Research

 

Three research themes are developed in the laboratory of Plant Physiology and Molecular Genetics, aiming at better understand the mechanisms of nutrient homeostasis or detoxification of non essential elements. We study  responses of plants to trace metallic elements excess (I), magnesium homeostasis (II) and nitrogen use efficiency (III).

I. Responses of plant to trace metallic elements excess

  • A. Cadmium accumulation and tolerance in plants
Trace metallic elements like cadmium, mercury and lead are not essential and can be toxic for living organisms even at low concentrations. Cadmium is one of the most important environmental pollutants and one of the most toxic heavy metals. Cadmium accumulation in soils originates from atmospheric deposition, application of sewage sludge, phosphate fertilisers, and industrial activities (cadmium is a common plastic stabiliser). Cadmium toxicity is thought to be due to its ability to replace Ca++ or Zn++ in the proteins as well as high reactivity with thiol groups. (reviewed in Verbruggen et al. 2009 a Current opinion in Plant Biology, 12:364-372)

Accumulation of cadmium by plants is a major route for entry in the food chain. A better understanding of the underlying mechanisms of metal uptake and processing by plants can help to devise remedy to avoid accumulation of toxic heavy metals in crops.  On the other hand, the use of plants represents a promising strategy to decontaminate or stabilise polluted soils. This strategy is called phytoremediation. Yet, its development is limited by our current knowledge of the biochemical, physiological and ecological processes involved in metal tolerance and accumulation in plants.

Plants differ profoundly in their sensitivity towards metals and the extent to which the metal ions are taken up or excluded from cells, and further distributed in plants. Elevated concentrations of potentially toxic trace metals in the soil represent a selection force that lead to the evolution of locally adapted populations. Two strategies of adaptation have evolved namely the exclusion and the hyperaccumulation. The last strategy consists in accumulation in the aerial parts, for example above 100 mg kg-1 Cd per shoot dry weight, or above 1000 mg kg-1 Ni, or above 10.000 mg kg-1 Zn. Those plants are named hyperaccumulators and represent fascinating study cases to understand adaptation to extreme environments as well as mechanisms of tolerance and accumulation..
Our research aims at a better understanding of processes underpinning cadmium tolerance and accumulation in hyperaccumulators. These processes are poorly understood both at the cellular and at the organismic levels. Main steps of the tolerance are transport, chelation, trafficking and sequestration processes. (Verbruggen et al.2009)

Fig.1 Overview of our current understanding of adaptations to hyperaccumulate metals, in particular Zn. REF Verbruggen et al. 2009 b New Phytologist

Our model species are Arabidopsis halleri and Noccaea caerulescens (previously named Thlaspi caerulescens), are two Cd hyperaccumulators. Both are close relatives of Arabidopsis thaliana for wich molecular tools have been largely developped. Furthermore this project uses crosses between metallicolous and non metallicolous populations as well as a collection of metallicolous populations with contrasting tolerance and metal accumulation capacities.

Several strategies are being developed in our laboratory to identify genes involved in Cd tolerance and hyperaccumulation.

Screening of cadmium resistant yeasts

Yeast have been transformed with cDNA libraries of Thlaspi caerulescens and Arabidopsis halleri. Several plant cDNAs enhancing cadmium tolerance in yeast have been identified.
Growth of S. cerevisiae (strain BY474)

Dilution of S cerevisiae BY474, transformed with empty (pYX212) or recombinant plasmid, spotted onto growth medium containing 20 µM Cd. 3 clones from the Cd screening are presented. Several Thlaspi Caerulescens genes, TcHMA4, TcMT1, TcMT2, TcMT3, identified in the screen have been further analyzed PhD thesis of Lucia Rubio.

Bernard et al. 2004, Roosens et al. 2004, Roosens et al. 2005, Meyer et al. 2011

 

Comparison of Thlaspi caerulescens populations

Populations originating from heavy metal polluted or non polluted soils, have been studied in collaboration with Prof. A. Smith (University of Oxford). (Roosens et al. 2003)

Populations with contrasting Cd tolerance and accumulation capacities are being further studied.

