeostasis using on-going projects in the host-laboratory. The second objective was to characterize a new mutant identified during a previous mutant screen (European project funded by a Marie Curie IIF fellowship: grant agreement number: 219457, Project acronym: CENTZIN) and understand the role of the mutated gene in Fe and Zn homeostasis. Finally, the last objective was to perform a different screen to identify new mutants involved in Fe homeostasis.
• A summary description of project context and objectives
A number of genes acting in iron and zinc homeostasis of Arabidopsis have been identified and functionally characterised at the molecular level. In previous work, a strong focus has been on membrane transport proteins acting in root metal uptake, vacuolar storage and re-mobilization, and short-and long-distance metal distribution in the plant. An understanding of the roles of the low-molecular-weight metal chelator nicotianamine and membrane transporters of nicotianamine and its metal complexes is emerging. Finally, the function and regulation of ferritin, a chloroplastic iron storage protein, has been elucidated in much detail. Recently published results have highlighted that metal homeostasis defects often manifest themselves in dramatically altered metal distribution between or within tissues or even at the sub-cellular level, which can explain major developmental defects such as male sterility. Furthermore, altered metal speciation is known to have dramatic effects on its intra- and inter-cellular as well as long-distance mobility of metals. However, in many cases methodological difficulties have either prevented or limited the resolution, precision or reliability of metal localization and in planta metal speciation in the past.
Although a number of metal homeostasis factors have been identified and characterized, the pathways involved in their regulation are less well understood and constitute a major current research topic. Only a couple of transcription factors have been identified that contribute to the transcriptional regulation of iron deficiency responses, and, apart from the well-characterized bHLH protein FIT (basic helix-loop-helix protein FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), our understanding of their individual roles and how they integrate into a regulatory network remain sketchy. The reason for this is probably that a number of genes of the regulatory pathways remain unidentified. Thus, the identification of additional genes contributing to iron-dependent regulation is critical for the construction of a comprehensive model of iron homeostasis in the future. Furthermore, it is now clear that iron-dependent regulation cannot be understood in isolation: the transcriptional response to iron deficiency includes increases in transcript levels of genes not acting in Fe acquisition, but in Zn detoxification instead. These Zn detoxification genes are transcriptionally responsive also to excess Zn. This common regulation could either be the result of overlapping or converging molecular regulatory pathways of responses to iron deficiency and excess zinc, or of converging physiological consequences of both nutrient regimes. Because of the lack of selectivity of IRT1 (Iron-Regulated Transporter 1), the major root iron uptake system of A. thaliana, Fe deficiency causes enhanced Zn influx into roots and thus the risk of Zn toxicity. Conversely, exposure to excess Zn causes iron deficiency by interfering with iron movement inside the plant and possibly also through competition during transport steps inside the plant and the formation of metalloproteins. Recent work suggests that all three of the described scenarios are relevant in vivo. To gain further insight into common and distinct regulatory pathways in iron and zinc homeostasis, it was proposed to follow a forward genetic approach combined with physiology and molecular biology, in a powerful combination with advanced imaging techniques to analyze more precisely metal localization and local speciation phenotypes.
One of the innovative aspects of the project was the use of cutting-edge imaging techniques to gain new insights of metal localization and speciation in Arabidopsis thaliana. Up to now, iron and zinc were mainly detected by histochemical techniques. Zinc can be visualized by adding fluorophores such as Zinpyr or Zinquin, and iron by the adjunction of Perls’stain. The resulting fluorescent metal-ligand complexes are then visualized by confocal laser scanning microscopy (CLSM). Quantification of metals in bulk tissues is commonly achieved employing digests of plant tissues (seed, root, stem or leaf) through the technique inductively coupled plasma mass spectrometry (ICP-MS) or atomic emission spectrometry (ICP-AES).
In this project, we went further than these well-known techniques and employed recently established physics techniques based on the interaction between radiation and matter to obtain complementary data on metal partitioning, speciation and local quantification in plants. For this purpose, experiments have been conducted on large instruments such as synchrotrons and nuclear microprobes. Their use in plant biology has been expanding as shown by several recent reviews.
