Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress.

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.

Contrasting cadmium resistance strategies in two metallicolous populations of Arabidopsis halleri.

While cadmium (Cd) tolerance is a constitutive trait in the Arabidopsis halleri species, Cd accumulation is highly variable. Recent adaptation to anthropogenic metal stress has occurred independently within the genetic units of A. halleri and the evolution of different mechanisms involved in Cd tolerance and accumulation has been suggested. To gain a better understanding of the mechanisms underlying Cd tolerance and accumulation in A. halleri, ionomic inductively coupled plasma mass spectrometry (ICP-MS), transcriptomic (RNA sequencing) and metabolomic (high-performance liquid chromatography-mass spectrometry) profiles were analysed in two A. halleri metallicolous populations from different genetic units (PL22 from Poland and I16 from Italy). The PL22 and I16 populations were both hypertolerant to Cd, but PL22 hyperaccumulated Cd while I16 behaved as an excluder both in situ and when grown hydroponically. The observed hyperaccumulator vs excluder behaviours were paralleled by large differences in the expression profiles of transporter genes. Flavonoid-related transcripts and metabolites were strikingly more abundant in PL22 than in I16 shoots. The role of novel A. halleri candidate genes possibly involved in Cd hyperaccumulation or exclusion was supported by the study of corresponding A. thaliana knockout mutants. Taken together, our results are suggestive of the evolution of divergent strategies for Cd uptake, transport and detoxification in different genetic units of A. halleri.

Potential preadaptation to anthropogenic pollution: evidence from a common quantitative trait locus for zinc and cadmium tolerance in metallicolous and nonmetallicolous accessions of Arabidopsis halleri.

As a drastic environmental change, metal pollution may promote the rapid evolution of genetic adaptations contributing to metal tolerance. In Arabidopsis halleri, genetic bases of zinc (Zn) and cadmium (Cd) tolerance have been uncovered only in a metallicolous accession, although tolerance is species-wide. The genetic determinants of Zn and Cd tolerance in a nonmetallicolous accession were thus investigated for the first time. The genetic architecture of tolerance was investigated in a nonmetallicolous population (SK2) by using first backcross progeny obtained from crosses between SK2 and Arabidopsis lyrata petraea, a nonmetallophyte species. Only one significant and common quantitative trait locus (QTL) region was identified explaining 22.6% and 31.2% of the phenotypic variation for Zn and Cd tolerance, respectively. This QTL co-localized with HEAVY METAL ATPASE 4 (AhHMA4), which was previously validated as a determinant of Zn and Cd tolerance in a metallicolous accession. Triplication and high expression of HMA4 were confirmed in SK2. In contrast, gene duplication and high expression of METAL TOLERANT PROTEIN 1A (MTP1A), which was previously associated with Zn tolerance in a metallicolous accession, were not observed in SK2. Overall, the results support the role of HMA4 in tolerance capacities of A. halleri that may have pre-existed in nonmetallicolous populations before colonization of metal-polluted habitats. Preadaptation to metal-contaminated sites is thus discussed.

Transcriptomic analysis supports the role of CATION EXCHANGER 1 in cellular homeostasis and oxidative stress limitation during cadmium stress.

Investigation of genetic determinants of Cd tolerance in the Zn/Cd hyperaccumulator Arabidopsis halleri allowed the identification of the vacuolar Ca(2+)/H(+) exchanger encoding CAX1 gene. CAX1 was proposed to interfere with the positive feedback loop between Reactive Oxygen Species (ROS) production and Cd-induced cytosolic Ca(2+) spikes, especially at low external Ca(2+) supply. In this study expression of genes involved in ROS homeostasis, cell wall composition, apoplastic pH regulation and Ca(2+) homeostasis were monitored in Arabidopsis thaliana wild-type and cax1-1 knock-out mutant and in Arabidopsis halleri wild-type exposed to cadmium or in control conditions. Clustering the outputs of the expression analysis in a gene co-expression network revealed that CAX1 and genes involved in Ca(2+) cellular homeostasis, apoplastic pH and oxidative stress response were highly correlated in A. thaliana, but not in A. halleri. Many of the studied genes were already highly expressed in A. halleri and/or their expression was not modified by exposure to Cd. The results further supported the role of CAX1 in the regulation of cytosolic ROS accumulation as well as the existence of different cell wall modifications strategies in response to Cd in Arabidopsis thaliana and halleri.

A Comparative Study of Ethylene Emanation upon Nitrogen Deficiency in Natural Accessions of Arabidopsis thaliana.