Three populations originated from St Félix-de-Pallière, Puente Basadre and Prayon (from left to right) grown on high Cd levels.

Study of Cd hyperaccumulation in Arabidopsis halleri

Crosses have been made between A.halleri (Cd and Zn tolerant and hyperaccumulator) and A.lyrata (non tolerant and non hyperaccumulator) (P. Saumitou-Laprade, Lille). A F1 has been back-crossed with A. lyrata. The back-cross progeny has been characterised for Cd tolerance and accumulation and three quantitative trait loci explaining more than 80% of the Cd tolerance character have been identified. (Bert et al. 2003, Willems et al. 2007,  Courbot et al. 2007 )

The major QTL for Cd tolerance co-localizes is the HMA4 a gene encoding a heavy metal pump, involved in the cellular efflux of Zn++ , Cd++ and their translocation from the root to the shoot.

A F2 population was also characterized. QTL Analysis for Cd accumulation allowed the identification of one locus explaining 20% of the Cd accumulation character and many loci with small additional effects ( Willems et al. 2010). The same population was used to study Zn accumulation (GEPV, University of Lille) (Frérot et al. 2010).

cDNA-AFLP analysis and characterization of genes highly expressed in Cd tolerant genotypes

cDNA-AFLP analysis has been applied on extreme genotypes of the BC1 progeny for Cd tolerance (Craciun et al.2006)
“Transcript-derived-DNA-fragments” visualised in the cDNA-AFLP that correlate with Cd tolerance are studied. The assignment of a function is greatly facilitated by the high homology (95% identity at the DNA level are predicted) with Arabidopsis thaliana.

Click on the picture to enlarge transcript profile in Cd sensible and resistant plants

Overexpression of selected genes in Arabidopsis thaliana

Selected genes are overexpressed in Arabidopsis thaliana, which is the easiest transformable plant since transformation can be done by flower dipping. As A. thaliana is very sensitive to cadmium, effect of gene overexpression on Cd tolerance can be easily measured. If enhanced cadmium accumulation and/or tolerance is observed, application will be foreseen in cultivated species.

Overexpression of selected Thlaspi caerulescens cDNAs in Nicotiana plumbaginifolia

Selected genes are overexpressed in Nicotiana plumbaginifolia, which is a high biomass easy transformable species. Better growth on contamined soils of Brussels could be observed for some lines (Alban Heudiard).

  • B. COPPER ACCUMULATION AND TOLERANCE IN PLANTS

A new research has been recently developed on plant responses to Cu excess.
(PhD thesis of Alfred Cubaka-Kagale, François Chipeng, Hélène Lequeux)

We have started a research on copper tolerance within the PIC project “Remedlu” in Katanga (RD Congo) coordinated by Pierre Meerts. The so-called copper arc in Katanga and Zambia (Central Africa) is one of the most mineralized regions in the world.  In particular copper in soil can be as high as 50.000 ppm while normal soils contain around 10 ppm – 25 ppm. The flora is there unique, characterized by a high degree of endemism.  We have studied Haumaniastrum katangense, called the copper flower, which shows extreme tolerance to copper and high accumulation capacity (PhD thesis of François Chipeng, Chipeng et al. 2009). We have demonstrated a higher requirement for copper for normal growth. Surplus copper was also required for cultivating H. katangense in sterile conditions, suggesting that Cu excess may be necessary for needs other than pathogen defence. The endophytic community of H. katangense and Crepidorhopalon tenuis, an other cuprophyte of the copper arc, was studied in collaboration with the Institut de Recherches Microbiologiques JMW/Laboratoire de Microbiologie and Max Mergeay (CEN/SCK), as a first step to evaluate their potential contribution to plant adaptation to copper excess (PhD thesis of Alfred Cubaka ; Cubaka et al. 2010). Bacteria with outstanding resistance to copper have been identified in the endophytes of both cuprophytes and in their rhizosphere (manuscript in preparation).