Synchrotron radiation-based micro X-ray fluorescence (µXRF) in scanning mode permits to obtain two-dimensional (2D) elemental distribution maps. The main advantages of this technique are its sensitivity (in the sub-milligram per kilogram range) and the multi-elemental nature of the information collected. Moreover, the spatial resolution is about 1 µm x 1 µm, and new beamlines are under construction with spatial resolution in the nanometer range, much higher than that obtained by other techniques such as laser-ablation ICP-MS (LA-ICP-MS) that is about 10 µm x 10 µm. µXRF resolution criteria permit to study metal partitioning in plants at the cellular scale. For instance, µXRF has been used with success to determine nutrient distribution in the barley grain. Distribution maps show that Fe, Mn, Cu, Zn and K were mainly present in the envelope of the seed. Another study has demonstrated that TiO2 nanoparticles were taken up by wheat plantlets and transferred to the shoot. µXRF based on synchrotron radiation has thus proven its efficiency to image metallic elements in plants.
Performing µXRF on synchrotron beamlines often permits to couple a micro X-ray absorption spectroscopy (µXAS) analysis in situ (LUCIA, SOLEIL, France or ID21, ESRF, France for instance). µXAS is a spatially resolved technique, informative on metal speciation (oxidation state and binding environment of the element of interest). In addition to elemental distribution, the speciation of metals is of vital importance in understanding their metabolism in plants as well as the toxicity of contaminants and the availability of micronutrients. Moreover, with recent development of cryo-stages on synchrotron beamlines, artefacts such as speciation changes or metal remobilization can now be avoided. µXAS has mainly been used on bulk samples (freeze-dried plant tissues, mixed and pressed as a pellet), but a few research teams have already performed the analysis in situ on µXRF maps. The combination of synchrotron radiation-based µXRF and µXAS has thus been used in plant biology by only a few research groups in the world, but with promising results. For instance, in wheat plantlets exposed to TiO2, µXRF maps of roots have shown that Ti was internalized in roots and found inside the vascular cylinder. By using µXAS analysis on Ti hot spots located on µXRF maps, the authors demonstrated that Ti speciation inside roots, and later inside leaves, is not modified.
Synchrotron based-techniques are very powerful to image metals in plant tissue at a sub-micrometer spatial resolution, but they are not quantitative. To have access to this information, we analyzed plant sections by microparticle-induced X-ray emission (µPIXE) coupled to Rutherford backscattering spectroscopy (RBS) on nuclear microprobes. This technique permits local elemental quantification within plant organs and even at the sub-cellular scale: in the nucleus or in the call wall for instance. It is thus much more spatially resolved than classical ICP. For instance, a µPIXE/RBS study was performed on Hybanthus floribundus exposed to Ni. ICP-AES measurements show that leaves contain 7.8 g Ni kg-1 dry matter (DM). Ni concentration in the whole leaf section measured by µPIXE/RBS gave the same result: 7.8 g Ni kg-1 DM. Actually, this global concentration averages over vast differences ranging from 1.0 g Ni kg-1 DM in the adaxial epidermis to 8.6 g Ni kg-1 DM in the vascular tissue. The µPIXE/RBS technique is thus very well suited to measure local concentrations of elements. Moreover, during nuclear microprobe experiments, a spectrum of the whole periodic table (from sodium to uranium) is recorded, allowing the extraction of images for each element and thus co-localization analyses.
In summary, the proposed project entailed a multidisciplinary approach combining physics techniques with molecular biology and genetics for an insightful, innovative and original approach in plant biology which is powerful to understand molecular mechanisms of metal homeostasis.
Objective A
The central goal of the project was to implement comparative metal localization, speciation and local quantification in wild type and mutant Arabidopsis plants for the elucidation of the functions of individual genes. According to beamtime allocation specific questions related to the topic of this project have been addressed in a set of characterized genotypes (see results).