An original approach to develop sustainable agriculture with less nitrogen fertilizer inputs is to tackle the cross-talk between nitrogen nutrition and plant growth regulators. In particular the gaseous hormone, ethylene, is a prime target for that purpose. The variation of ethylene production in natural accessions of the model species Arabidopsis thaliana was explored in response to the nitrate supply. Ethylene was measured with a laser-based photoacoustic detector. First, experimental conditions were established with Columbia-0 (Col-0) accession, which was grown in vitro on horizontal plates across a range of five nitrate concentrations (0.5, 1, 2.5, 5, or 10 mM). The concentrations of 1 and 10 mM nitrate were retained for further characterization. Along with a decrease of total dry biomass and higher biomass allocation to the roots, the ethylene production was 50% more important at 1 mM than at 10 mM nitrate. The total transcript levels of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASES (ACS) in roots and those of ACC OXIDASES (ACO) in shoots increased by 100% between the same treatments. This was mainly due to higher transcript levels of ACS6 and of ACO2 and ACO4 respectively. The assumption was that during nitrogen deficiency, the greater biomass allocation in favor of the roots was controlled by ethylene being released in the shoots after conversion of ACC originating from the roots. Second, biomass and ethylene productions were measured in 20 additional accessions. Across all accessions, the total dry biomass and ethylene production were correlated negatively at 1 mM but positively at 10 mM nitrate. Furthermore, polymorphism was surveyed in ACC and ethylene biosynthesis genes and gene products among accessions. Very few substitutions modifying the amino acids properties in conserved motifs of the enzymes were found in the accessions. Natural variation of ethylene production could be further explored to improve Nitrogen Use Efficiency (NUE), in particular by manipulating features like the biomass production and the timing of senescence upon nitrogen limitation.

CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.

Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants.

Intraspecific variability of cadmium tolerance and accumulation, and cadmium-induced cell wall modifications in the metal hyperaccumulator Arabidopsis halleri.

Certain molecular mechanisms of Cd tolerance and accumulation have been identified in the model species Arabidopsis halleri, while intraspecific variability of these traits and the mechanisms of shoot detoxification were little addressed. The Cd tolerance and accumulation of metallicolous and non-metallicolous A. halleri populations from different genetic units were tested in controlled conditions. In addition, changes in shoot cell wall composition were investigated using Fourier transform infrared spectroscopy. Indeed, recent works on A. halleri suggest Cd sequestration both inside cells and in the cell wall/apoplast. All A. halleri populations tested were hypertolerant to Cd, and the metallicolous populations were on average the most tolerant. Accumulation was highly variable between and within populations, and populations that were non-accumulators of Cd were identified. The effect of Cd on the cell wall composition was quite similar in the sensitive species A. lyrata and in A. halleri individuals; the pectin/polysaccharide content of cell walls seems to increase after Cd treatment. Nevertheless, the changes induced by Cd were more pronounced in the less tolerant individuals, leading to a correlation between the level of tolerance and the extent of modifications. This work demonstrated that Cd tolerance and accumulation are highly variable traits in A. halleri, suggesting adaptation at the local scale and involvement of various molecular mechanisms. While in non-metallicolous populations drastic modifications of the cell wall occur due to higher Cd toxicity and/or Cd immobilization in this compartment, the increased tolerance of metallicolous populations probably involves other mechanisms such as vacuolar sequestration.

Tolerance to cadmium in plants: the special case of hyperaccumulators.

On sols highly polluted by trace metallic elements the majority of plant species are excluders, limiting the entry and the root to shoot translocation of trace metals. However a rare class of plants called hyperaccumulators possess remarkable adaptation because those plants combine extremely high tolerance degrees and foliar accumulation of trace elements. Hyperaccumulators have recently gained considerable interest, because of their potential use in phytoremediation, phytomining and biofortification. On a more fundamental point of view hyperaccumulators of trace metals are case studies to understand metal homeostasis and detoxification mechanisms. Hyperaccumulation of trace metals usually depends on the enhancement of at least four processes, which are the absorption from the soil, the loading in the xylem in the roots and the unloading from the xylem in the leaves and the detoxification in the shoot. Cadmium is one of the most toxic trace metallic elements for living organisms and its accumulation in the environment is recognized as a worldwide concern. To date, only nine species have been recognized as Cd hyperaccumulators that is to say able to tolerate and accumulate more than 0.01 % Cd in shoot dry biomass. Among these species, four belong to the Brassicaceae family with Arabidopsis halleri and Noccaea caerulescens being considered as models. An update of our knowledge on the evolution of hyperaccumulators will be presented here.

Plant science: the key to preventing slow cadmium poisoning.

Practically all human populations are environmentally exposed to cadmium (Cd), mostly through plant-derived food. A growing body of epidemiological evidence suggests that there is no margin of safety between current Cd exposure levels and the threshold for adverse health effects and, hence, there is an urgent need to lower human Cd intake. Here we review recent studies on rice (Oryza sativa) and Cd-hyperaccumulating plants that have led to important insights into the processes controlling the passage of Cd from the soil to edible plant organs. The emerging molecular understanding of Cd uptake, root retention, root-to-shoot translocation and grain loading will enable the development of low Cd-accumulating crops.

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens.

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd(2+), Cu(2+), Zn(2+) and Pb(2+), with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.

Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation.

There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Félix-de-Pallières, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Félix-de-Pallières) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.

Low magnesium status in plants enhances tolerance to cadmium exposure.