In parallel to the work on organisms adapted to extreme copper concentrations, the Arabidopsis was used as a model to understand the plant response to copper excess. Physiological responses have been published with a focus on the root architecture (Lequeux et al. 2010).  A screen of several mutagenized seed banks on high copper was performed. A copper sensitive mutant (cop29) was further investigated and is affected in an uncharacterized gene with a putative function in cell cycle (PhD thesis of Hélène Lequeux). The role of the COP29 gene is currently under investigation.

II. CONTRIBUTION TO THE STUDY OF MAGNESIUM DEFICIENCY IN PLANTS:

  • Major elements nutrition
Understanding how plants regulate the absorption, transport and cycling of essential ions can have major applications for the environment and human health. By ameliorating those processes, it is possible to develop crops that grow efficiently on nutrient-poor soils (reduced fertilizers input) and crop products with added nutritional value (biofortification). Two themes are focused on magnesium homeostasis and the impact of nitrogen availability on the root system in the model species Arabidopsis thaliana. On the one hand, the aim is to understand how plants acquire and regulate Mg content internally. Physiological and transcriptomic approaches have identified global transport of sugars and the circadian clock as early targets of Mg deficiency. On the other hand, the mechanisms that govern the plasticity of the root system in response to nitrate is studied (low concentrations stimulate the development of lateral roots, while consistent high levels have an inhibitory effect). We use direct and genetic approaches to the exploitation of natural variability in populations of Arabidopsis to identify genes that regulate root architecture. For those two lines of research, knowledge transfer is considered to agronomic species of the genus Brassica (cabbage, oilseed rape ...).

  • Magnesium
Magnesium has become a priority metal because in addition to being an essential element in plant cell biology, it is the 4th common cation in the human body and half of its dietary intake is of plant origin. Hypomagnesaemia is recognized as a global clinical problem.

  • Physiological characterization of Mg deficiency in plants
Magnesium deficiency in plants is a widespread problem, affecting productivity and quality in agriculture, horticulture and forestry. In arable land, the incidence of Mg deficiency symptoms is increasing, due to intensive harvesting, amplified rotations per site and unbalanced application of fertilizers lacking in secondary elements (e.g. calcium, magnesium).

The targets of Mg deficiency on the photosynthetic apparatus were studied in sugar beet: the two photosystems showed sharply contrasting responses during the acclimation process to low Mg supply (Planta 220: 344-355). Photosystem II was down-regulated through a loss of antenna, whereas photosystem I was most probably down-regulated through a more important loss of reaction centres. As sugars accumulated in leaves before any loss of photosynthetic activity, we also examined the impact of low Mg status on sugar partitioning. We showed that sucrose export was an early target of Mg deficiency in Arabidopsis (J Exp Bot 56: 2153-2161) and sugar beet (Planta 220: 441-449). Interesting findings were that in response to low Mg level, a sucrose/H+ symporter gene was induced, however without further enhancement of the protein level and the sucrose loading into the phloem. Finally, we also showed that deficiencies of other major elements (N, P, K) can be used to manipulate sugar export and to alter biomass allocation (Trends Plant Sci 11: 610-617; 12: 532-533).

  • Magnesium homeostasis in plants
Two approaches are used to expand knowledge on the mechanisms underlying Mg homeostasis in Arabidopsis, a domain that was relatively unexplored to that point. The research focus is on (i) the exploitation of ionomic variation and (ii) the identification of transcriptome changes related to Mg limitation.

(i) The first approach uses the variation of Mg concentration in Arabidopsis mutants and natural accessions as a source of diversity to find new genes and alleles controlling root and shoot Mg homeostasis.