Recently developed techniques, namely µXRF, µXAS, µPIXE/RBS and µFTIR have been employed to obtain new and original data to complete first data obtained on studied mutants. For all these techniques, sample preparation is a crucial step and has been deeply optimized. Indeed, it can lead to artefacts such as metal remobilization or changes in metal speciation. The best way to preserve elemental partitioning and speciation is to keep the sample in cryo-conditions during the whole process. To determine metal speciation, reference compounds have been prepared as pellets in cryo-conditions and analysed (Zn-citrate, Zn-glutathione, Zn-His2, Fe-citrate, Fe-glutathione, Fe-His2 in the form of Fe(II) and Fe(III), Fe-ferritin). µXAS data have been fitted using Athena software by achieving linear combination fits with these reference compounds. µXRF data analyses have been achieved using PyMca software to obtain element maps. Absolute concentrations were obtained by processing the data through SIMNRA and Gupix softwares allowing comparing metal concentrations in different tissues and/or genotypes.
Objective B
To advance the number of known and characterized gene functions influencing the regulation of zinc and iron homeostasis, a mutant identified earlier, which fails to increase ZIF1promoter-GUS activity in response to growth in excess Zn, have been studied. This mutant has been preliminarily characterized to confirm the regulation defect and demonstrate that root and shoot Zn accumulation are altered. The phenotypic characterization of the mutant encompassed ZIF1promoter-GUS activity analyses of seedlings grown on different metal media, metal quantification in root and shoot tissues, growth assays (root length, fresh foliar and root biomasses) and photosynthesis study under a range of Zn and Fe metal supplies. Experiments have also been carried out to try to phenotypically rescue the plant phenotype using supplements on both soil and agar substrates.
Objective C
We decided to perform the screening on an EMS-mutagenized population of an iron homeostasis mutant to advance our understanding of Fe homeostasis. This mutant is being extensively studied in the host laboratory. The first step of this objective was to optimize the screening process by developing the most efficient medium to detect abnormal phenotypes. Several tests were performed both on differently composed agar media and on different type of soils with modified pH.
Then a batch of seeds of a common mutant genotype was further EMS-mutagenized and sown on soil. M2 seeds were pooled and M2 plants are presently being screened in the host laboratory. Putative mutant individuals will be back-crossed, and selfed to obtain the M3 generation.
• A description of the main S&T results/foregrounds
Objective A: Imaging of well-known mutants
The main goal of this part was to collaborate with other researchers and to take advantage of spectroscopic techniques to advance their knowledge on their research projects. This part of the project has been very beneficial in terms of networking between the plant biologist community and the spectroscopic community (Table 1). This work has also led to the adaptation of some instruments (cryo set-up on MicroXAS beamline, SLS, Switzerland & micro focused beam on RUBION nuclear microprobe, Germany) towards biological questions. It has also been very valuable for the fellow. The fellowship permitted her to deepen her knowledge of spectroscopic techniques and sample preparation as well as exploring different and complementary questions on the topic of metal homeostasis in plants. Additionally, it was also the opportunity of meeting new actors of both communities which represent potential collaborations for the future.
Finally, it is worth mentioning that this objective has been much further developed than initially foreseen. Indeed, getting beamtime is not an automatic process; however, all our proposals have been successful, leading to the granting of a high number of beamtime shifts, which required preparation, presence time and time for data evaluation. Thus, the amount of imaging data obtained in this project has been far larger than expected, thus enabling the completion of publishable imaging datasets for far more publications than we could have hoped for at the outset.
All the beamtime obtained through proposal acceptance or collaborations has been shared between my own project (Objective B) and collaborators’ projects. The order of priority between samples first presented in the ZISPLOC project has evolved according to new results obtained in the meantime. Several shifts of beamtime had also been allocated to sample preparation optimization, with Arabidopsis seedlings being particularly small and fragile.