In a transcriptomic study of magnesium (Mg) starvation in Arabidopsis, we identified several genes that were differentially regulated which are involved in the detoxification process of nonessential heavy metals such as cadmium (Cd). We further tested the impact of low Mg status on Cd sensitivity in plants. Interestingly, a -Mg pretreatment of 7 d alleviated the bleaching of young leaves caused by Cd. No or little difference in Cd tissue concentration between the +Mg and -Mg plants was observed, suggesting that lower Cd toxicity was probably not attributable to modified root to shoot translocation. Mg deficiency also promoted an increase in the iron (Fe) concentration (up to one-fourth) in Cd-treated leaves. Because high Fe concentrations have previously been reported to prevent the harmful effects of Cd, we explored whether Fe homeostasis plays a role in the Mg-Cd interaction. A protective effect of -Mg pretreatment was also observed on Fe starvation. However, Fe foliar spray partially alleviated Cd-induced chloroses, while it almost completely restored chlorophyll content in Fe-deficient leaves. In conclusion, the protective effect of Mg against Cd toxicity could be attributable partly to the maintenance of Fe status but also to the increase in antioxidative capacity, detoxification and/or protection of the photosynthetic apparatus.

Quantitative trait loci analysis of mineral element concentrations in an Arabidopsis halleri x Arabidopsis lyrata petraea F2 progeny grown on cadmium-contaminated soil.

SUMMARY: This study describes the quantitative trait locus (QTL) analysis of cadmium (Cd), zinc (Zn), iron (Fe), potassium (K), magnesium (Mg) and calcium (Ca) accumulation in the pseudometallophyte Arabidopsis halleri under conditions of Cd excess using an interspecific A. halleri x Arabidopsis lyrata F(2) population. *Our data provide evidence for the implication of one major QTL in Cd hyperaccumulation in A. halleri, and suggests that Cd tolerance and accumulation are not independent in A. halleri. Moreover, the major loci responsible for Zn hyperaccumulation in the absence of Cd appear to be the same when Cd is present at high concentrations. *More than twofold higher Fe concentrations were measured in A. halleri shoots than in A. lyrata, suggesting a different regulation of Fe accumulation in the hyperaccumulator. *With the exception of Ca, the accumulation of Cd was significantly correlated with the accumulation of all elements measured in the F(2) progeny, suggesting pleiotropic gene action. However, QTL analysis identified pleiotropic QTLs only for Cd, Zn and Fe. Mg accumulation was negatively correlated with Cd accumulation, as well as with dry shoot biomass, suggesting that it might indicate cellular damage.

Mechanisms to cope with arsenic or cadmium excess in plants.

The metalloid arsenic and the heavy metal cadmium have no demonstrated biological function in plants. Both elements are highly toxic and of major concern with respect to their accumulation in soils, in the food-chain or in drinking water. Arsenate is taken up by phosphate transporters and rapidly reduced to arsenite, As(III). In reducing environments, As(III) is taken up by aquaporin nodulin 26-like intrinsic proteins. Cd(2+) enters the root via essential metal uptake systems. As(III) and Cd(2+) share some similarity between their toxicology and sequestration machineries. Recent progress in understanding the mechanisms of As and Cd uptake and detoxification is presented, including the elucidation of why rice takes up so much arsenic from soil and of mechanisms of As and Cd hypertolerance.

A major quantitative trait locus for cadmium tolerance in Arabidopsis halleri colocalizes with HMA4, a gene encoding a heavy metal ATPase.

Cadmium (Cd) tolerance seems to be a constitutive species-level trait in Arabidopsis halleri sp. halleri. Therefore, an interspecific cross was made between A. halleri and its closest nontolerant interfertile relative, Arabidopsis lyrata sp. petraea, and a first-generation backcross population (BC1) was used to map quantitative trait loci (QTL) for Cd tolerance. Three QTL were identified, which explained 43%, 24%, and 16% of the phenotypic variation in the mapping population. Heavy metal transporting ATPases4 (HMA4), encoding a predicted heavy metal ATPase, colocalized with the peak of the major QTL Cdtol-1 and was consequently further studied. HMA4 transcripts levels were higher in the roots and the shoots of A. halleri than in A. lyrata sp. petraea. Furthermore, HMA4 was also more highly expressed in all BC1 genotypes harboring the HMA4 A. halleri allele at the QTL Cdtol-1, independently of the presence of an A. halleri allele at the two other QTL. Overexpression of AhHMA4 in yeast (Saccharomyces cerevisiae) supported a role of HMA4 in zinc (Zn) and Cd transport by reducing the Cd and Zn contents of the yeast cells. In epidermal tobacco (Nicotiana tabacum) cells, AhHMA4:green fluorescent protein was clearly localized in the plasma membrane. Taken together, all available data point to the elevated expression of HMA4 P(1B)-type ATPase as an efficient mechanism for improving Cd/Zn tolerance in plants under conditions of Cd/Zn excess by maintaining low cellular Cd(2+) and Zn(2+) concentrations in the cytoplasm.

A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens.

Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation. We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T. caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae. This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase. The corresponding TcHMA4 full-length sequence was isolated from T. caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4). Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance. Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C. In planta, both HMA4 genes were expressed at a higher level in roots than in shoots. However, TcHMA4 shows a much higher constitutive expression than AtHMA4. Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character.