(ii) Because the knowledge about the impact of Mg deficiency on physiological processes was scarce, we proceeded to transcriptome analyses to provide non-biased hints about targets of Mg starvation. We published a thorough description of the transcriptomic responses (within hours or days before the outbreak of visual symptoms) of Mg deprivation (New Phytol 187: 119-131; 187: 132-144). Mg starvation triggered a temporal difference in the early transcriptomic response in organs, with a higher number of genes being differentially regulated in the root after 8h and in young mature leaves after 28h. Unlike other mineral deficiencies, putative Mg transporters genes were not induced (post-transcriptional induction cannot be excluded). Interestingly, the rhythmic expression of circadian clock component genes was altered in roots, while abscisic acid signalling was triggered in leaves. Delayed responses after 7 days of Mg starvation impacted on the abundance of 33 % of the transcripts in leaves and less than 2% of the transcripts in roots. The analysis confirmed the visual observation that Mg starvation affected the shoot more than the root in Arabidopsis. Re-supply of Mg restored initial patterns of gene expression for one-fifth of the transcripts in the leaves and half in the roots. Further study of the circadian rhythm confirmed the altered amplitude of clock genes expression while the phase was still maintained. Higher expression was observed of genes in the ethylene biosynthetic pathway, in the reactive oxygen species detoxification and in the photoprotection of the photosynthetic apparatus. Higher production of ethylene and altered levels of antioxidants supported those transcriptomic data.

We also identified several genes being differentially regulated by -Mg which are involved in the detoxification process of nonessential heavy metals such as cadmium. Therefore, we further tested the impact of a low Mg status on Cd sensitivity in plants. Interestingly, an Mg starvation pre-treatment alleviated the bleaching of young leaves caused by Cd toxicity (New Phytol 187: 132-144). The protective effect was not explained by a modified Cd translocation to the Mg-deficient shoots. It could be due to an increase of the anti-oxidative capacity, a protection of the chloroplast against Cd-induced injuries, a modification of Fe homeostasis to prevent Cd toxicity and/or a cytosolic efflux transport or vacuolar storage of Cd. The functional characterization of genes potentially involved in the tolerance phenotype is currently being undertaken.

Effects of Mg deficiency on photosystems

Visual symptoms of Mg deficiency in sugar beet. Mg deficiency appears first on the most recently developped leaves as chlorosis between the veins, which remind green. Brown spottings and necroses appear when deficiency is severe or under high light intensity.

The behaviour of PSII and PSI was assessed using direct and modulated fluorescence measurement, near-infrared absorbance changes and 77K fluorescence emission spectroscopy. An early effect on the maximum PSI oxidation rate could be identified before any decrease in the rate of PSII maximum quantum yield of primary photochemistry, PSII electron transport or any chlorophyll degradation. Also, a decrease in the amplitude fluorescence emission peak at 735 nm has been observed, suggesting an early effect of Mg deficiency on PSI. Concomitantly, sucrose accumulates in source leaves upon Mg deficiency as an early response. High sucrose levels are known to down-regulate the chloroplast electron chain transport. The delay of photosynthesis regulation in Mg-deficient plants may provoke an unbalance between light and dark reactions, so inducing oxidative stress and further leading to necrogenesis on leaves.

Effects of Mg deficiency on sucrose partitioning

Sucrose pathway from production to consumption and storage organs. In sugar beet, sucrose loading from the apoplasm to companion cell is operated trough sucrose/H+ symporter.

Impact of magnesium deficiency on plant metabolism is ill known. The adaptation mechanisms of the primary photochemical apparatus to the lack of magnesium is not the first target of magnesium deficiency. Although Mg is found at the centre of the tetrapyrol ring, the lowering in chlorophyll, as a result of the lack of this element could only be established after a decrease in assimilation rate had already occured. A major effect of magnesium deficiency seems to be inhibition of sucrose translocation. More recent cellular targets have been recently identified by a global transcriptonics studied (Hermans et al. a,b).

Fig. from Hermans et al, 2006

III. NITROGEN USE EFFICIENCY:

 

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