Results obtained here are confidential (only described in the last periodic report in section 3.2.2. work progress and achievements during the period) and will lead to the publication of 5 articles in high impact factor journals that will associate both communities (researchers from large instruments and plant biologists). This objective of the project will lead to the publication of 5 papers as co-authors in high impact factor journals (one is under review in Nature Plants, and one will be soon submitted to PNAS).
Objective B: Characterization of a new mutant
A batch of ZIF1pGUS seeds (background Columbia) was EMS mutagenized. A screening was performed on 4800 M2 seeds by germinating them onto 1xHoaglands (1xHG) with 30 µM ZnSO4. 14 d old seedlings were assessed for chlorosis (i.e. Zn sensitivity) and other phenotypes (such as root growth or leaf/cotyledon morphology). A single cotyledon was GUS-stained. Mutants were selected for chlorosis and lack of the appropriate transcriptional response (i.e. induction of ZIF1 and thus blue staining). From those mutants, one was selected and called roz1 (regulator of ZIF1) and three alleles have been discovered so far and confirmed by crossing:
- roz1-1: A1.116
- roz1-2: D3.115 and possibly D3.333
- roz1-3: E1.417
- Elemental distribution and metal speciation
Elemental distribution and speciation were studied on the roz1-2 mutant by µXRF and µXANES. Local concentration was determined by µPIXE on the roz1-3 mutant.
An experiment was performed at the Zn-edge at MicroXAS beamline (Swiss Light Source) but the data set is not complete due to time spent to optimize the cryogenic set-up. Nevertheless, partial results obtained show that Zn in roots was mainly detected in the vasculature and in the cell walls. In leaves, Zn was seen in trichomes as well as in hot spots throughout leaf cross-sections and especially in the epidermis for the mutant exposed to high concentrations of Zn. These results were also confirmed by µPIXE analysis on the Slovenian nuclear microprobe (in collaboration with Katharina Vogel-Mikus). No major differences were detected between WT and mutants (Figure 1).
Zn was not enough concentrated in tissues cultivated under basal conditions to be detected by this technique (with the actual setup) except in the tips of the leaves of the WT. In high Zn conditions, Zn was detected in the base of the trichomes (adaxial side) and as small dots on the abaxial side (stomata?). In the cross-sections it was observed as small dots in the epidermis (stomatal cells?). Same distribution patterns were shown on cross-sections in low Fe condition.
Seedlings were also analyzed at the Fe-K edge on ID21 beamline (European Synchrotron Radiation Facility). On this beamline, Zn is not reachable but Fe and lighter elements such as P, S, Cl (and Ca, K) that were not analyzable on MicroXAS (analysis chamber not under vacuum loss of light element signal) were accessible.
In whole roots, the main difference between WT and mutant is the accumulation of Mn in the quiescent center for the mutant whereas it is more concentrated in the root cap for the WT. When analyzing root cross-sections, differences were detected in Fe distribution as well; it was adsorbed on root epidermis for both genotypes but in the mutant Fe was also detected in the cell walls of parenchymal cells.
In whole leaves, Fe, Mn and P distributions seem to be altered in the mutant: these elements are more homogeneously distributed in the leaf whereas they are more concentrated in the trichomes in the WT. Analysis of leaf cross-sections demonstrated that Fe was mainly localized in cell walls and vasculature. It was also detected in the base of the trichome for the WT but not for the mutant (as already shown by analysis of whole leaves).
Fe speciation was analyzed in situ for roots and leaves. Several spectra were acquired per point of interest to increase the signal/noise ratio. Several points were analyzed per tissue and averaged in Table 2. Differences in Fe distribution between WT and mutant were detected mainly in the trichome and in cell walls in roots. However this difference in distribution does not seem to be linked with a different speciation. Fe(III) seems to be more predominant in the mutant (especially in leaf surface and root epidermis) than in the WT.
- Phenotypic characterization
Mutant seeds were grown on soil together with the WT. It appears that all alleles of roz1 seedlings experienced a delayed development (Figure 2), with mutants having rosette of few leaves when the WT was giving seeds.
The three alleles of roz1 were also germinated on Hoagland plates for 7 days and then transferred on plates on different media for 7 more days: Hoagland (1 µM Zn, 5 µM Fe), +Zn (30 µM ZnSO4) and -Fe (washed agar, no Fe). After 15 days, GUS staining wad performed on the first true leaves, fresh biomass was recorded, photosynthetic pigment concentrations were determined by spectrophotometry and elemental concentrations were assessed by ICP-AES.
As seen on Figure 3, roz1 mutants seemed to grow smaller and paler than the WT in all conditions even if the difference is less evident in –Fe condition.
The lack of induction of ZIF1pGUS was checked after exposure for 7 days in high Zn condition (Figure 4). Results were in agreement with the previously obtained ones: in high Zn in the WT the staining expanded over the vasculature but not in the mutant.
Determination of the fresh root and shoot biomasses confirmed those observations.
Likewise, chlorophyll a, chlorophyll b and carotenoid pigment concentrations in the leaves of the three (potentially four) alleles of roz1 mutants were decreased as compared with the WT. Again the –Fe condition resulted in comparatively sick seedlings for the WT and the mutants.
Elemental composition showed that roz mutants had higher P concentration in leaves, lower Mg in roots and lower K in both root and shoot. Again, differences between wild type and mutants were less pronounced upon cultivation under –Fe conditions. Impaired P metabolism has already been evidenced by the previous µXRF analysis. In basal condition, ICP results also confirmed the altered Mn partitioning seen by µXRF as well as Fe behaviour in roots. Moreover, in all alleles of roz1 mutant over-accumulated Zn in both root and shoot.
Nucleic DNA was extracted to identify the exact location of the mutation in the roz1-1 mutant. Results are not yet available.
Different phenotypic rescue experiments were pursued (adding Fe or Zn-rich solution or on high pH soil) but none of these were conclusive.
Objective C: New screening
Towards objective C, screening conditions have been optimized to permit to faster the screening process afterwards. Several conditions have been tested comparing wild-type and a previously characterized iron-deficient mutant to obtain an efficient Fe-depleted medium: on plates (by addition of different concentrations of ferrozine) or on soil (by increasing the soil pH).
Plate conditions were too stringent for the irt1 mutant. A protocol was established to screen for Fe deficiency symptoms on soil (Figure 5). The intermediate pH level was chosen since it permitted to maximize differences between the WT and the irt1 phenotype with no dead plants. Higher pH was obtained through the addition of 10 g CaCO3, 5 g CaO and 5 g K2HPO4 for 500 g of soil.
Seeds of irt1 were EMS-mutagenized and sown on normal minitray soil for seed production and watered with sequestrene to avoid an overly dramatic Fe deficiency. M2 seeds were collected and are being cultivated on modified minitray soil for a first screen. Mutagenesis and screening were performed together with Dr Maria Bernal, who will pursue the project in the future.
• The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
First of all, this fellowship has been a unique opportunity for the fellow to broaden her knowledge and increase her employability. Indeed, the fellow terminated the contract two months before its expiration because she got a permanent position as scientist at the French centre for scientific research (CNRS). She is now developing a new activity and starting her own group.
This fellowship also contributes to networking inside and outside Europe. The fellow is involved in drafting a proposal for a European project (ETN) with the host institute that will be submitted next year. She will also continue to work with previous colleagues who are moving to Belgium and Brasil.
Another goal of this fellowship was to bridge the gap between the plant biologist and the spectroscopist communities. This goal was achieved with great success. In collaboration with scientists at synchrotrons and nuclear microprobes, developments have been made on beamlines to make them suitable for biological samples (cryo-stage at microXAS beamline in the Swiss Light Synchrotron, micro-focused beam at the nuclear microprobe in the Ruhr Univeristät Bochum, important novel software developments), and people working there have been sensitized to biological issues (fragile, inhomogeneous and diluted samples). In particular, this has been highlighted by the RUBION day (annual day of the nuclear microprobe in the Ruhr University Bochum) which was entitled this year “material science meets life science” where Prof. Ute Kraemer was invited to give the keynote lecture (1h30) to the user’s community based on the fellow’s work.
Results obtained during beamtime sessions will permit to publish research articles in journal with high impact factor (one article is now under review in the Nature Plants journal) and will give a large visibility to this work and the combination of both molecular biology and spectroscopy as a powerful tool to answer biological questions and address the role of genes in metal homeostasis. This will contribute to giving Europe a leading position in this field worldwide. Some other new collaborations were started thanks to this fellowship and will be pursued after the end of the contract.
From a scientific point of view, the fellowship permitted to gain a deeper understanding on the role of some genes in metal homeostasis in plants. In particular one of those genes was implied in Pb homeostasis in the hyperaccumulating species A. halleri. This new piece of information will help going toward more efficient phytoremediation technologies to depollute metal contaminated sites. Moreover, the lab is also very interested in the phytomining concept. Phytomining consists in growing hyperaccumulator plants on contaminated sites and being able to recycle the accumulated metal to re-use it for industrial applications. Indeed, starting from June 2015, the patent protecting this technology will fall into the public domain and research will have an important role to improve the efficiency of phytomining. This application is very promising and raises a lot of public attention. Prof. Kraemer’s lab is a key player in that area as proven by the journal, TV and radio teams received in the lab (Süddeutsche Zeitung Magazin, Motherboard online magazine, radio eins rbb Wissenschaft, DRadio Wissen Grünstreifen, Die große Show der Naturwunder on ARD national television; all accessible through http://www.ruhr-uni-bochum.de/pflaphy/Seiten_en/index_e.html(opens in new window)) .
Progress has also been made on the implication of a specific gene in Zn loading into developing seeds. Understanding the mechanisms of metal accumulation in plants will help for the rational design of strategies for the molecular-assisted breeding of crop species with enhanced essential mineral content (biofortification). This issue is of crucial importance since the World Health Organization estimates that two billion people suffer from Zn deficiency, making Zn deficiency one of the leading human nutritional disorder.
Communication was also developed for the public at large with the publication of postcard in the special program “die Welt unter der Lupe” of the Ruhr University Bochum. This initiative led to the publication of a postcard per month presenting a microscopic image and distributed inside the University but also in the centre of the city. Likewise, some images from experiments performed at ESRF were published on the ESRF website under the rubric “Beauty of Science” (http://www.esrf.eu/home/news/beauty-of-science/content-news/beauty-of-science/beauty-of-trichomes.html(opens in new window)) and were also showcased on the lab website (http://www.ruhr-uni-bochum.de/pflaphy/Seiten_en/index_e.html(opens in new window)). The work performed by the fellow was mentioned on an article published in the ESRF News (number 67, July 2014, p 9) and presented to the imaging communities of the ESRF as an invited talk during the ESRF User’s meeting in 2014.
Prof. Ute Kraemer’s lab in general is very active in the communication towards the general public around the topics that represent a question of general interest and has even developed a Twitter account in English to make available latest information (@PlantAdaptation) to a large population.
Finally, every year the lab organises an Open day at the Lehrstuhl für Pflanzenphysiologie to promote science in general and plant science in particular to students at the Ruhr Universität Bochum. It is particularly interesting since people from the lab come from different scientific communities (bioinformatics, spectroscopy, ecology, molecular biology) and different cultures/countries (Brasil, India, Spain, Poland, Egypt, France, Australia,…). Indeed, a survey carried out in the lab showed that this highly international and multidisciplinary team is an asset to attract students.
Very importantly, the fellow has participated in/presented at the FESPB/EPSO conference in Dublin (22-26 June 2014). This allowed her to present her results to a large community of researchers and engage in scientific exchange and networking.
• The address of the project public website, if applicable as well as relevant contact details.
http://www.ecolab.omp.eu/profils/Larue_Camille(opens in new window)
